G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
fibrinogen gamma chain
G00000055 (Mus musculus)

Databases (6)

ENSG00000171557 (Ensembl human gene)
2266 (Entrez Gene)
FGG (GeneCards)
134850 (OMIM)
Marker Symbol
HGNC:3694 (HGNC)
Protein Sequence
P02679 (UniProt)

Literature (168)

Pubmed - other

  • Interaction between fibrinogen and IL-6 genetic variants and associations with cardiovascular disease risk in the Cardiovascular Health Study.

    Carty CL, Heagerty P, Heckbert SR, Jarvik GP, Lange LA, Cushman M, Tracy RP and Reiner AP

    Department of Epidemiology, University of Washington, Seattle, USA. calyca@u.washington.edu

    The inflammatory cytokine interleukin-6 (IL-6) is a main regulator of fibrinogen synthesis, though its interaction with fibrinogen genes (FGA, FGB, FGG) and subsequent impact on cardiovascular disease (CVD) risk is not well-studied. We investigated joint associations of fibrinogen and IL6 tagSNPs with fibrinogen concentrations, carotid intima-media thickness, and myocardial infarction or ischemic stroke in 3900 European-American Cardiovascular Health Study participants. To identify combinations of genetic main effects and interactions associated with outcomes, we used logic regression. We also evaluated whether the relationship between fibrinogen SNPs and fibrinogen level varied by IL-6 level using linear regression models with multiplicative interaction terms. Combinations of fibrinogen and IL6 SNPs were significantly associated with fibrinogen level (p < 0.005), but not with other outcomes. Fibrinogen levels were higher in individuals having FGB1437 (rs1800790) and lacking FGA6534 (rs6050) minor alleles; these SNPs interacted with IL6 rs1800796 to influence fibrinogen level. Marginally significant (p= 0.03) interactions between IL-6 level and FGA and FGG promoter SNPs associated with fibrinogen levels were detected. We identified potential gene-gene interactions influencing fibrinogen levels. Although IL-6 responsive binding sites are present in fibrinogen gene promoter regions, we did not find strong evidence of interaction between fibrinogen SNPs and IL6 SNPs or levels influencing CVD.

    Funded by: NHLBI NIH HHS: N01 HC-55222, N01 HC015103, N01 HC035129, N01 HC045133, N01-HC-75150, N01-HC-85079, N01-HC-85080, N01-HC-85081, N01-HC-85082, N01-HC-85083, N01-HC-85084, N01-HC-85085, N01-HC-85086, N01HC55222, N01HC75150, N01HC85079, N01HC85086, R01 HL071862, R01 HL071862-05A1, T32 HL007902, T32 HL007902-10, U01 HL080295, U01 HL080295-01

    Annals of human genetics 2010;74;1;1-10

  • The pleiotropic role of the fibrinogen gamma' chain in hemostasis.

    Uitte de Willige S, Standeven KF, Philippou H and Ariëns RA

    Division of Cardiovascular & Diabetes Research, Section on Mechanisms of Thrombosis, Faculty of Medicine and Health, University of Leeds, Leeds LS2 9JT, United Kingdom. s.uittedewillige@leeds.ac.uk

    A fraction of fibrinogen contains a differently spliced gamma chain called gamma', which presents itself mainly as heterodimer with the common gammaA chain as gammaA/gamma' fibrinogen. The gamma' chain differs from the gammaA chain in its C-terminus and has important functional implications for fibrinogen. The presence of the gamma' chain modulates thrombin and FXIII activity, influences clot architecture, and eliminates a platelet-binding site. Associations of gammaA/gamma' fibrinogen levels with arterial and venous thrombosis have been reported, indicating that the functional effects of gammaA/gamma' fibrinogen may contribute to the pathology of thrombosis. This review summarizes the key biologic aspects of this interesting variant of fibrinogen and discusses inconsistencies in current reports.

    Blood 2009;114;19;3994-4001

  • Impaired protofibril formation in fibrinogen gamma N308K is due to altered D:D and "A:a" interactions.

    Bowley SR, Okumura N and Lord ST

    Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.

    "A:a" knob-hole interactions and D:D interfacial interactions are important for fibrin polymerization. Previous studies with recombinant gammaN308K fibrinogen, a substitution at the D:D interface, showed impaired polymerization. We examined the molecular basis for this loss of function by solving the crystal structure of gammaN308K fragment D. In contrast to previous fragment D crystals, the gammaN308K crystals belonged to a tetragonal space group with an unusually long unit cell (a = b = 95 A, c = 448.3 A). Alignment of the normal and gammaN308K structures showed the global structure of the variant was not changed and the knob "A" peptide GPRP was bound as usual to hole "a". The substitution introduced an elongated positively charged patch in the D:D region. The structure showed novel, symmetric D:D crystal contacts between gammaN308K molecules, indicating the normal asymmetric D:D interface in fibrin would be unstable in this variant. We examined GPRP binding to gammaN308K in solution by plasmin protection assay. The results showed weaker peptide binding, suggesting that "A:a" interactions were altered. We examined fibrin network structures by scanning electron microscopy and found the variant fibers were thicker and more heterogeneous than normal fibers. Considered together, our structural and biochemical studies indicate both "A:a" and D:D interactions are weaker. We conclude that stable protofibrils cannot assemble from gammaN308K monomers, leading to impaired polymerization.

    Funded by: NHLBI NIH HHS: HL 31048, R01 HL031048, R01 HL031048-18, R01 HL031048-19, R01 HL031048-20; NIBIB NIH HHS: P30 EB009998

    Biochemistry 2009;48;36;8656-63

  • Fibrinogen alpha and gamma genes and factor VLeiden in children with thromboembolism: results from 2 family-based association studies.

    Nowak-Göttl U, Weiler H, Hernandez I, Thedieck S, Seehafer T, Schulte T and Stoll M

    Pediatric Hematology/Oncology, University of Muenster, Muenster, Germany.

    Previous case-control studies showed that genetic variation in the fibrinogen gamma gene (FGG) increased the risk for deep vein thrombosis (VT) in adults. We investigated the association between the fibrinogen alpha (FGA) and FGG haplotypes, the factor V(Leiden)-mutation, and pediatric VT and thromboembolic stroke (TS) in 2 independent study samples. Association analysis revealed that the FGA-H1 and FGG-H2 haplotypes were significantly overtransmitted to VT patients (FGA-H1, P = .05; FGG: H2, P = .032). In contrast, the FGG-H3 haplotype was undertransmitted (P = .022). In an independent study sample, FGA-H1 (P = .008) and FGG-H2 (P = .05) were significantly associated with TS. The association of FGA and FGG haplotypes with VT was more pronounced in FV(Leiden)-negative families (FGA-H1, P = .001; FGG-H2, P = .001), indicating a genetic interaction between both risk factors. The risk-conferring FGG-H2 and the protective FGG-H3 haplotypes correlated with low (FGG-H2) and high (FGG-H3) levels of the gamma' chain variant, respectively. These results provide independent and novel evidence that FGA-H1 and FGG-H2 variants are associated with an increased risk of VT and TS in children. The observed negative correlation of genetic VT risk with the plasma levels of the fibrinogen gamma' variant suggests that FGG-H2 and -H3 haplotypes modify thrombosis risk by controlling the level of this FGG splice isoform.

    Blood 2009;114;9;1947-53

  • Genetic variation in the fibrinogen-alpha and fibrinogen-gamma genes in relation to arterial stiffness: the Rotterdam Study.

    Sie MP, Isaacs A, de Maat MP, Mattace-Raso FU, Uitterlinden AG, Kardys I, Hofman A, Hoeks AP, Reneman RS, van Duijn CM and Witteman JC

    Department of Epidemiology & Biostatistics, Erasmus Medical Center, Rotterdam, The Netherlands.

    Objective: Arterial stiffness increases with age and predicts cardiovascular disease. Fibrinogen is an acute-phase protein and some studies showed an association with arterial stiffness. We studied genetic variation in the fibrinogen-alpha (FGA) and fibrinogen-gamma (FGG) genes, by means of single nucleotide polymorphisms (FGA: -58 G/A, 1374 G/A, 1526 T/C, 312 Thr/Ala, and FGG: 4288 G/A, 6326 G/A, 7792 T/C) and resultant haplotypes in relation to arterial stiffness.

    Methods: The present study (n = 3891) was embedded in the Rotterdam Study. Associations of the fibrinogen level, genotypes and haplotypes with aortic stiffness (pulse wave velocity), carotid stiffness (distensibility coefficient) and pulse pressure were investigated in men and women by analyses of variance, linear regression and by haplotype analyses. Analyses were adjusted for age, mean arterial pressure, heart rate, known cardiovascular risk factors and atherosclerosis.

    Results: Genotype analyses yielded associations of FGA-58 G/A (P = 0.040, for trend) and FGA-1526 T/C (P = 0.004, for trend) with the fibrinogen levels, but no consistent associations with arterial stiffness, in women. FGA-haplotype 4 was associated with the fibrinogen level (P = 0.02) in women. FGA-haplotype 3 and FGG-haplotype 2 were associated with aortic stiffness (P = 0.05) in women. No associations were found in men.

    Conclusion: Findings indicate that the fibrinogen level and genetic variation in the FGA and FGG genes may influence arterial stiffness in women.

    Journal of hypertension 2009;27;7;1392-8

  • Fibrinogen gamma gene 3'-end polymorphisms and risk of venous thromboembolism in the African-American and Caucasian population.

    Uitte de Willige S, Pyle ME, Vos HL, de Visser MC, Lally C, Dowling NF, Hooper WC, Bertina RM and Austin H

    Einthoven Laboratory for Experimental Vascular Medicine, Department of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, the Netherlands.

    Genetic determinants of venous thromboembolism (VTE) in the African-American population are poorly characterised. It was recently shown that fibrinogen gamma gene (FGG) polymorphisms 10034C>T and 9340T>C influence VTE risk in the Caucasian population. In the African-American population these polymorphisms are common, with allele frequencies above 25%. Here we evaluated whether these and other FGG 3'-end polymorphisms were associated with VTE risk in the African-American population and aimed to replicate the association in the Caucasian population. We examined 557 Caucasian patients and 678 Caucasian controls, and 537 African-American patients and 586 African-American controls from the ;Genetic Attributes and Thrombosis Epidemiology' (GATE) study. In the African-American population, 10034C>T and 9340T>C marginally influenced VTE-risk, with a 20% increase in risk for 10034TT carriers and a 20% reduction in risk for 9340CC carriers. In the Caucasian population, 10034TT was associated with a 1.7-fold increase in risk, which increased to 2.1-fold for idiopathic VTE patients. 9340CC significantly reduced VTE risk approximately two-fold. In conclusion, both FGG polymorphisms 10034C>T and 9340T>C influence VTE-risk, with the strongest effects observed in the Caucasian population, confirming previous data on these polymorphisms in this population.

    Thrombosis and haemostasis 2009;101;6;1078-84

  • Fibrinogen Krakow: a novel hypo/dysfibrinogenemia mutation in fibrinogen gamma chain (Asn325Ile) affecting fibrin clot structure and function.

    Undas A, Zdziarska J, Iwaniec T, Stepien E, Skotnicki AB, de Moerloose P and Neerman-Arbez M

    Institute of Cardiology, Jagiellonian University School of Medicine, 80 Pradnicka St., 31-202 Cracow, Poland. mmundas@cyf-kr.edu.pl

    Thrombosis and haemostasis 2009;101;5;975-6

  • Prothrombotic genetic variants and atherosclerosis in patients with cerebral ischemia of arterial origin.

    Pruissen DM, Kappelle LJ, Rosendaal FR, Algra A and SMART Study Group

    Department of Neurology, Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Utrecht, The Netherlands.

    Objective: Prothrombotic genetic variants associated with arterial disease probably affect arterial thrombus formation but may also promote atherosclerosis. We hypothesized that specific prothrombotic variants lead to advanced atherosclerosis in patients with cerebral ischemia of arterial origin.

    We included 689 patients with nondisabling cerebral ischemia of arterial origin. Twenty-two variants in 14 genes were genotyped. None of the variants was associated with carotid intima-media thickness or younger age at the occurrence of cerebral ischemia. Factor V Leiden (mean prevalence difference 25%; 95% CI 11-40) and the glycoprotein 1b-alpha Thr145Met variant (mean prevalence difference 12%; 95% CI 2.3-22) were associated with symptomatic carotid stenosis. After accounting for multiple testing by determination of the false discovery rate, only the association between factor V Leiden and symptomatic carotid stenosis remained present.

    Conclusions: Prothrombotic genetic variants showed no consistent association with three markers of advanced atherosclerosis in patients with cerebral ischemia of arterial origin. This study does not support the hypothesis that prothrombotic genetic variants have a direct role in the pathogenesis of atherosclerosis.

    Atherosclerosis 2009;204;1;191-5

  • Candidate genetic variants in the fibrinogen, methylenetetrahydrofolate reductase, and intercellular adhesion molecule-1 genes and plasma levels of fibrinogen, homocysteine, and intercellular adhesion molecule-1 among various race/ethnic groups: data from the Women's Genome Health Study.

    Albert MA, Pare G, Morris A, Rose L, Buring J, Ridker PM and Zee RY

    Center for Cardiovascular Disease Prevention, Donald W. Reynolds Center for Cardiovascular Disease Research, Brigham and Women's Hospital, Harvard Medical School, Boston MA, USA. maalbert@partners.org

    Background: Although inflammation is a core element of atherogenesis and plasma levels of fibrinogen (FGB), homocysteine, and intercellular adhesion molecule-1 (ICAM-1) differ by race/ethnicity, little is known about the role of genetic polymorphisms in the FGB, methylenetetrahydrofolate reductase (MTHFR), and ICAM-1 genes in determining plasma levels of these biomarkers. We examined the relationship between specific polymorphisms in the FGB, homocysteine, and ICAM-1 genes and their respective inflammatory biomarker concentrations at baseline in women from different race/ethnic groups.

    Methods: We genotyped specific polymorphisms in FGB (-455G>A/rs1800790), MTHFR (677C>T/rs1801133), and ICAM-1 (Lys56Met/rs5491 and Gly241Arg/rs1799969) at baseline and evaluated their relationship with respective inflammatory biomarker levels in 25,565 white, 476 African-American (black), 277 Hispanic, and 370 Asian women participating in the Women's Genome Health Study.

    Results: Overall, the minor allele frequencies for -455G>A were similar among white, Hispanic, and Asian women (17.2%-21.9%) but significantly lower in black women (6.6%, P < .001). The minor allele was associated with elevated FGB levels only in whites and Asians. After adjustment for age, body mass index, smoking, postmenopausal status, diabetes, hormone replacement therapy use, hypertension, and education, black women had the highest FGB levels compared to other race/ethnic groups. The minor allele frequency of the MTHFR 677C>T polymorphism was lowest in blacks (blacks 12.1%, whites 33.1%, Hispanics 39.0%, Asians 24.0%), and the T allele was only significantly associated with homocysteine levels in white women. Among whites, Hispanics, and Asians, the Lys56Met polymorphism was rare compared to the frequency in blacks (P < .001). Neither the Lys56Met nor Gly241Arg polymorphisms were common in Asians. Nonetheless, both polymorphisms were generally associated with lower ICAM-1 levels; the lowest levels were observed in black women.

    Conclusion: We found significant associations between certain candidate genetic polymorphisms and baseline plasma levels of FGB, homocysteine, and ICAM-1 in women from various race/ethnic groups. The present investigation is hypothesis generating and suggests genetic determination of differential concentrations of these atherosclerosis-related inflammatory biomarkers differ among various race/ethnic groups.

    Funded by: NCI NIH HHS: R01 CA047988, R01 CA047988-09, R01 CA047988-10, R01 CA047988-11, R01 CA047988-12, R01 CA047988-13, R01 CA047988-14, R01 CA047988-15, R01 CA047988-16, R01 CA047988-17, R01 CA047988-18; NHLBI NIH HHS: R01 HL043851, R01 HL043851-09, R01 HL043851-10

    American heart journal 2009;157;4;777-83.e1

  • Novel loci, including those related to Crohn disease, psoriasis, and inflammation, identified in a genome-wide association study of fibrinogen in 17 686 women: the Women's Genome Health Study.

    Danik JS, Paré G, Chasman DI, Zee RY, Kwiatkowski DJ, Parker A, Miletich JP and Ridker PM

    Center for Cardiovascular Disease Prevention, Donald W. Reynolds Center for Cardiovascular Research, and Translational Medicine Division, Brigham and Women's Hospital, 900 Commonwealth Ave. East, Boston, MA 02215, USA.

    Background: Fibrinogen is a multifunctional circulating glycoprotein involved in wound healing, thrombosis, platelet aggregation, and inflammation, and elevated levels predict vascular disease. Despite evidence of crucial biological function and moderate heritability, comprehensive analysis of the influence of genetic variation on fibrinogen is not available.

    To address this issue, we undertook a genome-wide association study evaluating the potential relationships between 337 343 single-nucleotide polymorphisms (SNPs) and plasma fibrinogen levels among 17 686 apparently healthy women participating in the Women's Genome Health Study. As C-reactive protein is also an inflammatory marker known to predict cardiovascular diseases, we compared the determinants of fibrinogen levels with those of C-reactive protein. Four novel loci were identified, in addition to the fibrinogen gene cluster, which were associated with fibrinogen levels at genome-wide levels of significance (range of probability values from 8.82 x 10(-09) to 8.04 x 10(-39)). Two of the loci are related to common chronic inflammatory diseases: the first, at locus 5q31.1 (SLC22A5, SLC22A4, IRF1), lies immediately adjacent to a locus linked to Crohn disease (P value for lead SNP, 1.24 x 10(-12)) and the second, at locus 17q25.1 (CD300LF, SLC9A3R1, NAT9), has been associated with psoriasis (P value for lead SNP, 7.72 x 10(-11)). A third locus at 1q21.3 (IL6R) lies within the interleukin 6 receptor gene, a critical component of the inflammatory cascade (P value for lead SNP, 1.80 x 10(-11)). A novel locus at 2q34 (CPSI) participates in the urea cycle (P=8.82 x 10(-09)). The majority of implicated SNPs showed little evidence of dual association with C-reactive protein levels.

    Conclusions: A genome-wide survey of the human genome identifies novel loci related to common chronic inflammatory diseases as genetic determinants of fibrinogen levels, in addition to loci that relate to the inflammatory cascade, the urea cycle, and the fibrinogen gene cluster.

    Funded by: NCI NIH HHS: R01 CA047988, R01 CA047988-09, R01 CA047988-10, R01 CA047988-11, R01 CA047988-12, R01 CA047988-13, R01 CA047988-14, R01 CA047988-15, R01 CA047988-16, R01 CA047988-17, R01 CA047988-18; NHLBI NIH HHS: K08 HL076443-01, K08 HL076443-02, K08 HL076443-03, K08 HL076443-04, K08 HL076443-05, R01 HL043851, R01 HL043851-09, R01 HL043851-10, R01 HL080467, R01 HL080467-01, R01 HL080467-02, R01 HL080467-03

    Circulation. Cardiovascular genetics 2009;2;2;134-41

  • Fibrinogen-gamma C-terminal fragments induce endothelial barrier dysfunction and microvascular leak via integrin-mediated and RhoA-dependent mechanism.

    Guo M, Daines D, Tang J, Shen Q, Perrin RM, Takada Y, Yuan SY and Wu MH

    Division of Research, Department of Surgery, University of California Davis School of Medicine, Sacramento, CA 95817, USA.

    Objectives: The purposes of this study were to characterize the direct effect of the C-terminal fragment of fibrinogen gamma chain (gammaC) on microvascular endothelial permeability and to examine its molecular mechanism of action.

    Intravital microscopy was performed to measure albumin extravasation in intact mesenteric microvasculature, followed by quantification of hydraulic conductivity in single perfused microvessels. Transendothelial electric resistance was measured in microvascular endothelial cells in combination with immunoblotting and immunocytochemistry. The results show that gammaC induced time- and concentration-dependent increases in protein transvascular flux and water permeability and decreases in endothelial barrier function, coupled with Rho GTPase activation, myosin light chain phosphorylation, and stress fiber formation. Depletion of RhoA via siRNA knockdown or pharmacological inhibition of RhoA signaling attenuated gammaC-induced barrier dysfunction. Imaging analyses demonstrated binding of gammaC to endothelial cells; the interaction was inhibited during blockage of the alphavbeta3 integrin. Furthermore, in vivo experiments showed that the microvascular leak response to gammaC was attenuated in integrin beta3(-/-) animals.

    Conclusion: Fibrinogen-gamma C terminus directly interacts with the microvascular endothelium causing fluid and protein leak. The endothelial response to gammaC involves an integrin receptor-mediated RhoA-dependent signaling pathway that leads to paracellular hyperpermeability.

    Funded by: NHLBI NIH HHS: HL-061507, HL-070752, HL-073324, R01 HL061507, R01 HL061507-11, R01 HL070752, R01 HL070752-07, R01 HL073324, R01 HL073324-06

    Arteriosclerosis, thrombosis, and vascular biology 2009;29;3;394-400

  • Association study between variants in the fibrinogen gene cluster, fibrinogen levels and hypertension: results from the MONICA/KORA study.

    Kolz M, Baumert J, Gohlke H, Grallert H, Döring A, Peters A, Wichmann HE, Koenig W and Illig T

    Department of Internal Medicine II - Cardiology, University of Ulm Medical Center, Robert-Koch Str. 8, D-89081 Ulm, Germany.

    Previous studies reported a gender-specific association between plasma fibrinogen concentrations and incident hypertension. We systematically analysed polymorphisms and haplotypes across the fibrinogen gene cluster with fibrinogen levels and assessed their contribution to prevalent hypertension in 2,200 men and 2,159 women from the population-based MONICA/KORA Augsburg study. Eleven tagging single nucleotide polymorphisms (SNPs) were systematically selected in the three fibrinogen genes and haplotypes were reconstructed. The minor alleles of two SNPs, rs2227401 (FGB) and rs2070016 (FGA) and the haplotypes tagged by those variants, were significantly associated with higher fibrinogen concentrations in both, men and women, explaining 1% of the total variance of fibrinogen concentrations. In addition, a FGG haplotype, tagged by rs1049636, was associated with lower concentrations of fibrinogen in women, but not in men. Regarding hypertension, we detected a significant association with a FGA promoter variant (rs2070008) in women only, whereas fibrinogen haplotypes were not associated with hypertension after correction for multiple comparisons in either men or women. In conclusion, our results suggest that variants in all three fibrinogen genes are significantly associated with differences in fibrinogen concentrations with modest contribution to phenotypic variance. It is likely that other genetic variants outside the fibrinogen gene loci are involved in the regulation of fibrinogen concentrations. In addition, one FGA promoter variant was significantly associated with hypertension in women. Confirmation of these findings by future studies is warranted.

    Thrombosis and haemostasis 2009;101;2;317-24

  • Fibrinogen has chaperone-like activity.

    Tang H, Fu Y, Cui Y, He Y, Zeng X, Ploplis VA, Castellino FJ and Luo Y

    National Engineering Laboratory for Anti-tumor Protein Therapeutics Tsinghua University, Beijing 100084, China.

    Partially or completely unfolded polypeptides are highly prone to aggregation due to nonspecific interactions between their exposed hydrophobic surfaces. Extracellular proteins are continuously subjected to stresses conditions, but the existence of extracellular chaperones remains largely unexplored. The results presented here demonstrate that one of the most abundant extracellular proteins, fibrinogen has chaperone-like activity. Fibrinogen can specifically bind to nonnative form of citrate synthase and inhibit its thermal aggregation and inactivation in an ATP-independent manner. Interestingly, fibrinogen maintains thermal-denatured luciferase in a refolding competent state allowing luciferase to be refolded in cooperation with rabbit reticulocyte lysate. Fibrinogen also inhibits fibril formation of yeast prion protein Sup35 (NM). Furthermore, fibrinogen rescues thermal-induced protein aggregation in the plasma of fibrinogen-deficient mice. Our studies demonstrate the chaperone-like activity of fibrinogen, which not only provides new insights into the extracellular chaperone protein system, but also suggests potential diagnostic and therapeutic approaches to fibrinogen-related pathological conditions.

    Funded by: NHLBI NIH HHS: HL073750, P01 HL073750, P01 HL073750-01A19002

    Biochemical and biophysical research communications 2009;378;3;662-7

  • Thrombosis risk modification in transgenic mice containing the human fibrinogen thrombin-binding gamma' chain sequence.

    Mosesson MW, Cooley BC, Hernandez I, Diorio JP and Weiler H

    Blood Research Institute, BloodCenter of Wisconsin, Milwaukee, WI 53201-2178, USA. michael.mosesson@bcw.edu

    Thrombin binding activity in murine fibrin (Antithrombin I) is restricted to its E domains inasmuch as murine gamma' chains (mu-gamma') do not bind thrombin. This feature prompted us to produce a 'gain-of-function' transgenic mouse in which the wild-type (WT) C-terminal mu-gamma' chain fibrinogen sequence had been replaced with the C-terminal thrombin-binding human gamma' sequence.

    Results: This procedure resulted in a murine fibrinogen species containing chimeric hu-gamma' chains (hu-gamma' fibrinogen). As anticipated, thrombin bound to WT fibrin at a single class of sites, whereas thrombin binding to heterodimeric hu-gamma'-containing fibrin was increased, reflecting its content of hu-gamma' chains. In an electrolytically-induced femoral vein thrombosis injury model, we found no differences in the volume of thrombus generation between WT and heterozygous hu-gamma' mice. However, heterozygous factor (F) V Leiden (FVL(+/-)) mice developed greater thrombus volumes than did WT controls (P < 0.01). In doubly heterozygous FVL(+/-), hu-gamma' mice, thrombus formation was reduced to WT levels (P < 0.05).

    Conclusions: Murine hu-gamma' fibrinogen down-regulates venous thrombosis in the presence of another known thrombosis risk factor, FV Leiden. This finding indicates that hu-gamma' chain-containing fibrinogen is a thrombosis risk modifier.

    Funded by: NHLBI NIH HHS: R01 HL-70627

    Journal of thrombosis and haemostasis : JTH 2009;7;1;102-10

  • Serum fibrinogen levels are an independent predictor of mortality in patients with chronic kidney disease (CKD) stages 3 and 4.

    Goicoechea M, de Vinuesa SG, Gómez-Campderá F, Aragoncillo I, Verdalles U, Mosse A and Luño J

    Servicio de Nefrología, Hospital Universitario Gregorio Marañón, Madrid, Spain. albvia@terra.es

    Patients with chronic kidney disease have substantial risk for cardiovascular mortality, but the relative importance of traditional and novel risk factors is unknown. Several studies in hemodialysis patients have demon 1f40 strated that inflammatory markers are potent predictors of mortality, however there are scarce data about stable patients with moderate chronic kidney disease. 128 outpatients with estimated glomerular filtration of less than 60 ml/min per 1.73 m(2) were included in the study. Medical records about cardiovascular factors were recorded. Analytical parameters and inflammation markers were determined in baseline period. Participants were initially recruited from January 2002 to May 2002. The average length of follow-up in this longitudinal study was 5.5 years. Mortality and non-fatal cardiovascular events were the end points. Median follow-up was 67.8 months, all cause of mortality was 22.7%/(n=29) and non-fatal cardiovascular events were 39.1% (n=50). In multivariate analysis adjusting for demographic, cardiovascular and kidney disease factors, age hazard ratio (HR): 1.01, interval confidence (IC): 1.00-1.10), previous congestive heart failure (HR: 3.50, IC: 1.10-11.17) and both high CRP (HR: 3.48, IC: 1.53-7.59) and serum fibrinogen (HR: 1.45, IC: 1.04-1.50) were independent predictors of all-cause of mortality. Age (HR: 1.06, IC: 1.01-1.11), high CRP (HR: 2.80, IC: 1.60-4.90), cardiac troponin T (HR: 1.21, IC: 1.04-1.40) and previous coronary disease (HR: 2.67, IC: 1.28-5.54) but not serum fibrinogen were independent predictors of non-fatal cardiovascular events. Both high CRP and high serum fibrinogen levels and previous congestive heart failure measured in CKD stages 3 and 4, are independent risk factors for all-cause of mortality. High CRP but not high serum fibrinogen is a risk factor for non-fatal cardiovascular events. These results suggest that high CRP and high serum fibrinogen provide prognostic information in CKD patients.

    Kidney international. Supplement 2008;111;S67-70

  • Association between the SERPING1 gene and age-related macular degeneration: a two-stage case-control study.

    Ennis S, Jomary C, Mullins R, Cree A, Chen X, Macleod A, Jones S, Collins A, Stone E and Lotery A

    Genetic Epidemiology and Bioinformatics Group, University of Southampton, Human Genetics Division (Mp 808), Southampton General Hospital, Southampton, UK.

    Background: Age-related macular degeneration is the most prevalent form of visual impairment and blindness in developed countries. Genetic studies have made advancements in establishing the molecular cause of this disease, identifying mutations in the complement factor H (CFH) gene and a locus on chromosome 10 encompassing the HTRA1/LOC387715/ARMS2 genes. Variants in complement 3 (C3) and an HLA locus containing both factor B and C2 genes have also been implicated. We aimed to identify further genetic risk factors for this disease.

    Methods: We used a case-control study design in a UK sample of patients with age-related macular degeneration (n=479) and controls (n=479) and undertook a low-density screen of 32 genes using 93 single nucleotide polymorphisms (SNPs). Genes were selected as candidates on the basis of potential functional relevance to age-related macular degeneration. Significant initial findings were confirmed by replication in an independent US cohort of 248 unrelated patients with disease and 252 controls, and by high-density genotyping around association signals.

    Findings: The SNP variant rs2511989, located within intron six of the SERPING1 gene, showed highly significant genotypic association with age-related macular degeneration (uncorrected p=4.0x10(-5), corrected p=0.00372). We detected no evidence for association between disease and the other 31 candidate genes. The odds ratio for age-related macular degeneration in rs2511989 G/A heterozygotes compared with wild type G/G homozygotes was 0.63 (95% CI 0.47-0.84). A similar comparison of the A/A homozygotes with the wild type yielded an odds ratio of 0.44 (0.31-0.64). We replicated the observed genotypic association in a US cohort (p=0.008). Furthermore, a secondary high-density genotyping study across the SERPING1 gene region identified five additional SNP variants similarly associated with age-related macular degeneration (rs2244169, rs2511990, rs2509897, rs1005510, and rs2511988).

    Interpretation: Genetic variation in SERPING1 significantly alters susceptibility to age-related macular degeneration. SERPING1 encodes the C1 inhibitor, which has a crucial role in inhibition of complement component 1 (C1) and might implicate the classic pathway of complement activation in this disease.

    Funded by: Howard Hughes Medical Institute; NEI NIH HHS: EY-016822, EY-017451, R01 EY016822, R01 EY017451; Wellcome Trust: 076169

    Lancet (London, England) 2008;372;9652;1828-34

  • Congenital hypofibrinogenemia: characterization of two missense mutations affecting fibrinogen assembly and secretion.

    Platè M, Asselta R, Spena S, Spreafico M, Fagoonee S, Peyvandi F, Tenchini ML and Duga S

    Department of Biology and Genetics for Medical Sciences, University of Milan, Via Viotti, 3/5-20133 Milan, Italy.

    Congenital hypofibrinogenemia is a rare bleeding disorder characterized by abnormally low levels of fibrinogen in plasma, generally due to heterozygous mutations in one of the three fibrinogen genes (FGA, FGB, and FGG, coding for Aalpha, Bbeta, and gamma chain, respectively). Hypofibrinogenemic patients are usually asymptomatic, whereas individuals bearing similar mutations in the homozygous or compound heterozygous state develop a severe bleeding disorder: afibrinogenemia. The mutational spectrum of these quantitative fibrinogen disorders includes large deletions, point mutations causing premature termination codons, and missense mutations affecting fibrinogen assembly or secretion, distributed throughout the 50-kb fibrinogen gene cluster. In this study, we report the mutational screening of two unrelated hypofibrinogenemic patients leading to the identification of two missense mutations, one hitherto unknown (alphaCys45Phe), and one previously described (gammaAsn345Ser). The involvement of alphaCys45Phe and gammaAsn345Ser in the pathogenesis of hypofibrinogenemia was investigated by in-vitro expression experiments. Both mutations were demonstrated to cause a severe impairment of intracellular fibrinogen processing, either by affecting half-molecule dimerization (alphaCys45Phe) or by hampering hexamer secretion (gammaAsn345Ser).

    Blood cells, molecules & diseases 2008;41;3;292-7

  • gammaA/gamma' fibrinogen inhibits thrombin-induced platelet aggregation.

    Lovely RS, Rein CM, White TC, Jouihan SA, Boshkov LK, Bakke AC, McCarty OJ and Farrell DH

    Department of Biomedical Sciences, Missouri State University, Springfield, MO, USA.

    The minor gammaA/gamma' fibrinogen isoform contains a high affinity binding site for thrombin exosite II that is lacking in the major gammaA/gammaA fibrinogen isoform. We therefore investigated the biological consequences of the gamma' chain binding to thrombin. Thrombin-induced platelet aggregation was inhibited by gammaA/gamma' fibrinogen. Carboxyl terminal peptide fragment gamma'410-427 from the gamma' chain was also inhibitory, with an IC(50) of approximately 200 microM in whole plasma. Deletion of the peptide from either the amino or carboxyl end significantly decreased inhibition. In contrast to thrombin-induced platelet aggregation, aggregation induced by epinephrine, ADP, arachidonic acid, or SFLLRN peptide showed little inhibition by the gamma' peptide. The inhibition of thrombin-induced platelet aggregation was not due to direct inhibition of the thrombin active site, since cleavage of a small peptidyl substrate was 91% of normal even in the presence of 1 mM gamma'410-427. The gamma'410-427 peptide blocked platelet adhesion to immobilized thrombin under both static and flow conditions, blocked soluble thrombin binding to platelet GPIbalpha, and inhibited PAR1 cleavage by thrombin. These results suggest that the gamma' chain of fibrinogen inhibits thrombin-induced platelet aggregation by binding to thrombin exosite II. Thrombin that is bound to the gamma' chain is thereby prevented from activating platelets, while retaining its amidolytic activity.

    Funded by: NHLBI NIH HHS: F32 HL71463, K02HL04215, R21 HL75006, R41HL074535

    Thrombosis and haemostasis 2008;100;5;837-46

  • Fibrinogen-elongated gamma chain inhibits thrombin-induced platelet response, hindering the interaction with different receptors.

    Lancellotti S, Rutella S, De Filippis V, Pozzi N, Rocca B and De Cristofaro R

    Institute of Internal Medicine and Geriatrics, and Haemostasis Research Centre, Catholic University School of Medicine, 00168 Rome, Italy.

    The expression of the elongated fibrinogen gamma chain, termed gamma', derives from alternative splicing of mRNA and causes an insertion sequence of 20 amino acids. This insertion domain interacts with the anion-binding exosite (ABE)-II of thrombin. This study investigated whether and how gamma' chain binding to ABE-II affects thrombin interaction with its platelet receptors, i.e. glycoprotein Ibalpha (GpIbalpha), protease-activated receptor (PAR) 1, and PAR4. Both synthetic gamma' peptide and fibrinogen fragment D*, containing the elongated gamma' chain, inhibited thrombin-induced platelet aggregation up to 70%, with IC(50) values of 42+/-3.5 and 0.47+/-0.03 microm, respectively. Solid-phase binding and spectrofluorimetric assays showed that both fragment D* and the synthetic gamma' peptide specifically bind to thrombin ABE-II and competitively inhibit the thrombin binding to GpIbalpha with a mean K(i) approximately 0.5 and approximately 35 microm, respectively. Both these gamma' chain-containing ligands allosterically inhibited thrombin cleavage of a synthetic PAR1 peptide, of native PAR1 molecules on intact platelets, and of the synthetic chromogenic peptide D-Phe-pipecolyl-Arg-p-nitroanilide. PAR4 cleavage was unaffected. In summary, fibrinogen gamma' chain binds with high affinity to thrombin and inhibits with combined mechanisms the platelet response to thrombin. Thus, its variations in vivo may affect the hemostatic balance in arterial circulation.

    The Journal of biological chemistry 2008;283;44;30193-204

  • A novel Asp344Val substitution in the fibrinogen gamma chain (fibrinogen Caen) causes dysfibrinogenemia associated with thrombosis.

    Robert-Ebadi H, Le Querrec A, de Moerloose P, Gandon-Laloum S, Borel Derlon A and Neerman-Arbez M

    Division of Angiology and Haemostasis, University Hospital, Geneva, Switzerland.

    A 5-year-old boy was hospitalized for acute appendicitis. Routine preoperative hemostasis screening resulted in a diagnosis of dysfibrinogenemia. Fifteen days after the operation the patient was re-hospitalized for deep vein thrombosis. Genetic analysis of the fibrinogen genes revealed a novel missense mutation in exon 8 of fibrinogen gamma-chain gene (FGG): c.1031A>T, p.Asp344Val (p.Asp318Val in the mature chain) in heterozygosity. Interestingly, this same residue in the fibrinogen gamma chain was previously found to be mutated to a glycine (fibrinogen Giessen IV) in another young dysfibrinogenemia patient with thrombosis. The side chain of Asp344 (or Asp318) in the gamma chain is directly involved in binding to calcium. Abnormal polymerization of fibrin in fibrinogen Giessen IV and in the novel fibrinogen Caen described here could lead to the formation of abnormal clots leading to thrombosis, in addition to abnormal thrombin binding and decreased fibrinolysis.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2008;19;7;697-9

  • A genome-wide association study for late-onset Alzheimer's disease using DNA pooling.

    Abraham R, Moskvina V, Sims R, Hollingworth P, Morgan A, Georgieva L, Dowzell K, Cichon S, Hillmer AM, O'Donovan MC, Williams J, Owen MJ and Kirov G

    Department of Psychological Medicine, Cardiff University School of Medicine, Heath Park, Cardiff, CF14 4XN, UK. AbrahamRA@Cardiff.ac.uk

    Background: Late-onset Alzheimer's disease (LOAD) is an age related neurodegenerative disease with a high prevalence that places major demands on healthcare resources in societies with increasingly aged populations. The only extensively replicable genetic risk factor for LOAD is the apolipoprotein E gene. In order to identify additional genetic risk loci we have conducted a genome-wide association (GWA) study in a large LOAD case - control sample, reducing costs through the use of DNA pooling.

    Methods: DNA samples were collected from 1,082 individuals with LOAD and 1,239 control subjects. Age at onset ranged from 60 to 95 and Controls were matched for age (mean = 76.53 years, SD = 33), gender and ethnicity. Equimolar amounts of each DNA sample were added to either a case or control pool. The pools were genotyped using Illumina HumanHap300 and Illumina Sentrix HumanHap240S arrays testing 561,494 SNPs. 114 of our best hit SNPs from the pooling data were identified and then individually genotyped in the case - control sample used to construct the pools.

    Results: Highly significant association with LOAD was observed at the APOE locus confirming the validity of the pooled genotyping approach.For 109 SNPs outside the APOE locus, we obtained uncorrected p-values </= 0.05 for 74 after individual genotyping. To further test these associations, we added control data from 1400 subjects from the 1958 Birth Cohort with the evidence for association increasing to 3.4 x 10-6 for our strongest finding, rs727153.rs727153 lies 13 kb from the start of transcription of lecithin retinol acyltransferase (phosphatidylcholine - retinol O-acyltransferase, LRAT). Five of seven tag SNPs chosen to cover LRAT showed significant association with LOAD with a SNP in intron 2 of LRAT, showing greatest evidence of association (rs201825, p-value = 6.1 x 10-7).

    Conclusion: We have validated the pooling method for GWA studies by both identifying the APOE locus and by observing a strong enrichment for significantly associated SNPs. We provide evidence for LRAT as a novel candidate gene for LOAD. LRAT plays a prominent role in the Vitamin A cascade, a system that has been previously implicated in LOAD.

    Funded by: Medical Research Council: G0300429, G9810900

    BMC medical genomics 2008;1;44

  • Common genetic polymorphisms and haplotypes of fibrinogen alpha, beta, and gamma chains affect fibrinogen levels and the response to proinflammatory stimulation in myocardial infarction survivors: the AIRGENE study.

    Jacquemin B, Antoniades C, Nyberg F, Plana E, Müller M, Greven S, Salomaa V, Sunyer J, Bellander T, Chalamandaris AG, Pistelli R, Koenig W and Peters A

    Centre for Environmental Research, Municipal Institute of Medical Research, Barcelona, Spain.

    Objectives: This study was designed to investigate whether single nucleotide polymorphisms (SNPs) and haplotypes of the fibrinogen gene-cluster (fibrinogen chains alpha [FGA], beta [FGB], and gamma [FGG]) could explain the inter- and intraindividual variability of fibrinogen levels in patients with atherosclerosis. We also searched for genetic determinants affecting the responses of fibrinogen genes to proinflammatory stimulation.

    Background: The mechanisms regulating fibrinogen levels are not fully understood, and they are likely to be regulated by complex gene-environment interactions.

    Methods: In the AIRGENE study, 895 survivors of myocardial infarction from 5 European cities were followed prospectively for 6 to 8 months, and plasma fibrinogen, interleukin (IL)-6, and C-reactive protein levels were determined monthly. We analyzed 21 SNPs and the corresponding haplotypes in the 3 fibrinogen genes.

    Results: Eight SNPs in FGA and FGB were significantly associated with fibrinogen levels. Similarly, 2 different haplotypes in FGA and 3 in FGB were also associated with mean fibrinogen levels. The IL-6 levels had a significant impact on the associations between SNPs/haplotypes in FGA/FGB and fibrinogen levels. We also identified SNPs and haplotypes in FGA and FGB with strong impact on the intraindividual variability of fibrinogen during the follow-up period.

    Conclusions: We identified common SNPs and haplotypes on FGA/FGB genes, explaining the interindividual and intraindividual variability of fibrinogen levels, in patients with a history of myocardial infarction. We have also identified for the first time, SNPs/haplotypes on FGA/FGB whose effects on fibrinogen expression are modified by the underlying IL-6 levels. These findings may have an impact on risk stratification and the design of genetically guided therapeutic approaches in patients with advanced atherosclerosis.

    Journal of the American College of Cardiology 2008;52;11;941-52

  • Variation in fibrinogen FGG and FGA genes and risk of stroke: the Rotterdam Study.

    Cheung EY, Bos MJ, Leebeek FW, Koudstaal PJ, Hofman A, de Maat MP and Breteler MM

    Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands.

    Haplotypes of the fibrinogen gamma and alpha (FGG and FGA) genes are associated with the structure of the fibrin network and may therefore influence the risk of stroke. We investigated the relationship between common variation in these genes with ischemic and haemorrhagic stroke. The study was based on 6,275 participants of the prospective population-based Rotterdam Study who at baseline (1990-1993) were aged 55 years or over, free from stroke, and had successful assessment of at least one FGG or FGA single nucleotide polymorphisms (SNP). Common haplotypes were estimated using seven tagging SNPs across a 30 kb region containing the FGG and FGA genes. Follow-up for incident stroke was complete until January 1, 2005. Associations between constructed haplotypes and risk of stroke were estimated with an age- and sex-adjusted logistic regression model. We observed 668 strokes, of which 393 were ischemic and 62 haemorrhagic, during a median follow-up time of 10.1 years. FGG + FGA haplotype 3 (H3) was associated with an increased risk of ischemic stroke (odds ratio [OR] 1.36, 95% confidence interval [CI] 1.09-1.69) and the risk estimate for hemorrhagic stroke was 0.71 (95% CI 0.46-1.09) compared to the most frequent H1. The FGG and FGA genes were not associated with stroke or its subtypes when analyzed separately. In conclusion, risk of ischemic stroke was higher in FGG + FGA H3 than in H1. The results suggested that an opposite association may exist for haemorrhagic stroke.

    Thrombosis and haemostasis 2008;100;2;308-13

  • Haplotypes of the fibrinogen gene and cerebral small vessel disease: the Rotterdam scan study.

    van Oijen M, Cheung EY, Geluk CE, Hofman A, Koudstaal PJ, Breteler MM and de Maat MP

    Department of Epidemiology and Biostatistics, Erasmus Medical Centre, PO Box 2040, 3000 CA Rotterdam, The Netherlands.

    Objective: Fibrinogen levels and fibrinogen clot structure have been implicated in the pathogenesis of vascular disease. We examined fibrinogen levels and variation in fibrinogen genes (fibrinogen gamma (FGG), alpha (FGA) and beta (FGB)), which have been associated with fibrin clot structure and fibrinogen levels, in relation to cerebral small vessel disease (SVD).

    This study was performed as part of the Rotterdam Scan Study, a population based study in 1077 elderly patients undergoing cerebral MRI. Plasma fibrinogen levels and haplotypes were determined. We examined the association between fibrinogen levels and haplotype with silent brain infarcts and white matter lesions using logistic regression models. We constructed seven haplotypes (frequency >0.01) that describe the total common variation in the FGG and FGA genes. Haplotype 2 (GATAGTG) was associated with the presence of silent brain infarcts compared with the most frequent haplotype (GGTGGTA) (OR 1.41, 95% CI 1.03 to 1.94). Haplotype 3 (GGCGATA) was associated with periventricular white matter lesions in the highest tertile of the distribution (OR 1.40, 95% CI 1.01 to 1.92). No association was found between plasma fibrinogen levels and SVD.

    Conclusions: Our study provides evidence for an association of common variation in the FGG and FGA genes with cerebral SVD. It is possible that the structure of the fibrin clot rather than plasma fibrinogen levels plays a role in the pathogenesis of cerebral SVD.

    Journal of neurology, neurosurgery, and psychiatry 2008;79;7;799-803

  • Fibrinogen gene variation and ischemic stroke.

    Jood K, Danielson J, Ladenvall C, Blomstrand C and Jern C

    Institute of Neuroscience and Physiology, the Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden.

    Background: Plasma fibrinogen level and fibrin clot structure are heritable traits that may be of importance in the pathogenesis of ischemic stroke.

    Objectives: To investigate associations between variation in the fibrinogen gamma (FGG), alpha (FGA) and beta (FGB) genes, fibrinogen level, and ischemic stroke.

    Methods: The Sahlgrenska Academy Study on Ischemic Stroke comprises 600 cases and 600 matched population controls. Stroke subtypes were defined according to TOAST criteria. Plasma fibrinogen level was measured by an automated clot-rate assay. Eight tagging single nucleotide polymorphisms (SNPs) were selected to capture genetic variation in the FGA, FGG, and FGB genes.

    Results: Plasma fibrinogen was independently associated with overall ischemic stroke and all subtypes, both in the acute stage (P < 0.001) and at three-month follow-up (P < 0.05). SNPs belonged to two haplotype blocks, one containing the FGB gene and the other the FGG and FGA genes. FGB haplotypes were associated with fibrinogen level (P < 0.01), but not with ischemic stroke. In contrast, FGG/FGA haplotypes showed independent association to ischemic stroke but not to fibrinogen level. In an additive model with the most common FGG/FGA haplotype (A1) as reference, the adjusted odds ratios of ischemic stroke were 1.4 [95% confidence interval (95% CI) 1.1-1.8], P < 0.01, 1.4 (95% CI 1.0-1.8), P < 0.05, and 1.5 (95% CI 1.0-2.1), P < 0.05 for the A2, A3, and A4 FGG/FGA haplotypes, respectively.

    Conclusion: FGG/FGA haplotypes show association to ischemic stroke. This association is independent of fibrinogen level, thus suggesting that the association between ischemic stroke and variation at the FGG/FGA genes is mediated by qualitative rather than quantitative effects on fibrin(ogen).

    Journal of thrombosis and haemostasis : JTH 2008;6;6;897-904

  • Common genetic variants associated with plasma fibrin D-dimer concentration in older European- and African-American adults.

    Lange LA, Reiner AP, Carty CL, Jenny NS, Cushman M and Lange EM

    Department of Genetics and the Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, NC, USA. leslie_lange@med.unc.edu

    D-dimer is a hemostasis marker that reflects ongoing fibrin formation and degradation. There is significant inter-individual and inter-population variability in D-dimer concentration, but whether genetic factors underlie these differences is largely unknown. We hypothesized that common coagulation gene variants contribute to differences in circulating D-dimer concentration.

    Methods: The setting was European-American (EA; n = 1858) and African-American (AA; n = 327) unrelated older adults from the Cardiovascular Health Study (CHS), in which we genotyped SNPs in 42 genes related to blood coagulation and fibrinolysis.

    Results: Several fibrinogen gene polymorphisms, including the Thr312Ala Aalpha chain variant and the FGG-10034 C/T variant, were associated with approximately 20% higher plasma D-dimer levels in EA (false discovery rate < 5% for covariate-adjusted model). There was also some evidence that a Pro41Leu variant of the PLAU gene encoding urinary plasminogen activator and non-coding polymorphism of the plasminogen activator inhibitor type 1 gene (SERPINE1) were associated with higher plasma D-dimer in EA. There were no significant associations between the studied coagulation or fibrinolysis gene SNPs and plasma D-dimer levels in the smaller AA sample. However, each standard deviation increase in European ancestry assessed by ancestry-informative gene markers was associated with approximately 10% lower mean D-dimer levels in AA.

    Conclusions: Together, common coagulation/fibrinolysis gene SNPs explained only approximately 2% of the variance in plasma D-dimer levels in EA. These findings suggest that the association of D-dimer with risk of vascular outcomes may be mediated largely by environmental factors, other genes, and/or genetic interactions.

    Funded by: NHLBI NIH HHS: N01 HC-15103, N01 HC-55222, N01-HC-85079, N01-HC-85086, R01 HL-071862, U01 HL080295

    Journal of thrombosis and haemostasis : JTH 2008;6;4;654-9

  • Fibrinogen genes and myocardial infarction: a haplotype analysis.

    Koch W, Hoppmann P, Biele J, Mueller JC, Schömig A and Kastrati A

    Deutsches Herzzentrum München and 1. Medizinische Klinik, Klinikum rechts der Isar, Munich, Germany. wkoch@dhm.mhn.de

    Objective: Fibrinogen has a role in inflammatory processes and participates in atherosclerotic plaque formation. Despite intensive investigation, there is no clear evidence for a role of variations in the genes coding for the fibrinogen-alpha, fibrinogen-beta, and fibrinogen-gamma polypeptide chains in myocardial infarction. We examined the association of haplotypes in the 50-kb fibrinogen gene region with myocardial infarction in 2 large case-control samples.

    Study sample 1 consisted of 3657 patients with myocardial infarction and 1211 control individuals and sample 2 comprised 1392 patients and 1392 controls. Haplotypes were inferred from genotype analyses of tagging single nucleotide polymorphisms dispersed among the fibrinogen genes. The frequencies of these haplotypes were not significantly different between the case and control groups in either sample (P > or = 0.07). In addition, haplotypes specific for individual fibrinogen genes were analyzed. No substantial differences in the frequencies of these haplotypes were observed between the groups (P > or = 0.13). Finally, haplotypes composed of SNPs that exhibited relatively low pairwise allelic associations among each other were examined. The proportions of the haplotypes were not significantly different between cases and controls (P > or = 0.12).

    Conclusions: A haplotype analysis did not reveal a link between genetic variations in the fibrinogen gene region and myocardial infarction.

    Arteriosclerosis, thrombosis, and vascular biology 2008;28;4;758-63

  • Fibrinogen gamma' in ischemic stroke: a case-control study.

    Cheung EY, Uitte de Willige S, Vos HL, Leebeek FW, Dippel DW, Bertina RM and de Maat MP

    To determine the contribution of fibrinogen gamma' levels and FGG haplotypes to ischemic stroke.

    Methods: Associations between fibrinogen gamma' levels, fibrinogen gamma'/total fibrinogen ratio, and FGG haplotypes with the risk of ischemic stroke were determined in 124 cases and 125 controls.

    Results: Fibrinogen gamma'/total fibrinogen ratio was higher in patients than in controls during the acute phase of the stroke and lower in the convalescent phase 3 months after the stroke. FGG haplotype 3 (H3) was associated with a reduced risk of ischemic stroke (odds ratio 0.60; 95% CI, 0.38 to 0.94), but not with the fibrinogen gamma'/total fibrinogen ratio. In contrast, FGG-H2 was associated with a decreased fibrinogen gamma'/total fibrinogen ratio, but not with risk of stroke.

    Conclusions: Fibrinogen gamma'/total fibrinogen ratio is associated with ischemic stroke, especially in the acute phase of the disease. In addition, FGG-H3 haplotype appears to be protective against ischemic stroke.

    Stroke 2008;39;3;1033-5

  • Associations between common fibrinogen gene polymorphisms and cardiovascular disease in older adults. The Cardiovascular Health Study.

    Carty CL, Cushman M, Jones D, Lange LA, Hindorff LA, Rice K, Jenny NS, Durda JP, Walston J, Carlson CS, Nickerson D, Tracy RP and Reiner AP

    Cardiovascular Health Research Unit, Department of Epidemiology, University of Washington, 1760 Minor Avenue, Seattle, WA 98101, USA. calyca@u.washington.edu

    Elevated plasma fibrinogen is a risk factor for cardiovascular disease (CVD), but associations between fibrinogen single nucleotide polymorphisms (SNPs) and disease risk are inconsistent. We investigated whether common (> or = 5% minor allele frequency) variation in the fibrinogen genes (FGA, FGB, FGG) is associated with fibrinogen concentration, carotid artery intima-medial thickness (IMT) and risk of incident myocardial infarction (MI), ischemic stroke and CVD mortality in European- (EA) and African-descent (AA) adults (> or = 65 years) from the Cardiovascular Health Study. TagSNPs were genotyped in 3,969 EA and 719 AA free of MI or stroke at baseline. Race-specific models included multiple testing correction and adjustment for sex, age and site. Among EA, minor alleles of FGA3807, FGB1437 and FGG902 were associated with higher fibrinogen levels; whereas FGA251, FGA2224, FGA6534 and FGG10034 were associated with lower levels, p<0.004 for each. Strongest associations were seen for FGB1437; each additional copy of the minor allele was associated with 13 mg/dl (95%CI: 9-16) higher fibrinogen level. Similar trends in AA were not significant. Fibrinogen haplotypes were not significantly associated with internal or common carotid IMT. No associations with MI or CVD mortality were seen in EA, though FGB1038 and FGG902 were significantly associated with increased and decreased risk of stroke in men, respectively, as were related haplotypes. FGB1038 was also associated with CVD mortality in AA, HR = 1.9 (95%CI: 1.3-2.7). In conclusion, while fibrinogen genetic variation was strongly associated with fibrinogen levels, there was less evidence of association with the more complex outcomes of IMT and CVD events.

    Funded by: NHLBI NIH HHS: HL 071862, N01 HC 15103, N01 HC 35129, N01 HC 55222, N01 HC 85079, N01 HC 85080, N01 HC 85081, N01 HC 85082, N01 HC 85083, N01 HC 85084, N01 HC 85085, N01 HC 85086, U01 HL 080295

    Thrombosis and haemostasis 2008;99;2;388-95

  • Fibrinogen synthesized by cancer cells augments the proliferative effect of fibroblast growth factor-2 (FGF-2).

    Sahni A, Simpson-Haidaris PJ, Sahni SK, Vaday GG and Francis CW

    Hematology/Oncology Division, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA. abha_sahni@urmc.rochester.edu

    Background: Fibroblast growth factor (FGF)-2 is a critical growth factor in normal and malignant cell proliferation and tumor-associated angiogenesis. Fibrinogen and fibrin bind to FGF-2 and modulate FGF-2 functions. Furthermore, we have shown that extrahepatic epithelial cells are capable of endogenous production of fibrinogen.

    Objective: Herein we examined the role of fibrinogen and FGF-2 interactions on prostate and lung adenocarcinoma cell growth in vitro.

    Methods: Cell proliferation was measured by (3)H-thymidine uptake and the specificity of FGF-2-fibrinogen interactions was measured using wild-type and mutant FGF-2s, fibrinogen gamma-chain (FGG) RNAi and co-immunoprecipitation. Metabolic labeling, immunopurification and fluorography demonstrated de novo fibrinogen production.

    Results: FGF-2 stimulated DU-145 cell proliferation, whereas neither FGF-2 nor fibrinogen affected the growth of PC-3 or A549 cells. Fibrinogen augmented the proliferative effect of FGF-2 on DU-145 cells. The role of fibrinogen in FGF-2-enhanced DNA synthesis was confirmed using an FGF-2 mutant that exhibits no binding affinity for fibrinogen. FGG transcripts were present in PC-3, A549 and DU-145 cells, but only PC-3 and A549 cells produced detectable levels of intact protein. RNAi-mediated knockdown of FGG expression resulted in decreased production of fibrinogen protein and inhibited (3)H-thymidine uptake in A549 and PC-3 cells by 60%, which was restored by exogenously added fibrinogen. FGF-2 and fibrinogen secreted by the cells were present in the medium as a soluble complex, as determined by coimmunoprecipitation studies.

    Conclusions: These data indicate that endogenously synthesized fibrinogen promotes the growth of lung and prostate cancer cells through interaction with FGF-2.

    Funded by: NHLBI NIH HHS: HL30616

    Journal of thrombosis and haemostasis : JTH 2008;6;1;176-83

  • [Fibrinogen genes polymorphism in patients with ischemic stroke].

    Gusev EI, Favorova OO, Sudomoina MA, Martynov MIu, Serdiuk IE, Parfenov MG and Nikonova AA

    Zhurnal nevrologii i psikhiatrii imeni S.S. Korsakova 2008;108;4;91-8

  • Gender differences in genetic risk profiles for cardiovascular disease.

    Silander K, Alanne M, Kristiansson K, Saarela O, Ripatti S, Auro K, Karvanen J, Kulathinal S, Niemelä M, Ellonen P, Vartiainen E, Jousilahti P, Saarela J, Kuulasmaa K, Evans A, Perola M, Salomaa V and Peltonen L

    Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland. kaisa.silander@ktl.fi

    Background: Cardiovascular disease (CVD) incidence, complications and burden differ markedly between women and men. Although there is variation in the distribution of lifestyle factors between the genders, they do not fully explain the differences in CVD incidence and suggest the existence of gender-specific genetic risk factors. We aimed to estimate whether the genetic risk profiles of coronary heart disease (CHD), ischemic stroke and the composite end-point of CVD differ between the genders.

    We studied in two Finnish population cohorts, using the case-cohort design the association between common variation in 46 candidate genes and CHD, ischemic stroke, CVD, and CVD-related quantitative risk factors. We analyzed men and women jointly and also conducted genotype-gender interaction analysis. Several allelic variants conferred disease risk for men and women jointly, including rs1801020 in coagulation factor XII (HR = 1.31 (1.08-1.60) for CVD, uncorrected p = 0.006 multiplicative model). Variant rs11673407 in the fucosyltransferase 3 gene was strongly associated with waist/hip ratio (uncorrected p = 0.00005) in joint analysis. In interaction analysis we found statistical evidence of variant-gender interaction conferring risk of CHD and CVD: rs3742264 in the carboxypeptidase B2 gene, p(interaction) = 0.009 for CHD, and rs2774279 in the upstream stimulatory factor 1 gene, p(interaction) = 0.007 for CHD and CVD, showed strong association in women but not in men, while rs2069840 in interleukin 6 gene, p(interaction) = 0.004 for CVD, showed strong association in men but not in women (uncorrected p-values). Also, two variants in the selenoprotein S gene conferred risk for ischemic stroke in women, p(interaction) = 0.003 and 0.007. Importantly, we identified a larger number of gender-specific effects for women than for men.

    A false discovery rate analysis suggests that we may expect half of the reported findings for combined gender analysis to be true positives, while at least third of the reported genotype-gender interaction results are true positives. The asymmetry in positive findings between the genders could imply that genetic risk loci for CVD are more readily detectable in women, while for men they are more confounded by environmental/lifestyle risk factors. The possible differences in genetic risk profiles between the genders should be addressed in more detail in genetic studies of CVD, and more focus on female CVD risk is also warranted in genome-wide association studies.

    PloS one 2008;3;10;e3615

  • The functional role of an interleukin 6-inducible CDK9.STAT3 complex in human gamma-fibrinogen gene expression.

    Hou T, Ray S and Brasier AR

    Department of Biochemistry, and Sealy Center for Molecular Medicine, University of Texas Medical Branch, Galveston, Texas 77555-1060, USA.

    The signal transducer and activator of transcription 3 (STAT3) is an IL-6-inducible transcription factor that mediates the hepatic acute phase response (APR). Using gamma-fibrinogen (FBG) as a model of the APR, we investigated the requirement of an IL-6-inducible complex of STAT3 with cyclin-dependent kinase 9 (CDK9) on gamma-FBG expression in HepG2 hepatocarcinoma cells. IL-6 induces rapid nuclear translocation of Tyr-phosphorylated STAT3 that forms a nu f4e clear complex with CDK9 in nondenaturing co-immunoprecipitation and confocal colocalization assays. To further understand this interaction, we found that CDK9-STAT3 binding is mediated via both STAT NH2-terminal modulatory and COOH-terminal transactivation domains. Both IL-6-inducible gamma-FBG reporter gene and endogenous mRNA expression are significantly decreased after CDK9 inhibition using the potent CDK inhibitor, flavopiridol (FP), or specific CDK9 siRNA. Moreover, chromatin immunoprecipitation (ChIP) experiments revealed an IL-6-inducible STAT3 and CDK9 binding to the proximal gamma-FBG promoter as well as increased loading of RNA Pol II and phospho-Ser2 CTD Pol II on the TATA box and coding regions. Finally, FP specifically and efficiently inhibits association of phospho-Ser2 CTD RNA Pol II on the gamma-FBG promoter, indicating that CDK9 kinase activity mediates IL-6-inducible CTD phosphorylation. Our data indicate that IL-6 induces a STAT3.CDK9 complex mediated by bivalent STAT3 domains and CDK9 kinase activity is necessary for licensing Pol II to enter a transcriptional elongation mode. Therefore, disruption of IL-6 signaling by CDK9 inhibitors could be a potential therapeutic strategy for inflammatory disease.

    Funded by: NHLBI NIH HHS: R01 HL070925; NIEHS NIH HHS: P30 ES06676

    The Journal of biological chemistry 2007;282;51;37091-102

  • Genetic variation in 1253 immune and inflammation genes and risk of non-Hodgkin lymphoma.

    Cerhan JR, Ansell SM, Fredericksen ZS, Kay NE, Liebow M, Call TG, Dogan A, Cunningham JM, Wang AH, Liu-Mares W, Macon WR, Jelinek D, Witzig TE, Habermann TM and Slager SL

    Division of Epidemiology, Department of Health Sciences Research, Mayo Clinic College of Medicine, Rochester, MN 55905, USA. cerhan.james@mayo.edu

    Smaller-scale evaluations suggest that common genetic variation in candidate genes related to immune function may predispose to the development of non-Hodgkin lymphoma (NHL). We report an analysis of variants within genes associated with immunity and inflammation and risk of NHL using a panel of 9412 single-nucleotide polymorphisms (SNPs) from 1253 genes in a study of 458 patients with NHL and 484 frequency-matched controls. We modeled haplotypes and risk of NHL, as well as the main effects for all independent SNPs from a gene in multivariate logistic regression models; we separately report results for nonsynonymous (ns) SNPs. In gene-level analyses, the strongest findings (P < or = .001) were for CREB1, FGG, MAP3K5, RIPK3, LSP1, TRAF1, DUSP2, and ITGB3. In nsSNP analyses, the strongest findings (P < or = .01) were for ITGB3 L59P (odds ratio [OR] = 0.66; 95% confidence interval [CI] 0.52-0.85), TLR6 V427A (OR = 5.20; CI 1.77-15.3), SELPLG M264V (OR = 3.20; CI 1.48-6.91), UNC84B G671S (OR = 1.50; CI 1.12-2.00), B3GNT3 H328R (OR = 0.74; CI 0.59-0.93), and BAT2 V1883L (OR = 0.64; CI 0.45-0.90). Our results suggest that genetic variation in genes associated with immune response (TRAF1, RIPK3, BAT2, and TLR6), mitogen-activated protein kinase (MAPK) signaling (MAP3K5, DUSP2, and CREB1), lymphocyte trafficking and migration (B3GNT3, SELPLG, and LSP1), and coagulation pathways (FGG and ITGB3) may be important in the etiology of NHL, and should be prioritized in replication studies.

    Funded by: NCI NIH HHS: K07 CA094919, K07 CA94919, R01 CA092153, R01 CA92153, R25 CA092049

    Blood 2007;110;13;4455-63

  • Fibrinogen Tolaga Bay: a novel gammaAla341Val mutation causing hypofibrinogenaemia.

    Davis RL and Brennan SO

    Christchurch School of Medicine and Health Sciences, University of Otago, Christchurch, New Zealand. ryan.davis@chmeds.ac.nz

    Thrombosis and haemostasis 2007;98;5;1136-8

  • Fibrinogen angers with a new deletion (gamma GVYYQ 346-350) causes hypofibrinogenemia with hepatic storage.

    Dib N, Quelin F, Ternisien C, Hanss M, Michalak S, De Mazancourt P, Rousselet MC and Calès P

    Department of Hepato-Gastroenterology, University Hospital, Angers.

    Introduction: This study reports a family with chronically abnormal blood liver function tests (LFT) and congenital hypofibrinogenemia. The proposita had cirrhosis initially related to alcohol abuse and chronic viral hepatitis C (HCV), but abnormal LFT persisted even when alcohol intake was stopped and despite HCV treatment was efficient based on serum RNA negative testing.

    Results: Needle biopsy specimens of the proposita and her brother showed eosinophilic intra-cytoplasmic inclusions that reacted strongly with fibrinogen antisera on direct immunofluorescence. Electron microscopic examination showed that the rough endoplasmic reticulum was filled with inclusions that consisted of densely packed, curved tubular structures arranged in a fingerprint-like pattern. Coagulation studies revealed low functional and antigenic fibrinogen concentrations suggestive of hypofibrinogenemia. Amplification and DNA sequencing showed a heterozygous deletion of the a7690 to g7704 nucleotides of the gamma chain gene in the 3'end of exon 8 (g 7690_7704del14; Genbank access M10014); this deletion encompassed the splicing site at position 7703 and predicts in a new putative consensus splicing sequence (AATGgtatgtt). RNA was extracted from a liver specimen from the proposita's brother. The cDNA obtained by reverse transcription polymerase chain reaction confirmed the usage of a newly generated donor site at position 7688 of the genomic sequence resulting in an in-frame heterozygous 5 amino acid deletion (GVYYQ 346-350; p.G372_Q376del) and that this mutation is responsible for a new splicing site at position 7688 of the genomic sequence.

    Conclusion: we suggest that the molecular defect in fibrinogen Angers results in an impaired assembly and causes defective secretion and hepatic storage of fibrinogen.

    Journal of thrombosis and haemostasis : JTH 2007;5;10;1999-2005

  • Fibrinogen Leipzig II (gamma351Gly-->Ser and gamma82Ala-->Gly): hypodysfibrinogenaemia due to two independent amino acid substitutions within the same polypeptide chain.

    Meyer M, Dietzel H, Kaetzel R, Schmidt D, Liebscher K and Brennan SO

    Department of Biomedical Engineering and Biotechnology, University of Applied Sciences (FH), Carl-Zeiss-Promenade 2, 07745 Jena, Germany. michael.meyer@fh-jena.de

    Thrombosis and haemostasis 2007;98;4;903-5

  • Pseudo-exon activation caused by a deep-intronic mutation in the fibrinogen gamma-chain gene as a novel mechanism for congenital afibrinogenaemia.

    Spena S, Asselta R, Platé M, Castaman G, Duga S and Tenchini ML

    Department of Biology and Genetics for Medical Sciences, University of Milan, Milan, Italy.

    Congenital afibrinogenaemia, characterized by severe fibrinogen deficiency, is caused by mutations within FGA, FGB or FGG. Conventional sequencing of coding regions and splice signals of these three genes did not reveal any mutation in an afibrinogenaemic proband. After confirming disease co-segregation with the fibrinogen cluster, full intron sequencing was tackled leading to the identification of a novel transvertion within FGG intron 6 (IVS6-320A-->T). Its effect on mRNA processing was evaluated in-vitro: the in-frame inclusion of a 75-bp pseudo-exon carrying a premature stop was found, representing the first report of pseudo-exon activation as a mechanism leading to afibrinogenaemia.

    British journal of haematology 2007;139;1;128-32

  • Probing the beta-chain hole of fibrinogen with synthetic peptides that differ at their amino termini.

    Doolittle RF and Pandi L

    Department of Chemistry and Biochemistry and Division of Biology, University of California, San Diego, La Jolla, California 92093-0314, USA. rdoolittle@ucsd.edu

    In a recent report, we showed that alanine can replace glycine at the amino terminus of synthetic B-knobs that bind to human fibrin(ogen). We now report a survey of 13 synthetic peptides with the general sequence XHRPYam, all tested with regard to their ability to delay fibrinolysis in an in vitro system activated by t-PA, the results being used as measures of binding affinity to the betaC hole. Unexpectedly, some large and bulky amino acids, including methionine and arginine, are effective binders. Amino acids that branch at the beta carbon (valine, isoleucine, and threonine) do not bind effectively. Crystal structures were determined for two of the peptides (GHRPYam and MHRPYam) complexed with fibrin fragment D-dimer; the modeling of various other side chains showed clashing in the cases of beta-carbon substituents. The two crystal structures also showed that the enhanced binding observed with pentapeptides with carboxyl-terminal tyrosine, compared with that of their tetrapeptide equivalents, is attributable to an interaction between the tyrosine side chain and a guanidino group of a nearby arginine (beta406). The equivalent position in gamma-chains of human fibrin(ogen) is occupied by a lysine (gamma338), but in chicken and lamprey fibrin(ogen), it is an arginine, just as occurs in beta chains. Accordingly, the peptides GPRPam and GPRPYam, which are surrogate A-knobs, were tested for their influence on fibrin polymerization with fibrinogen from lamprey and humans. In lampreys, GPRPYam is a significantly better inhibitor, but in humans, it is less effective than GPRPam, indicating that in the lamprey system the same tyrosine-arginine interaction can also occur in the gamma-chain setting.

    Funded by: NHLBI NIH HHS: HL-81553

    Biochemistry 2007;46;35;10033-8

  • The chymotrypsin-like protease complex of Treponema denticola ATCC 35405 mediates fibrinogen adherence and degradation.

    Bamford CV, Fenno JC, Jenkinson HF and Dymock D

    Department of Oral and Dental Science, University of Bristol, Lower Maudlin St., Bristol BS1 2LY, United Kingdom.

    Treponema denticola is an anaerobic spirochete strongly associated with human periodontal disease. T. denticola bacteria interact with a range of host tissue proteins, including fibronectin, laminin, and fibrinogen. The latter localizes in the extracellular matrix where tissue damage has occurred, and interactions with fibrinogen may play a key role in T. denticola colonization of the damaged sites. T. denticola ATCC 35405 showed saturable binding of fluid-phase fibrinogen to the cell surface and saturable adherence to immobilized fibrinogen. Levels of fibrinogen binding were enhanced in the presence of the serine protease inhibitor phenylmethylsulfonyl fluoride. The Aalpha and Bbeta chains of fibrinogen, but not the gamma chains, were specifically recognized by T. denticola. Following fibrinogen affinity chromatography analysis of cell surface extracts, a major fibrinogen-binding component (polypeptide molecular mass, approximately 100 kDa), which also degraded fibrinogen, was purified. Upon heating at 100 degrees C, the polypeptide was dissociated into three components (apparent molecular masses, 80, 48, and 45 kDa) that did not individually bind or degrade fibrinogen. The native 100-kDa polypeptide complex was identified as chymotrypsin-like protease (CTLP), or dentilisin. In an isogenic CTLP(-) mutant strain, CKE, chymotrypsin-like activity was reduced >90% compared to that in the wild type and fibrinogen binding and hydrolysis were ablated. Isogenic mutant strain MHE, deficient in the production of Msp (major surface protein), showed levels of CTLP reduced 40% relative to those in the wild type and exhibited correspondingly reduced levels of fibrinogen binding and proteolysis. Thrombin clotting times in the presence of wild-type T. denticola cells, but not strain CKE (CTLP(-)) cells, were extended. These results suggest that interactions of T. denticola with fibrinogen, which may promote colonization and modulate hemostasis, are mediated principally by CTLP.

    Infection and immunity 2007;75;9;4364-72

  • Activation of NF-kappaB by IL-1beta blocks IL-6-induced sustained STAT3 activation and STAT3-dependent gene expression of the human gamma-fibrinogen gene.

    Albrecht U, Yang X, Asselta R, Keitel V, Tenchini ML, Ludwig S, Heinrich PC, Häussinger D, Schaper F and Bode JG

    Clinic for Gastroenterology, Hepatology and Infectiology, Heinrich-Heine-University, 40255 Düsseldorf, Germany.

    Despite the essential role of the fibrinogen gamma-chain as a blood clotting factor, the fibrinogen gamma-chain contains a number of interaction sites to recruit other factors such as leukocytes important for prevention of pathogen entry and propagation of the repair process. Interleukin-6 (IL-6) is known as the major inducer of gamma-fibrinogen synthesis in hepatocytes, whereas IL-1beta has been shown to act as a potent inhibitor of gamma-fibrinogen expression. Studies on the rat fibrinogen gamma-chain promoter suggest that nuclear factor (NF)-kappaB replaces the signal transducer and activator of transcription (STAT) 3 from binding to overlapping NF-kappaB/STAT3 binding sites within the 5' regulatory region of the rat gamma-chain gene promoter. However, despite its physiological relevance, the underlying mechanism responsible for the inhibitory effect of IL-1beta in humans is still not understood and apparently more complex. In contrast to the mechanism described for the rat gene our results indicate that IL-1beta suppresses the IL-6-induced activation of the human gamma-fibrinogen gene particularly by blocking the late phase STAT3-tyrosine phosphorylation NF-kappaB-dependently but independent from de novo protein synthesis. Consequently, blocking NF-kappaB activation restores specifically late phase STAT3 activation as well as the induction of the human gamma-fibrinogen gene. In contrast, specifically early STAT3 activation could be restored by a block of the p38 mitogen-activated protein kinase (p38(MAPK)) pathway. In summary, our results indicate that expression of the gamma-fibrinogen gene is mainly controlled by the strength of late phase STAT3 activation, which in turn is negatively regulated by the extent of IL-1beta-mediated NF-kappaB activity.

    Cellular signalling 2007;19;9;1866-78

  • Clinical and biological features of 3 cases of hypofibrinogenemia associated with three different mutations (gamma Ala341Thr, Bbeta Tyr326Cys and Aalpha Asp496Asn).

    Hanss M, Chevreaud C, French P, Négrier C and de Mazancourt P

    Thrombosis and haemostasis 2007;98;3;689-91

  • Fibrinogen Columbus: a novel gamma Gly200Val mutation causing hypofibrinogenemia in a family with associated thrombophilia.

    Davis RL, Mosesson MW, Kerlin BA, Canner JA, Ruymann FB and Brennan SO

    Fibrinogen is an essential component of the coagulation cascade and the acute phase response. The native 340 kDa molecule has a symmetrical trinodular structure composed of a central E-domain connected to outer D-domains by triple helical coiled-coils.1 Several mutations known to cause hypofibrinogenemia occur within the C-terminal gammaD-domain and have helped to elucidate the structurally and functionally important areas of this domain.2-5 Here we report the identification of a novel point mutation gammaG200V (fibrinogen Columbus) causing hypofibrinogenemia and co-segregating with three genetic thrombophilia risk factors.

    Funded by: NHLBI NIH HHS: R01 HL070627

    Haematologica 2007;92;8;1151-2

  • Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme.

    Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH and Coulombe B

    Laboratory of Gene Transcription and Proteomics Discovery Platform, Institut de Recherches Cliniques de Montréal, Montréal, QC, Canada.

    We have performed a survey of soluble human protein complexes containing components of the transcription and RNA processing machineries using protein affinity purification coupled to mass spectrometry. Thirty-two tagged polypeptides yielded a network of 805 high-confidence interactions. Remarkably, the network is significantly enriched in proteins that regulate the formation of protein complexes, including a number of previously uncharacterized proteins for which we have inferred functions. The RNA polymerase II (RNAP II)-associated proteins (RPAPs) are physically and functionally associated with RNAP II, forming an interface between the enzyme and chaperone/scaffolding proteins. BCDIN3 is the 7SK snRNA methylphosphate capping enzyme (MePCE) present in an snRNP complex containing both RNA processing and transcription factors, including the elongation factor P-TEFb. Our results define a high-density protein interaction network for the mammalian transcription machinery and uncover multiple regulatory factors that target the transcription machinery.

    Funded by: Canadian Institutes of Health Research: 14309-3, 82851-1

    Molecular cell 2007;27;2;262-74

  • Fibrinogen gamma-A chain precursor in CSF: a candidate biomarker for Alzheimer's disease.

    Lee JW, Namkoong H, Kim HK, Kim S, Hwang DW, Na HR, Ha SA, Kim JR and Kim JW

    Department of Neurology, Bobath Memorial Hospital, Bundang-Gu, Seongnam, Gyungki-do, Korea. marineultrablue@yahoo.co.kr <marineultrablue@yahoo.co.kr&gt;

    Background: Cerebrospinal fluid (CSF) may be valuable for exploring protein markers for the diagnosis of Alzheimer's disease (AD). The prospect of early detection and treatment, to slow progression, holds hope for aging populations with increased average lifespan. The aim of the present study was to investigate candidate CSF biological markers in patients with mild cognitive impairment (MCI) and AD and compare them with age-matched normal control subjects.

    Methods: We applied proteomics approaches to analyze CSF samples derived from 27 patients with AD, 3 subjects with MCI and 30 controls. The AD group was subdivided into three groups by clinical severity according to clinical dementia rating (CDR), a well known clinical scale for dementia.

    Results: We demonstrated an elevated level of fibrinogen gamma-A chain precursor protein in CSF from patients with mild cognitive impairment and AD compared to the age-matched normal subjects. Moreover, its expression was more prominent in the AD group than in the MCI and correlated with disease severity and progression. In contrast, fibrinogen gamma-A chain precursor protein was detected very low in the age-matched normal group.

    Conclusion: These findings suggest that the CSF level of fibrinogen gamma-A chain precursor may be a candidate biomarker for AD.

    BMC neurology 2007;7;14

  • Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen gamma'/gammaA mRNA ratio.

    Uitte de Willige S, Rietveld IM, De Visser MC, Vos HL and Bertina RM

    Department of Hematology, Hemostasis and Thrombosis Research Center, Leiden University Medical Center, Leiden, The Netherlands.

    Background: Fibrinogen gamma haplotype 2 (FGG-H2) is associated with reduced fibrinogen gamma' levels and fibrinogen gamma'/total fibrinogen ratios and with an increased deep-venous thrombosis (DVT) risk. Two FGG-H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T and 10034C>T, are located in the region of alternative FGG pre-mRNA processing. 10034C>T is located in a GT-rich downstream sequence element (DSE) that comprises a putative cleavage stimulation factor (CstF) binding site.

    Objectives: To investigate the functionality of SNPs 9615C>T and 10034C>T, and the importance of the DSE containing 10034C>T.

    Methods: Different minigene constructs containing FGG exon 9, intron 9, exon 10 and the 3' region were transiently transfected into HepG2 cells and quantitative real-time polymerase chain reaction was used to measure relative polyadenylation (pA) signal usage (pA1/pA2 ratio).

    Results: Compared with the reference construct CC (9615C-10034C; FGG-H1; pA1/pA2 ratio set at 100%), the pA1/pA2 ratio of construct TT (9615T-10034T; FGG-H2) was 1.4-fold decreased (71.5%, P = 0.015). The pA1/pA2 ratio of construct CT (9615C-10034T) was almost 1.2-fold decreased (85.3%, P = 0.001), whereas the pA1/pA2 ratio of construct TC (9615T-10034C) did not differ significantly from the reference construct (101.6%, P = 0.890). Functionality of the putative CstF binding site was confirmed using constructs in which this site was deleted or its sequence altered by point mutations.

    Conclusions: SNP 10034C>T is located in a GT-rich DSE involved in regulating the usage of the pA2 signal of FGG, which may represent a CstF binding site. We propose that the 10034C>T change is the functional variation in FGG-H2 that is responsible for the reduction in the fibrinogen gamma'/total fibrinogen ratio and the increased DVT risk.

    Journal of thrombosis and haemostasis : JTH 2007;5;6;1243-9

  • Elevated plasma fibrinogen gamma' concentration is associated with myocardial infarction: effects of variation in fibrinogen genes and environmental factors.

    Mannila MN, Lovely RS, Kazmierczak SC, Eriksson P, Samnegård A, Farrell DH, Hamsten A and Silveira A

    Department of Medicine, Atherosclerosis Research Unit, King Gustaf V Research Institute, Karolinska Institutet, Solna, Sweden. maria.nastase.mannila@ki.se

    Background: Fibrinogen gamma', a fibrinogen gamma-chain variant generated via alternative mRNA processing, has been associated with susceptibility to thrombotic disease.

    Objective: The present case-control study searched for potential determinants of the plasma fibrinogen gamma' concentration and examined the relationship between this variant and risk of myocardial infarction (MI).

    The Stockholm Coronary Artery Risk Factor study, comprising 387 postinfarction patients and 387 healthy individuals, was employed. The fibrinogen gamma (FGG) 9340T > C [rs1049636], fibrinogen alpha (FGA) 2224G > A [rs2070011] and fibrinogen beta (FGB) 1038G > A [rs1800791] polymorphisms were determined. The plasma fibrinogen gamma' concentration was measured by enzyme-linked immunosorbent assay. The multifactor dimensionality reduction method was used for interaction analyses on risk of MI.

    Results: The FGG 9340T > C and FGA 2224G > A polymorphisms, total plasma concentrations of fibrinogen, insulin and high-density lipoprotein, and gender appeared to be independent determinants of plasma fibrinogen gamma' concentration in patients, and the corresponding determinants in controls included FGG 9340T > C and FGA 2224G > A polymorphisms and plasma fibrinogen concentration. An elevated plasma fibrinogen gamma' concentration proved to be an independent predictor of MI [adjusted odds ratio (OR) (95% CI): 1.24 (1.01, 1.52)]. The plasma fibrinogen gamma' concentration was involved in a high-order interaction with total plasma fibrinogen and the FGG 9340T > C and FGA 2224G > A polymorphisms, associated with a further increased risk of MI [OR (95% CI): 3.22 (2.35, 4.39)].

    Conclusions: Plasma fibrinogen gamma' concentration influences the risk of MI, and this relationship seems to be strengthened by the presence of an elevated total plasma fibrinogen concentration and the FGG 9340T and FGA 2224G alleles.

    Funded by: NHLBI NIH HHS: 1 R21 HL75006

    Journal of thrombosis and haemostasis : JTH 2007;5;4;766-73

  • The association between fibrinogen haplotypes and myocardial infarction in men is partly mediated through pleiotropic effects on the serum IL-6 concentration.

    Mannila MN, Eriksson P, Leander K, Wiman B, de Faire U, Hamsten A and Silveira A

    Department of Medicine, Atherosclerosis Research Unit, King Gustaf V Research Institute, Karolinska University Hospital, Solna, Sweden. maria.nastase.mannila@ki.se

    Fibrinogen haplotypes have been associated with risk of myocardial infarction (MI), independently of plasma fibrinogen concentration, and experimental data indicate that fibrinogen exerts pleiotropic effects on interleukin 6 (IL-6) production. Also, the coagulation factor XIII (gene symbol F13A1) Val34Leu haplotype tag single nucleotide polymorphism (htSNP) has been reported to exert pleiotropic effects on serum IL-6 concentration and to be associated with risk of MI. Therefore, in the present case-control study (a substudy to the Stockholm Heart Epidemiology Program), the effects of the fibrinogen gamma (FGG) 9340T>C [rs1049636], fibrinogen alpha (FGA) 2224G>A [rs2070011] and F13A1 Val34Leu [rs5985] htSNPs on concentrations of plasma fibrinogen and serum IL-6 and risk of MI were assessed.

    Results: There were no associations between these SNPs and the plasma fibrinogen concentration. In contrast, in male controls the FGA 2224G>A htSNP was significantly associated with serum IL-6 concentration (P < 0.05). Also, in men the FGG-FGA*1 haplotype (containing the major FGG 9340T and FGA 2224G alleles) was associated with increased risk of MI [adjusted odds ratio (OR) 95% confidence interval (CI): 1.29 (1.02, 1.62)] and with higher IL-6 concentrations, whereas the least common FGG-FGA*4 haplotype (containing the minor FGG 9340C and FGA 2224A alleles) conferred lowered risk [adjusted OR (95% CI): 0.70 (0.57, 0.86)] and lowered IL-6 concentrations. In women, fibrinogen haplotypes were not associated with risk of MI after adjusting for cardiovascular risk factors.

    Conclusion: In healthy men, fibrinogen haplotypes are associated with serum IL-6 concentrations in a manner consistent with their impact on MI risk.

    Journal of internal medicine 2007;261;2;138-47

  • Fibrinogen gene haplotypes in relation to risk of coronary events and coronary and extracoronary atherosclerosis: the Rotterdam Study.

    Kardys I, Uitterlinden AG, Hofman A, Witteman JC and de Maat MP

    Department of Epidemiology and biostatistics, Erasmus Medical Center, Rotterdam, The Netherlands.

    Fibrin network structure has been correlated with coronary disease. Fibrinogen gamma and alpha (FGG and FGA) gene haplotypes (chromosome 4q28) may be associated with fibrin network structure, and thereby with rigidity of the fibrin clot and sensitivity of the fibrin clot to the fibrinolytic system. Through these mechanisms they may influence risk of cardiovascular disease. We set out to investigate the relation between combined fibrinogen FGG and FGA gene haplotypes, representing the common variation of the fibrinogen FGG and FGA genes, coronary events and measures of coronary and extracoronary atherosclerosis. The study was embedded in the Rotterdam Study, a prospective population-based study among men and women aged >or=55 years. Common haplotypes were studied using seven tagging SNPs across a 30-kb region with the FGG and FGA genes. Incident coronary events were registered, and carotid intima-media thickness, carotid plaques, ankle-arm index, aortic calcification and coronary calcification were assessed. Seven haplotypes with frequencies >1% covered 97.5% of the genetic variation. In 5,667 participants without history of coronary heart disease (CHD), 733 CHD cases occurred during a median follow-up time of 11.9 years. Fibrinogen gene haplotypes were not associated with coronary events. Fibrinogen gene haplotypes did not show a consistent association with measures of coronary and extracoronary atherosclerosis. In conclusion, fibrinogen FGG and FGA gene haplotypes are not associated with coronary events, coronary atherosclerosis or extracoronary atherosclerosis. Confirmation of these findings by future population-based studies is warranted.

    Thrombosis and haemostasis 2007;97;2;288-95

  • Polymerization of fibrin: Direct observation and quantification of individual B:b knob-hole interactions.

    Litvinov RI, Gorkun OV, Galanakis DK, Yakovlev S, Medved L, Shuman H and Weisel JW

    Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, 1040 BRB II/III, 421 Curie Blvd, Philadelphia, PA 19104-6058, USA. litvinov@mail.med.upenn.edu

    The polymerization of fibrin occurs primarily through interactions between N-terminal A- and B-knobs, which are exposed by the cleavage of fibrinopeptides A and B, respectively, and between corresponding a- and b-holes in the gamma- and beta-modules. Of the potential knob-hole interactions--A:a, B:b, A:b, and B:a--the first has been shown to be critical for fibrin formation, but the roles of the others have remained elusive. Using laser tweezers-based force spectroscopy, we observed and quantified individual B:b and A:b interactions. Both desA-fibrin with exposed A-knobs and desB-fibrin bearing B-knobs interacted with fragment D from the gammaD364H fibrinogen containing b-holes but no functional a-holes. The strength of single B:b interactions was found to be 15 to 20 pN, approximately 6-fold weaker than A:a interactions. B:b binding was abrogated by B-knob mimetic peptide, the (beta15-66)2 fragment containing 2 B-knobs, and a monoclonal antibody against the beta15-21 sequence. The interaction of desB-fibrin with fragment D containing a- and b-holes produced the same forces that were insensitive to A-knob mimetic peptide, suggesting that B:a interactions were absent. These results directly demonstrate for the first time B:b binding mediated by natural B-knobs exposed in a fibrin monomer.

    Funded by: NHLBI NIH HHS: HL-30954, HL-31048, HL-56051, R01 HL030954, R01 HL031048, R01 HL056051

    Blood 2007;109;1;130-8

  • The fibrinogen gamma (FGG) 10034C>T polymorphism is associated with venous thrombosis.

    Grünbacher G, Weger W, Marx-Neuhold E, Pilger E, Köppel H, Wascher T, März W and Renner W

    Clinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University Graz, 8036 Graz Austria.

    Introduction: Thrombin-induced conversion of fibrinogen to fibrin plays an essential role in hemostasis and results in the stabilization of thrombi. Elevated plasma fibrinogen levels have been associated with both increased plasma viscosity and platelet aggregability. Recently, a haplotype-tagging single nucleotide polymorphism characterized by a C to T substitution at nucleotide 10034 of the fibrinogen gamma gene (FGG 10034C>T, rs2066865), has been proposed as a novel risk factor for deep venous thrombosis (DVT). Aim of the present study was to provide further data on the role of the FGG 10034C>T polymorphism for DVT.

    FGG genotypes were determined by 5'-exonuclease assay (TaqMan) in 358 patients with documented DVT and a total of 783 control subjects.

    Results: In a multivariate analysis adjusting for age, sex, presence of factor V Leiden and carriage of prothrombin 20210A, homozygosity for the FGG 10034 TT genotype yielded an odds ratio of 2.01 (95% CI 1.23-3.31; p=0.006) for DVT.

    Conclusions: Our data confirm the primary finding that the FGG 10034C>T polymorphism is associated with DVT risk.

    Thrombosis research 2007;121;1;33-6

  • Matrix-specific suppression of integrin activation in shear stress signaling.

    Orr AW, Ginsberg MH, Shattil SJ, Deckmyn H and Schwartz MA

    Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA.

    Atherosclerotic plaque develops at sites of disturbed flow. We previously showed that flow activates endothelial cell integrins, which then bind to the subendothelial extracellular matrix (ECM), and, in cells on fibronectin or fibrinogen, trigger nuclear factor-kappaB activation. Additionally, fibronectin and fibrinogen are deposited into the subendothelial ECM at atherosclerosis-prone sites at early times. We now show that flow activates ECM-specific signals that establish patterns of integrin dominance. Flow induced alpha2beta1 activation in cells on collagen, but not on fibronectin or fibrinogen. Conversely, alpha5beta1 and alphavbeta3 are activated on fibronectin and fibrinogen, but not collagen. Failure of these integrins to be activated on nonpermissive ECM is because of active suppression by the integrins that are ligated. Protein kinase A is activated specifically on collagen and suppresses flow-induced alphavbeta3 activation. Alternatively, protein kinase Calpha is activated on fibronectin and mediates alpha2beta1 suppression. Thus, integrins actively cross-inhibit through specific kinase pathways. These mechanisms may determine cellular responses to complex extracellular matrices.

    Funded by: NHLBI NIH HHS: HL56595, R01 HL056595, R01 HL075092, R01 HL75092

    Molecular biology of the cell 2006;17;11;4686-97

  • The COOH-terminal globular domain of fibrinogen gamma chain suppresses angiogenesis and tumor growth.

    Akakura N, Hoogland C, Takada YK, Saegusa J, Ye X, Liu FT, Cheung AT and Takada Y

    Department of Dermatology, University of California-Davis Medical Center, Sacramento, CA 95817, USA.

    Fibrinogen is a major plasma protein (350 kDa) that induces proliferative signals by serving as a scaffold to support the binding of growth factors and to promote the cellular responses of adhesion, proliferation, and migration during wound healing, angiogenesis, and tumor growth. Fibrin(ogen) degradation products generated during fibrinolysis are implicated in tissue injury. The fibrinogen gamma chain has a COOH-terminal globular domain (gamma C, residues 151-411 of the gamma chain, 30 kDa) to which several integrin cell adhesion receptors (e.g., platelet alpha(IIb)beta(3), endothelial alpha(v)beta(3), and leukocyte alpha(M)beta(2)) bind. Integrins play a critical role in signal transduction from fibrin(ogen). We found that gamma C and its truncation mutant (designated gamma C399tr), with a deletion of the COOH-terminal 12 residues, induced apoptosis of endothelial cells and blocked tube formation of endothelial cells. DLD-1 human colon cancer cells that secrete gamma C or gamma C399tr grew at similar levels in vitro but grew much slower in vivo than mock-transfected cells. The recombinant purified gamma C399tr fragment markedly suppressed tumor growth, development of intratumoral vasculature, and tumor metastasis in vivo in the highly metastatic Met-1 breast cancer model. The determinant responsible for binding to endothelial cells is cryptic in native fibrinogen but is exposed in gamma C and gamma C399tr. These results suggest that fibrinogen has a novel cryptic determinant, which can exert apoptosis-inducing activity on endothelial cells when exposed, and polypeptides containing this determinant have therapeutic potential.

    Funded by: NCI NIH HHS: CA093373-04/10, CA113298; NIGMS NIH HHS: GM047157

    Cancer research 2006;66;19;9691-7

  • Analysis of fibrinogen variants at gamma387Ile shows that the side chain of gamma387 and the tertiary structure of the gammaC-terminal tail are important not only for assembly and secretion of fibrinogen but also for lateral aggregation of protofibrils and XIIIa-catalyzed gamma-gamma dimer formation.

    Kani S, Terasawa F, Yamauchi K, Tozuka M and Okumura N

    Laboratory of Clinical Chemistry, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan.

    To examine the role of fibrinogen gamma-chain residue 387Ile in the assembly and secretion of this multichain protein, we synthesized a series of variants with substitution at gamma387 by Arg, Leu, Met, Ala, or Asp. Only the variant gamma387Asp showed impaired synthesis in the cells and very low secretion into the medium. In addition, we performed thrombin-catalyzed fibrin polymerization and factor (F) XIIIa-catalyzed cross-linking of the gamma-chain for 4 variants. The degree of lateral aggregation of protofibrils into fibrin fibers was slightly reduced for gamma387Arg and Ala, and moderately reduced for gamma387Leu and Met. Although the FXIIIa-catalyzed cross-linking for all of the variants was slower than that for gamma387Ile, that of gamma387Arg was much more markedly impaired than that of the others. In summary, our studies demonstrated that the specific residue at gamma387 or the conformation of gamma388-411 residues, but not the length of the gammaC tail, is critical for fibrinogen assembly and subsequent secretion. Moreover, this residue or the conformation is also important for not only the lateral aggregation of fibrin polymers but also the FXIIIa-catalyzed cross-linking of the gamma-chain. Interestingly, our results clearly indicate that the conformations critical for these 2 functions are different from each other.

    Blood 2006;108;6;1887-94

  • Association between patterns of nucleotide variation across the three fibrinogen genes and plasma fibrinogen levels: the Coronary Artery Risk Development in Young Adults (CARDIA) study.

    Reiner AP, Carty CL, Carlson CS, Wan JY, Rieder MJ, Smith JD, Rice K, Fornage M, Jaquish CE, Williams OD, Tracy RP, Lewis CE, Siscovick DS, Boerwinkle E and Nickerson DA

    Departments of Epidemiology and Genome Sciences, University of Washington, Seattle, WA 98101-1448, USA. apreiner@u.washington.edu

    Background: Previous genotype-phenotype association studies of fibrinogen have been limited by incomplete knowledge of genomic sequence variation within and between major ethnic groups in FGB, FGA, and FGG.

    Methods: We characterized the linkage disequilibrium patterns and haplotype structure across the human fibrinogen gene locus in European- and African-American populations. We analyzed the association between common polymorphisms in the fibrinogen genes and circulating levels of both 'functional' fibrinogen (measured by the Clauss clotting rate method) and total fibrinogen (measured by immunonephelometry) in a large, multi-center, bi-racial cohort of young US adults.

    Results: A common haplotype tagged by the A minor allele of the well-studied FGB-455 G/A promoter polymorphism (FGB 1437) was confirmed to be strongly associated with increased plasma fibrinogen levels. Two non-coding variants specific to African-American chromosomes, FGA 3845 A and FGG 5729 G, were each associated with lower plasma fibrinogen levels. In European-Americans, a common haplotype tagged by FGA Thr312Ala and several other variant alleles across the fibrinogen gene locus was strongly associated with decreased fibrinogen levels as measured by functional assay, but not by immunoassay. Overall, common polymorphisms within the three fibrinogen genes explain < 2% of the variability in plasma fibrinogen concentration.

    Conclusions: In young adults, fibrinogen multi-locus genotypes are associated with plasma fibrinogen levels. The specific single nucleotide polymorphism and haplotype patterns for these associations differ according to population and also according to phenotypic assay. It is likely that a substantial proportion of the heritable component of plasma fibrinogen concentration is due to genetic variation outside the three fibrinogen genes.

    Funded by: NHLBI NIH HHS: HL66642, HL66682, HL71017, N01-HC-05187, N01-HC-45134, N01-HC-48047, N01-HC-48048, N01-HC-48049, N01-HC-48050, N01-HC-95095

    Journal of thrombosis and haemostasis : JTH 2006;4;6;1279-87

  • Common genetic variation in five thrombosis genes and relations to plasma hemostatic protein level and cardiovascular disease risk.

    Kathiresan S, Yang Q, Larson MG, Camargo AL, Tofler GH, Hirschhorn JN, Gabriel SB and O'Donnell CJ

    National Heart, Lung, and Blood Institute Framingham Heart Study, Framingham, MA 01702-5827, USA.

    Objective: We undertook a linkage disequilibrium (LD)-based genetic approach to investigate the hypothesis that common sequence variants in 5 thrombosis genes influence plasma hemostatic protein levels or risk of cardiovascular disease (CVD).

    In a reference panel, we characterized LD structure at the fibrinogen gene cluster (fibrinogen-beta[FGB], FGA, and FGG), factor VII (F7), and tissue plasminogen activator (PLAT) loci. Forty-one tag single nucleotide polymorphisms (SNPs) were genotyped in 1811 unrelated Framingham Heart Study participants. There were significant associations of 9 FGB SNPs with fibrinogen level (minimum P=0.002) and of 7 F7 SNPs and factor VII level (minimum P<0.0001). SNPs at the PLAT locus were not associated with PLAT level. In stepwise analysis, a single FGB variant explained 1% of the residual variance in fibrinogen level, and 2 F7 SNPs together explained 10% of the residual variance in factor VII level. Two PLAT haplotypes were associated with CVD (multivariable-adjusted global P=0.0004).

    Conclusions: A comprehensive survey of common sequence variation demonstrates that cis-regulatory SNPs explain a modest proportion of the residual variance in circulating fibrinogen and factor VII level and PLAT haplotypes increase the risk of CVD. Additional studies are warranted to confirm the association of PLAT sequence variation and risk of CVD.

    Funded by: NHLBI NIH HHS: HL66582, N01-HC-25195

    Arteriosclerosis, thrombosis, and vascular biology 2006;26;6;1405-12

  • Cell type-specific differential induction of the human gamma-fibrinogen promoter by interleukin-6.

    Duan HO and Simpson-Haidaris PJ

    Department of Medicine/Hematology-Oncology Division, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642, USA.

    During an acute phase response, interleukin-6 (IL-6) and glucocorticoids up-regulate expression of the three fibrinogen (FBG) genes (fga, fgb, and fgg) in liver and lung epithelium; however, little constitutive lung expression occurs. Recently, we showed that the magnitude of Stat3 binding to three IL-6 motifs on the human gammaFBG promoter correlates negatively with their functional activity in hepatocytes, although these cis-elements are critical for promoter activity. We determined the role of IL-6-receptor-gp130-Stat3 signaling in IL-6 activation of the gammaFBG promoter in liver and lung epithelial cells. Although IL-6 induced gammaFBG promoter activity approximately 30-fold in HepG2 cells, it was increased only 2-fold in lung A549 cells. Equivalent production of gp130 was demonstrated in both cell types by Western blotting; however, lower production of both IL-6-receptor and Stat3 explains, in part, reduced activity of the gammaFBG promoter in lung cells. Dexamethasone potentiated IL-6 induction of the gammaFBG promoter 2.3-fold in both HepG2 and A549 cells for a combined increase in promoter activity of 70-fold or 4.5-fold, respectively. Dexamethasone potentiation is likely due to the induction of IL-6-receptor expression as well as prolonged intensity and duration of Stat3 activation. By circumventing IL-6-receptor-gp130-coupled signaling with ectopic expression of the granulocyte colony-stimulating factor receptor (GCSFR)-gp130(133) chimeric receptor, overexpression of Stat3 induced gammaFBG promoter activity 30-fold in A549 cells. Together, the data suggest tissue-specific differences in IL-6-receptor-gp130-coupled signaling, thereby limiting the extent of Stat3 activation and gammaFBG expression during lung inflammation.

    Funded by: NHLBI NIH HHS: P01-HL30616, R01-HL50616

    The Journal of biological chemistry 2006;281;18;12451-7

  • p130Cas: a versatile scaffold in signaling networks.

    Defilippi P, Di Stefano P and Cabodi S

    Department of Genetics, Biology and Biochemistry, University of Turin, Via Santena 5 bis 10126 Turin, Italy. paola.defilippi@unito.it

    The Cas family of multiadaptor and scaffold molecules has an essential role in intracellular signaling events. Although these proteins do not have enzymatic or transcriptional activity, they spatially and temporally control signaling events through their ability to undergo changes in phosphorylation and to associate with effectors proteins in multimolecular complexes. The involvement of p130Cas in cell motility as a component of the integrin signaling machinery is well established. Here, we discuss recent developments that highlight a fundamental role in cell transformation and microbial pathogenesis and the implications of these developments on p130Cas function under normal and pathological conditions.

    Trends in cell biology 2006;16;5;257-63

  • Epistatic and pleiotropic effects of polymorphisms in the fibrinogen and coagulation factor XIII genes on plasma fibrinogen concentration, fibrin gel structure and risk of myocardial infarction.

    Mannila MN, Eriksson P, Ericsson CG, Hamsten A and Silveira A

    King Gustaf V Research Institute, Karolinska University Hospital Solna, S-171 76 Stockholm, Sweden, Maria.Nastase.Mannila@ki.se

    An intricate interplay between the genes encoding fibrinogen gamma (FGG), alpha (FGA) and beta (FGB), coagulation factor XIII (F13A1) and interleukin 6 (IL6) and environmental factors is likely to influence plasma fibrinogen concentration, fibrin clot structure and risk of myocardial infarction (MI). In the present study, the potential contribution of SNPs harboured in the fibrinogen, IL6 and F13A1 genes to these biochemical and clinical phenotypes was examined. A database and biobank based on 387 survivors of a first MI and population-based controls were used. Sixty controls were selected according to FGG 9340T > C [rs1049636] genotype for studies on fibrin clot structure using the liquid permeation method. The multifactor dimensionality reduction method was used for interaction analyses. We here report that the FGA 2224G > A [rs2070011] SNP (9.2%), plasma fibrinogen concentration (13.1%) and age (8.1%) appeared as independent determinants of fibrin gel porosity. The FGA 2224G > A SNP modulated the relation between plasma fibrinogen concentration and fibrin clot porosity. The FGG-FGA*4 haplotype, composed of the minor FGG 9340C and FGA 2224A alleles, had similar effects, supporting its reported protective role in relation to MI. Significant epistasis on plasma fibrinogen concentration was detected between the FGA 2224G > A and F13A1 Val34Leu [rs5985] SNPs (p < 0.001). The FGG 9340T > C and FGB 1038G > A [rs1800791] SNPs appeared to interact on MI risk, explaining the association of FGG-FGB haplotypes with MI in the absence of effects of individual SNPs. Thus, epistatic and pleiotropic effects of polymorphisms contribute to the variation in plasma fibrinogen concentration, fibrin clot structure and risk of MI.

    Thrombosis and haemostasis 2006;95;3;420-7

  • Elucidation of N-glycosylation sites on human platelet proteins: a glycoproteomic approach.

    Lewandrowski U, Moebius J, Walter U and Sickmann A

    Protein Mass Spectrometry and Functional Proteomics Group, Rudolf Virchow Center for Experimental Biomedicine, Versbacher Strasse 9, 97078 Wuerzburg, Germany.

    Among known platelet proteins, a prominent and functionally important group is represented by glycoprotein isoforms. They account e.g. for secretory proteins and plasma membrane receptors including integrins and glycoprotein VI as well as intracellular components of cytosol and organelles including storage proteins (multimerin 1 etc.). Although many of those proteins have been studied for some time with regard to their function, little attention has been paid with respect to their glycosylation sites. Here we report the analysis of N-glycosylation sites of human platelet proteins. For the enrichment of glycopeptides, lectin affinity chromatography as well as chemical trapping of protein bound oligosaccharides was used. Therefore, concanavalin A was used for specific interaction with carbohydrate species along with periodic acid oxidation and hydrazide bead trapping of glycosylated proteins. Derivatization by peptide:N-glycosidase F yielded deglycosylated peptides, which provided the basis for the elucidation of proteins and their sites of modification. Using both methods in combination with nano-LC-ESI-MS/MS analysis 70 different glycosylation sites within 41 different proteins were identified. Comparison with the Swiss-Prot database established that the majority of these 70 sites have not been specifically determined by previous research projects. With this approach including hydrazide bead affinity trapping, the immunoglobulin receptor G6f, which is known to couple to the Ras-mitogen-activated protein kinase pathway in the immune system, was shown here for the first time to be present in human platelets.

    Molecular & cellular proteomics : MCP 2006;5;2;226-33

  • Haplotypes of the fibrinogen gamma gene do not affect the risk of myocardial infarction.

    Uitte DE Willige S, Doggen CJ, DE Visser MC, Bertina RM and Rosendaal FR

    Journal of thrombosis and haemostasis : JTH 2006;4;2;474-6

  • A cluster of basic amino acid residues in the gamma370-381 sequence of fibrinogen comprises a binding site for platelet integrin alpha(IIb)beta3 (glycoprotein IIb/IIIa).

    Podolnikova NP, Gorkun OV, Loreth RM, Yee VC, Lord ST and Ugarova TP

    Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Lerner Research Institute, Cleveland, Ohio 44195, USA.

    Adhesive interactions of platelet integrin alpha(IIb)beta3 with fibrinogen and fibrin are central events in hemostasis and thrombosis. However, the mechanisms by which alpha(IIb)beta3 binds these ligands remain incompletely understood. We have recently demonstrated that alpha(IIb)beta3 binds the gamma365-383 sequence in the gammaC-domain of fibrin(ogen). This sequence contains neither the AGDV nor the RGD recognition motifs, known to bind alpha(IIb)beta3, suggesting the different specificity of the integrin. Here, using peptide arrays, mutant fibrinogens, and recombinant mutant gammaC-domains, we have examined the mechanism whereby alpha(IIb)beta3 binds gamma365-383. The alpha(IIb)beta3-binding activity was localized within gamma370-381, with two short sequences, gamma370ATWKTR375 and gamma376WYSMKK381, being able to independently bind the integrin. Furthermore, recognition of alpha(IIb)beta3 by gamma370-381 depended on four basic residues, Lys373, Arg375, Lys380, and Lys381. Simultaneous replacement of these amino acids and deletion of the gamma408AGDV411 sequence in the recombinant gammaC-domain resulted in the loss of alpha(IIb)beta3-mediated platelet adhesion. Confirming the critical roles of the identified residues, abnormal fibrinogen Kaiserslautern, in which gammaLys380 is replaced by Asn, demonstrated delayed clot retraction and impaired alpha(IIb)beta3 binding. Also, a mutant recombinant fibrinogen modeled after the naturally occurring variant Osaka V (gammaArg375 --> Gly) showed delayed clot retraction and reduced binding to purified alpha(IIb)beta3. These results identify the gamma370-381 sequence of fibrin(ogen) as the binding site for alpha(IIb)beta3 involved in platelet adhesion and clot retraction and define the new recognition specificity of this integrin.

    Funded by: NHLBI NIH HHS: HL 63199

    Biochemistry 2005;44;51;16920-30

  • Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen gamma' levels.

    Uitte de Willige S, de Visser MC, Houwing-Duistermaat JJ, Rosendaal FR, Vos HL and Bertina RM

    Hemostasis and Thrombosis Research Center, Department of Hematology (C2-R), Leiden University Medical Center, Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.

    We investigated the association between haplotypes of fibrinogen alpha (FGA), beta (FGB), and gamma (FGG), total fibrinogen levels, fibrinogen gamma' (gammaA/gamma' plus gamma'/gamma') levels, and risk for deep venous thrombosis. In a population-based case-control study, the Leiden Thrombophilia Study, we typed 15 haplotype-tagging single nucleotide polymorphisms (htSNPs) in this gene cluster. None of these haplotypes was associated with total fibrinogen levels. In each gene, one haplotype increased the thrombosis risk approximately 2-fold. After adjustment for linkage disequilibrium between the genes, only FGG-H2 homozygosity remained associated with risk (odds ratio [OR], 2.4; 95% confidence interval [95% CI], 1.5-3.9). FGG-H2 was also associated with reduced fibrinogen gamma' levels and reduced ratios of fibrinogen gamma' to total fibrinogen. Multivariate analysis showed that reduced fibrinogen gamma' levels and elevated total fibrinogen levels were both associated with an increased risk for thrombosis, even after adjustment for FGG-H2. A reduced fibrinogen gamma' to total fibrinogen ratio (less than 0.69) also increased the risk (OR, 2.4; 95% CI, 1.7-3.5). We propose that FGG-H2 influences thrombosis risk through htSNP 10034C/T [rs2066865] by strengthening the consensus of a CstF site and thus favoring the formation of gammaA chain above that of gamma' chain. Fibrinogen gamma' contains a unique high-affinity, nonsubstrate binding site for thrombin, which seems critical for the expression of the antithrombin activity that develops during fibrin formation (antithrombin 1).

    Blood 2005;106;13;4176-83

  • Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry.

    Liu T, Qian WJ, Gritsenko MA, Camp DG, Monroe ME, Moore RJ and Smith RD

    Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99354, USA.

    The enormous complexity, wide dynamic range of relative protein abundances of interest (over 10 orders of magnitude), and tremendous heterogeneity (due to post-translational modifications, such as glycosylation) of the human blood plasma proteome severely challenge the capabilities of existing analytical methodologies. Here, we describe an approach for broad analysis of human plasma N-glycoproteins using a combination of immunoaffinity subtraction and glycoprotein capture to reduce both the protein concentration range and the overall sample complexity. Six high-abundance plasma proteins were simultaneously removed using a pre-packed, immobilized antibody column. N-linked glycoproteins were then captured from the depleted plasma using hydrazide resin and enzymatically digested, and the bound N-linked glycopeptides were released using peptide-N-glycosidase F (PNGase F). Following strong cation exchange (SCX) fractionation, the deglycosylated peptides were analyzed by reversed-phase capillary liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Using stringent criteria, a total of 2053 different N-glycopeptides were confidently identified, covering 303 nonredundant N-glycoproteins. This enrichment strategy significantly improved detection and enabled identification of a number of low-abundance proteins, exemplified by interleukin-1 receptor antagonist protein (approximately 200 pg/mL), cathepsin L (approximately 1 ng/mL), and transforming growth factor beta 1 (approximately 2 ng/mL). A total of 639 N-glycosylation sites were identified, and the overall high accuracy of these glycosylation site assignments as assessed by accurate mass measurement using high-resolution liquid chromatography coupled to Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR) is initially demonstrated.

    Funded by: NCRR NIH HHS: P41 RR018522, RR18522; NIGMS NIH HHS: U54 GM-62119-02, U54 GM062119

    Journal of proteome research 2005;4;6;2070-80

  • Fibrin but not adsorbed fibrinogen supports fibronectin assembly by spread platelets. Effects of the interaction of alphaIIb beta3 with the C terminus of the fibrinogen gamma-chain.

    Cho J, Degen JL, Coller BS and Mosher DF

    Molecular and Cellular Pharmacology Program and Department of Medicine, University of Wisconsin-Madison School of Medicine, Madison, Wisconsin 53706, USA.

    We investigated the assembly of soluble fibronectin by lysophosphatidic acid-activated platelets adherent to fibrinogen or fibrin. More fibronectin was assembled by activated platelets spread on fibrin matrices than by platelets spread on adsorbed fibrinogen. The difference between platelets adherent to fibrinogen and fibrin occurred under both static and flow conditions. Similar differences were seen in binding of the 70-kDa N-terminal fragment of fibronectin that recognizes fibronectin assembly sites on adherent cells. Antibody and peptide blocking studies demonstrated that alphaIIb beta3 integrin mediates platelet adhesion to fibrinogen, whereas both alphav beta3 and alphaIIb beta3 mediate platelet adhesion to fibrin. The hypothesis that engagement of the C-terminal QAGDV sequence of the fibrinogen gamma-chain by alphaIIb beta3 inhibits the ability of the platelet to assemble fibronectin was tested by several experiments. Activated platelets adherent to adsorbed mutant fibrinogen lacking the QAGDV sequence (gammadelta5FG) were assembly-competent, as were platelets adherent to adsorbed normal fibrinogen that had been pretreated with the 7E9 antibody to the C terminus of the gamma-chain. Moreover, adsorbed normal fibrinogen but not gammadelta5FG suppressed the ability of co-adsorbed fibronectin to direct assembly of soluble fibronectin by spread platelets. The suppressive effect was lost when a surface of co-adsorbed fibronectin and fibrinogen was pretreated with 7E9. These results support a model in which the engagement of alphaIIb beta3 by the C-terminal sequence of the fibrinogen gamma-chain initiates signals that suppress subsequent fibronectin assembly by spread platelets. This interaction is less dominant when platelets adhere to fibrin, resulting in enhanced fibronectin assembly.

    The Journal of biological chemistry 2005;280;42;35490-8

  • Towards a proteome-scale map of the human protein-protein interaction network.

    Rual JF, Venkatesan K, Hao T, Hirozane-Kishikawa T, Dricot A, Li N, Berriz GF, Gibbons FD, Dreze M, Ayivi-Guedehoussou N, Klitgord N, Simon C, Boxem M, Milstein S, Rosenberg J, Goldberg DS, Zhang LV, Wong SL, Franklin G, Li S, Albala JS, Lim J, Fraughton C, Llamosas E, Cevik S, Bex C, Lamesch P, Sikorski RS, Vandenhaute J, Zoghbi HY, Smolyar A, Bosak S, Sequerra R, Doucette-Stamm L, Cusick ME, Hill DE, Roth FP and Vidal M

    Center for Cancer Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115, USA.

    Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.

    Funded by: NCI NIH HHS: R33 CA132073; NHGRI NIH HHS: P50 HG004233, R01 HG001715, RC4 HG006066, U01 HG001715; NHLBI NIH HHS: U01 HL098166

    Nature 2005;437;7062;1173-8

  • Hypofibrinogenaemia caused by a novel FGG missense mutation (W253C) in the gamma chain globular domain impairing fibrinogen secretion.

    Vu D, de Moerloose P, Batorova A, Lazur J, Palumbo L and Neerman-Arbez M

    Department of Genetic Medicine and Development, University Medical School, Geneva, Switzerland.

    Background: Inherited disorders of fibrinogen are rare and affect either the quantity (hypofibrinogenaemia and afibrinogenaemia) or the quality of the circulating fibrinogen (dysfibrinogenaemia). Extensive allelic heterogeneity has been found for all three disorders: in congenital afibrinogenaemia >30 mutations, the majority in FGA, have been identified in homozygosity or in compound heterozygosity. Several mutations have also been identified in patients with hypofibrinogenaemia; many of these are heterozygous carriers of afibrinogenaemia null mutations.

    Objective: To report the case of a patient from Slovakia diagnosed with hypofibrinogenaemia characterised by fibrinogen concentrations of around 0.7 g/l.

    Results: The patient was found to be heterozygous for a novel missense mutation W253C (W227C in the mature protein) in the C-terminal globular domain of the fibrinogen gamma chain. Co-expression of the W253C FGG mutant cDNA (fibrinogen Bratislava) in combination with wild-type FGA and FGB cDNAs showed that fibrinogen molecules containing the mutant gamma chain can assemble intracellularly but are not secreted into the media, confirming the causative nature of the identified mutation.

    Conclusions: Current analysis of fibrinogen Bratislava indicates that the domains important for the processes of hexamer assembly and hexamer secretion should not be considered as strictly restricted to one or other fibrinogen chain.

    Journal of medical genetics 2005;42;9;e57

  • Integrins and Src: dynamic duo of adhesion signaling.

    Shattil SJ

    Hematology-Oncology Division, Department of Medicine, University of California-San Diego, La Jolla, CA 92093, USA. sshattil@ucsd.edu

    Src family protein tyrosine kinases (SFKs) play important roles downstream of integrin adhesion receptors, and they are necessary for the generation of "outside-in signals" that regulate cytoskeletal organization, cell motility and gene expression in response to cell adhesion. One relatively under-explored facet of this relationship is the possible physical interaction of integrins with SFKs. Recently, it has been established that beta3 integrins and c-Src can interact directly, and this pool of c-Src is activated by cell adhesion to initiate outside-in signaling in platelets, osteoclasts and cells of the vasculature. Here, the biochemical basis for and biological significance of this integrin-SFK interaction is summarized, and I propose a general mechanism for initiation of outside-in integrin signaling.

    Trends in cell biology 2005;15;8;399-403

  • In vitro expression demonstrates impaired secretion of the gammaAsn319, Asp320 deletion variant fibrinogen.

    Kani S, Terasawa F, Lord ST, Tozuka M, Ota H, Okumura N and Katsuyama T

    Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto, 390-8621, Japan.

    The hypodysfibrinogenemia Otsu is caused by the two-residue deletion, gammaAsn319 and gammaAsp320. Analysis of plasma or purified fibrinogen from the heterozygous propositus revealed that the amount of variant gamma-chain was lower than that of normal gamma-chain. In order to examine the basis for this difference, we transfected Chinese hamster ovary cells and established stable cell lines that expressed both chains, gammaDelta/gammaN, only the normal chain, gammaN, and only the variant chain, gammaDelta. We measured fibrinogen concentration of confluent cultures by ELISA. We found the ratios of the concentrations in the media to the concentrations in the cell lysates of gammaDelta, gammaDelta/gammaN, and gammaN-cells were 0.42, 0.60, and 1.00, respectively. We measured the concentrations of the gammaDelta and gammaN chains by densitometric analysis of samples following separation by SDS-PAGE and found the fraction of gammaDelta-chains in cell lysates was always greater than the fraction in the respective culture media. We examined the kinetics of fibrinogen synthesis, assembly and secretion in pulse-chase experiments, and found that the gammaDelta-chain was assembled into intact fibrinogen at a rate similar to assembly of the gammaN-chain into normal fibrinogen, but was secreted into the medium at a slightly slower rate than normal fibrinogen. Considered together, these experiments indicate secretion of the variant fibrinogen was slightly impaired. These results suggest that the reduced level of gammaDelta319,320 fibrinogen in the plasma of the Otsu patient arises from modestly impaired secretion of this variant fibrinogen.

    Funded by: NHLBI NIH HHS: R01 HL68836

    Thrombosis and haemostasis 2005;94;1;53-9

  • Fibrinogen Philadelphia, a hypodysfibrinogenemia characterized by abnormal polymerization and fibrinogen hypercatabolism due to gamma S378P mutation.

    Keller MA, Martinez J, Baradet TC, Nagaswami C, Chernysh IN, Borowski MK, Surrey S and Weisel JW

    Department of Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA.

    Fibrinogen Philadelphia, a hypodysfibrinogenemia described in a family with a history of bleeding, is characterized by prolonged thrombin time, abnormal fibrin polymerization, and increased catabolism of the abnormal fibrinogen. Turbidity studies of polymerization of purified fibrinogen under different ionic conditions reveal a reduced lag period and lower final turbidity, indicating more rapid initial polymerization and impaired lateral aggregation. Consistent with this, scanning and transmission electron microscopy show fibers with substantially lower average fiber diameters. DNA sequence analysis of the fibrinogen genes A, B, and G revealed a T>C transition in exon 9 resulting in a serine-to-proline substitution near the gamma chain C-terminus (S378P). The S378P mutation is associated with fibrinogen Philadelphia in this kindred and was not found in 10 controls. This region of the gamma chain is involved in fibrin polymerization, supporting this as the polymerization defect causing the mutation. Thus, this abnormal fibrinogen is characterized by 2 unique features: (1) abnormal polymerization probably due to a major defect in lateral aggregation and (2) hypercatabolism of the mutant protein. The location, nature, and unusual characteristics of this mutation may add to our understanding of fibrinogen protein interactions necessary for normal catabolism and fibrin formation.

    Funded by: NHLBI NIH HHS: HL059227, HL30954

    Blood 2005;105;8;3162-8

  • Regulation of gamma-fibrinogen chain expression by heterogeneous nuclear ribonucleoprotein A1.

    Xia H

    Lindsley F. Kimball Research Institute, New York Blood Center, New York, New York 10021, USA. hxia@nybloodcenter.org

    Earlier studies showed that HepG2 cells stably transfected with any one fibrinogen chain cDNA enhanced the expression of the other two fibrinogen chains. In this report, a regulatory element "TGCTCTC" in the gamma-fibrinogen promoter region, -322 to -316, is identified, which is involved in increased expression of gamma chain in HepG2 cells that are transfected with Bbeta fibrinogen cDNA. By electrophoretic mobility shift assay, three DNA-protein complexes were found to form with the regulatory element. The amount of the protein complexes that bind with the regulatory element was much reduced in HepG2 cells transfected with Bbeta cDNA. By DNA-affinity chromatography, mass spectrometry, and supershift assay, human heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) was identified as a component of the complexes. Overexpression of hnRNP A1 suppressed basal gamma-fibrinogen transcription. These results indicate that the basal expression of gamma-fibrinogen is regulated by a constitutive transcriptional repressor protein, hnRNP A1, and the decreased binding activity of hnRNP A1 leads to the overexpression of gamma chain in HepG2 cells that overexpress the Bbeta chain.

    The Journal of biological chemistry 2005;280;13;13171-8

  • gammaAla82Gly represents a common fibrinogen gamma-chain variant in Caucasians.

    Ivaskevicius V, Jusciute E, Steffens M, Geisen C, Hanfland P, Wienker TF, Seifried E and Oldenburg J

    Institute for Transfusion Medicine and Immune Haematology, DRK Blood Donor Service Baden-Württemberg/Hessen, Sandhofstrasse 1, D-60528 Frankfurt am Main, Germany.

    Screening of 200 blood donors for the presence of polymorphisms in three fibrinogen genes (FGA, FGB, FGG), revealed two individuals with a heterozygous missense mutation (c.323C > G, gammaAla82Gly) in the FGG gene. This mutation has been reported previously to cause mild hypofibrinogenaemia. Analysis of an additional 416 blood donors showed two more heterozygous gammaAla82Gly mutations, resulting in an overall gammaAla82Gly allele frequency of 0.0032. Haplotype analysis demonstrated that the gammaAla82Gly mutation originated from a common founder. From these data we estimated that homozygous individuals for gammaAla82Gly should occur at a frequency of 1: 95 000, suggesting that hypofibrinogenaemia represents a more frequent condition in the population than so far believed.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2005;16;3;205-8

  • Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway.

    Carlsson F, Sandin C and Lindahl G

    Department of Medical Microbiology, Dermatology and Infection, Lund University, Sölvegatan 23, SE-22362 Lund, Sweden.

    Human fibrinogen (Fg) binds to surface proteins expressed by many pathogenic bacteria and has been implicated in different host-pathogen interactions, but the role of bound Fg remains unclear. Here, we analyse the role of Fg bound to Streptococcus pyogenes M protein, a major virulence factor that confers resistance to phagocytosis. Studies of the M5 system showed that a chromosomal mutant lacking the Fg-binding region was completely unable to resist phagocytosis, indicating that bound Fg plays a key role in virulence. Deposition of complement on S. pyogenes occurred via the classical pathway even under non-immune conditions, but was blocked by M5-bound Fg, which reduced the amount of classical pathway C3 convertase on the bacterial surface. This property of M protein-bound Fg may explain its role in phagocytosis resistance. Previous studies have shown that many M proteins do not bind Fg, but interfere with complement deposition and phagocytosis by recruiting human C4b-binding protein (C4BP), an inhibitor of the classical pathway. Thus, all M proteins may share ability to recruit a human plasma protein, Fg or C4BP, which inhibits complement deposition via the classical pathway. Our data identify a novel function for surface-bound Fg and allow us to propose a unifying mechanism by which M proteins interfere with innate immunity.

    Molecular microbiology 2005;56;1;28-39

  • Contribution of haplotypes across the fibrinogen gene cluster to variation in risk of myocardial infarction.

    Mannila MN, Eriksson P, Lundman P, Samnegård A, Boquist S, Ericsson CG, Tornvall P, Hamsten A and Silveira A

    Atherosclerosis Research Unit, Karolinska Institutet, King Gustaf V Research Institute, Karolinska University Hospital, Solna, Stockholm, Sweden. Maria.Nastase.Mannila@medks.ki.se

    Fibrinogen has consistently been recognized as an independent predictor of myocardial infarction (MI). Multiple mechanisms link fibrinogen to MI; therefore disentangling the factors underlying variation in plasma fibrinogen concentration is essential. Candidate regions in the fibrinogen gamma (FGG), alpha (FGA) and beta (FGB) genes were screened for single nucleotide polymorphisms (SNPs). Several novel SNPs were detected in the FGG and FGA genes in addition to the previously known SNPs in the fibrinogen genes. Tight linkage disequilibrium extending over various physical distances was observed between most SNPs. Consequently, eight SNPs were chosen and determined in 377 postinfarction patients and 387 healthy individuals. None of the SNPs were associated with plasma fibrinogen concentration or MI. Haplotype analyses revealed a consistent pattern of haplotypes associated with variation in risk of MI. Of the four haplotypes inferred using the FGA -58G>A and FGG 1299 +79T>C SNPs, the most frequent haplotype, FGG-FGA*1 (prevalence 46.6%), was associated with increased risk of MI (OR 1.51; 95%CI 1.18, 1.93), whereas the least frequent haplotype, FGG-FGA*4 (11.8%), was associated with lower risk of MI (OR 0.79 95%CI 0.64, 0.98). In conclusion, fibrinogen haplotypes, but not SNPs in isolation, are associated with variation in risk of MI.

    Thrombosis and haemostasis 2005;93;3;570-7

  • Interaction of fibrin(ogen) with leukocyte receptor alpha M beta 2 (Mac-1): further characterization and identification of a novel binding region within the central domain of the fibrinogen gamma-module.

    Yakovlev S, Zhang L, Ugarova T and Medved L

    University of Maryland School of Medicine, Rockville, Maryland 20855, USA.

    The fibrinogen gamma-module sequences, gamma190-202 or P1, and gamma377-395 or P2, were implicated in interaction with the alpha(M)I-domain of the leukocyte receptor alpha(M)beta(2). P1 is an integral part of the gamma-module central domain, while P2 is inserted into this domain forming an antiparallel beta-strand with P1. We hypothesized earlier that separation of P2 from P1 may regulate interaction of fibrin(ogen) with leukocytes during the inflammatory response. To test the relative contributions of these sequences to the interaction and the effect of their separation, we prepared the recombinant gamma-module (gamma148-411) and its halves, gamma148-286 and gamma287-411 fragments containing P1 and P2, respectively, and evaluated their affinities for the recombinant alpha(M)I-domain. In a solid-phase binding assay, the immobilized gamma-module exhibited high affinity for alpha(M)I (K(d) = 22 nM), while the affinities of the isolated gamma148-286 and gamma287-411 halves were much lower (K(d)'s = 521 and 194 nM, respectively), indicating that both halves contribute to the interaction in a synergistic manner. This is consistent with the above hypothesis. Further, we prepared the recombinant gamma148-191 and gamma192-286 fragments corresponding to the NH(2)-terminal and central domains, respectively, as well as gamma148-226 containing P1, and tested their interaction with alpha(M)I. The immobilized gamma192-286 fragment bound to alpha(M)I with K(d) = 559 nM, while both gamma148-191 and gamma148-226 failed to bind suggesting that P1 does not contribute substantially to the binding and that the binding occurs mainly through the gamma227-286 region. To further localize a putative binding sequence, we cleaved gamma192-286 and analyzed the resulting peptides. The only alpha(M)I-binding activity was associated with the gamma228-253 peptide, indicating that this region of the central domain contains a novel alpha(M)beta(2)-binding sequence.

    Funded by: NHLBI NIH HHS: HL-56051, HL-61589, HL-63199, P01 HL054710, R01 HL056051, R01 HL061589

    Biochemistry 2005;44;2;617-26

  • Expression and analysis of a split premature termination codon in FGG responsible for congenital afibrinogenemia: escape from RNA surveillance mechanisms in transfected cells.

    Neerman-Arbez M, Germanos-Haddad M, Tzanidakis K, Vu D, Deutsch S, David A, Morris MA and de Moerloose P

    Department of Genetic Medicine and Development, University Medical School, Geneva, Switzerland. marguerite.arbez@medecine.unige.ch

    Congenital afibrinogenemia, the most severe form of fibrinogen deficiency, is characterized by the complete absence of fibrinogen. The disease is caused by mutations in 1 of the 3 fibrinogen genes FGG, FGA, and FGB, clustered on the long arm of human chromosome 4. The majority of cases are due to null mutations in the FGA gene although one would expect the 3 genes to be equally implicated. However, most patients studied so far are white, and therefore the identification of causative mutations in non-European families is necessary to establish if this finding holds true in all ethnic groups. In this study, we report the identification of a novel nonsense mutation (Arg134Xaa) in the FGG gene responsible for congenital afibrinogenemia in 10 patients from Lebanon. Expression studies in COS-7 cells demonstrated that the Arg134Xaa codon, which is encoded by adjacent exons (TG-intron 4-A) affected neither mRNA splicing nor stability, but led to the production of an unstable, severely truncated fibrinogen gamma chain that is not incorporated into a functional fibrinogen hexamer.

    Blood 2004;104;12;3618-23

  • Hypofibrinogenaemia associated with a novel heterozygous gamma289 Ala -->Val substitution (fibrinogen Dorfen).

    Dear A, Brennan SO, Dempfle CE, Kirschstein W and George PM

    Molecular Pathology Laboratory, Christchurch School of Medicine and Health Sciences, PO Box 4345, Christchurch, New Zealand. amy.dear@chmeds.ac.nz.

    The molecular basis of hypofibrinogenaemia was investigated in a 34-year-old woman and her 10-year-old daughter. DNA sequencing revealed a single heterozygous GCC-->GTC transition in exon 8 of the fibrinogen gamma ?gene in both subjects, predicting a novel gamma289 Ala-->Val substitution. Examination of fibrinogen gamma ?chains by electrospray ionization mass spectrometry failed to detect the variant chain in plasma fibrinogen. Further evidence for its non-expression came from tryptic peptide mapping. The mutation predicts a mass increase of 28 Da in peptide T32, but only the normal (M + 2H) ion was detected at 1418 m/z in the proposita. Our finding that gamma289 is an important determinant of plasma fibrinogen levels highlights the role of mutational analysis in defining structurally important regions of the fibrinogen molecule. This case suggests that the highly conserved Ala(289) is important in maintaining structure of the "a" polymerization site via hydrogen bonding to Thr(371).

    Thrombosis and haemostasis 2004;92;6;1291-5

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Genetic and environmental determinants of fibrin structure and function: relevance to clinical disease.

    Scott EM, Ariëns RA and Grant PJ

    Academic Unit of Molecular Vascular Medicine, Martin Wing, The General Infirmary at Leeds, Leeds, LS1 3EX, UK.

    The formation of a fibrin clot is one of the key events in atherothrombotic vascular disease. The structure of the fibrin clot and the genetic and environmental factors that modify it have effects on its biological function. Alterations in fibrin structure and function have implications for the clinical presentation of vascular disease. This review briefly describes the key features involved in the formation of a fibrin clot, its typical structure, and function. This is followed by a review of the current literature on genetic and environmental influences on fibrin structure/function and the relationship to clinical disease. The formation of a fibrin clot is one of the key events in atherothrombotic vascular disease. This review discusses how genetic and environmental factors alter fibrin structure and function and the implications this has for the clinical presentation of vascular disease.

    Arteriosclerosis, thrombosis, and vascular biology 2004;24;9;1558-66

  • Investigation of residues in the fibrin(ogen) gamma chain involved in tissue plasminogen activator binding and plasminogen activation.

    Wilhelm SE, Lounes KC and Lord ST

    Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.

    In order to characterize tissue plasminogen activator (t-PA) binding to gamma-chain residues in fibrinogen, we generated variant fibrinogens substituting alanine for gamma D316, gamma D318, gamma D320, and gamma K321. We measured thrombin-catalyzed polymerization and found normal polymerization with gamma K321A, no polymerization with gamma D316A, and, as reported by Lounes et al. in 2002, impaired polymerization with gamma D318A and gamma D320A. We measured t-PA binding in a solid-phase assay, and t-PA activity by the generation of plasmin. Comparing normal fibrin with fibrinogen, we found a seven-fold increase in binding and a two-fold increase in activity. Binding to all variant fibrinogens was the same as normal. In contrast, t-PA binding to all variant fibrins was weaker than binding to normal fibrin, 2.5-fold for gamma K321A, seven-fold for gamma D320A and 10-fold for gamma D316A and gamma D318A. Plasmin generation in the presence of variant fibrinogens was similar, although not identical, to normal, and plasmin generation in the presence of variant fibrins was impaired for the Asp to Ala variants. As the three variants with the weakest t-PA binding and least activity also showed impaired polymerization, our results support previous findings demonstrating the DD:E complex, found in the normal fibrin polymer, is necessary for the fibrin enhanced binding of t-PA and activation of plasminogen.

    Funded by: NHLBI NIH HHS: HL 31048

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 2004;15;6;451-61

  • Comparison of thrombin-catalyzed fibrin polymerization and factor XIIIa-catalyzed cross-linking of fibrin among three recombinant variant fibrinogens, gamma 275C, gamma 275H, and gamma 275A.

    Hirota-Kawadobora M, Terasawa F, Suzuki T, Tozuka M, Sano K and Okumura N

    Department of Pathology, Shinshu University School of Medicine, Shinshu University Hospital, Japan.

    We have previously reported that recombinant gamma 275Cys fibrinogen exhibits a marked impairment of functions as well as aberrant fibrin clot and bundle structures, as compared with wild-type, gamma 275Arg, and plasma fibrinogen from a heterozygous proband. Since gamma Arg275His mutations have also been reported in 10 families, we synthesized recombinant gamma 275His fibrinogen and gamma 275Ala fibrinogen (as a control) and analyzed and compared them with gamma 275Cys and gamma 275Arg.

    Methods: A variant gamma-chain expression plasmid was transfected into Chinese hamster ovary cells expressing normal human fibrinogen A alpha- and B beta-chains. After purification of the recombinant variant fibrinogens, we performed functional analyzes for thrombin-catalyzed fibrin polymerization and factor XIIIa (FXIIIa)-catalyzed gamma-gamma dimer formation from fibrin or fibrinogen and also ultrastructural analysis of fibrin clots and bundles.

    Results: By comparison with both gamma 275His and gamma 275Ala fibrinogens, recombinant gamma 275Cys fibrinogen exhibited a more impaired gamma-gamma dimer formation from fibrin or fibrinogen, a more aberrant fibrin clot structure, and thicker fibers in fibrin bundles. In 1 : 1 mixtures of gamma 275Arg and gamma 275Cys fibrinogens or gamma 275Arg and gamma 275His fibrinogens, thrombin-catalyzed fibrin polymerization and both fibrin clot and fiber structures showed some compensation (as compared with gamma 275Cys or gamma 275His alone).

    Conclusion: These results strongly suggest that an amino acid substitution of gamma 275Arg alone disrupts D:D interactions in thrombin-catalyzed fibrin polymerization and the formation of fibrin bundles and fibrin clots. Moreover, the existence of a subsequent disulfide-linked Cys in gamma 275C fibrinogen augments the impairment caused by a His or Ala substitution.

    Journal of thrombosis and haemostasis : JTH 2004;2;8;1359-67

  • Screening for N-glycosylated proteins by liquid chromatography mass spectrometry.

    Bunkenborg J, Pilch BJ, Podtelejnikov AV and Wiśniewski JR

    MDS Proteomics Odense, Denmark.

    In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.

    Proteomics 2004;4;2;454-65

  • Fibrinogen C-terminal peptidic sequences (Haptides) modulate fibrin polymerization.

    Marx G, Ben-Moshe M, Magdassi S and Gorodetsky R

    HAPTO Biotech (Israel) Ltd. PO Box 12275, Jerusalem 91121, Israel 91121. gmarx@hapto.co.il

    We previously described synthetic peptides of 19-21 amino acid residues, homologous to the C-termini of fibrinogen Fib(340) and Fib(420), from the beta-chain (Cbeta), the extended alphaE chain (CalphaE) and near the end of the gamma-chain (preCgamma) which elicited attachment (haptotactic) responses from mesenchymal cells. We named these haptotactic peptides -Haptides. The effects of Haptides on fibrin clot formation was evaluated and their possible effects on platelet aggregation was examined. The Haptides Cbeta,CalphaE and preCgamma, (2-10 micro M) increased fibrin clot turbidity and also decreased thrombin-induced clotting time. Higher concentrations (>120 micro M of Cbeta or preCgamma) induced fibrinogen precipitation even without thrombin. These precipi-tates exhibited different ultrastructure from thrombin-induced fibrin by scanning and transmission microscopy. C-terminal peptides of the other fibrinogen chains exerted no such effects. Sepharose beads covalently coated with either whole fibrinogen or Haptides (SB-Fib or SB-Haptide) highly adsorbed free (FITC) Haptides. In aqueous solution, Haptides formed nano-par-ticles with average size of approximately 150 nm in diameter. We suggest that such positively charged aggregates could serve to nucleate and accelerate fibrin gel formation. These results also indicate that Cbeta and preCgamma sequences within fibrin(ogen) participate in the docking and condensation of fibrin(ogen) during its assembly into a fibrin clot. By contrast, Haptides up to 100 micro M did not bind to platelets, and had no effect on platelet aggregation. Our findings highlight the roles of the C-terminal sequences of the beta and gamma chains in fibrin(ogen) polymerization as well as in cell attachment.

    Thrombosis and haemostasis 2004;91;1;43-51

  • Substitution of the gamma-chain Asn308 disturbs the D:D interface affecting fibrin polymerization, fibrinopeptide B release, and FXIIIa-catalyzed cross-linking.

    Okumura N, Gorkun OV, Terasawa F and Lord ST

    Laboratory of Clinical Chemistry, Department of Biomedical Laboratory Sciences, School of Health Sciences, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan. nobuoku@gipac.shinshu-u.ac.jp

    Crystallographic structures indicate that gamma-chain residue Asn308 participates in D:D interactions and indeed substitutions of gammaAsn308 with lysine or isoleucine have been identified in dysfibrinogens with impaired polymerization. To probe the role of Asn308 in polymerization, we synthesized 3 variant fibrinogens: gammaAsn308 changed to lysine (gammaN308K), isoleucine (gammaN308I), and alanine (gammaN308A). We measured thrombin-catalyzed polymerization by turbidity, fibrinopeptide release by high-performance liquid chromatography, and factor XIIIa-catalyzed cross-linking by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the absence of added calcium, polymerization was clearly impaired with all 3 variants. In contrast, at 0.1 mM calcium, only polymerization of gammaN308K remained markedly abnormal. The release of thrombin-catalyzed fibrinopeptide B (FpB) was delayed in the absence of calcium, whereas at 1 mM calcium FpB release was delayed only with gammaN308K. Factor XIIIa-catalyzed gamma-gamma dimer formation was delayed with fibrinogen (in absence of thrombin), whereas with fibrin (in presence of thrombin) gamma-gamma dimer formation of only gammaN308K was delayed. These data corroborate the recognized link between FpB release and polymerization. They show fibrin cross-link formation likely depends on the structure of protofibrils. Together, our results show substitution of Asn308 with a hydrophobic residue altered neither polymer formation nor polymer structure at physiologic calcium concentrations, whereas substitution with lysine altered both.

    Funded by: NHLBI NIH HHS: HL-31048

    Blood 2004;103;11;4157-63

  • Functional analysis of interleukin 6 response elements (IL-6REs) on the human gamma-fibrinogen promoter: binding of hepatic Stat3 correlates negatively with transactivation potential of type II IL-6REs.

    Duan HO and Simpson-Haidaris PJ

    Department of Medicine, University of Rochester School of Medicine and Dentistry Rochester, New York 14642, USA.

    Several families of transcription factors play important roles in modulating liver-specific gene expression during an acute phase response (APR). Stat3/APR factor is the main transactivator of gene expression by the interleukin (IL)-6 family of cytokines signaling through gp130. During an APR, fibrinogen (FBG) genes are coordinately up-regulated by IL-6 and glucocorticoids. Except for rat gammaFBG, attempts to demonstrate direct binding of IL-6-activated Stat3 to FBG CTGGGAA promoter elements have not been successful. Herein we show the presence of three functional type II IL-6 response elements (IL-6REs) on the human gammaFBG promoter and that the magnitude of Stat3 binding to these elements correlates negatively with their functional activity in reporter gene assays. Stat3-specific binding to gammaFBG IL-6REs was confirmed by cross-competition with alpha2-macroglobulin IL-6RE and specific interactions with anti-Stat3 in electrophoretic mobility shift assays. All type II IL-6REs contributed to full promoter activity; however, transactivation from Site II at -306 to -301 was strongest. In contrast to a previous report, IL-6 failed to induce activation of serum amyloid A-activating factor-1/c-Myc-associated zinc finger protein (SAF-1/MAZ), and mutation of the SAF-1RE had little effect on IL-6 induction of gammaFBG promoter activity. In the absence of a functional glucocorticoid receptor response element, dexamethasone potentiated IL-6-induced gammaFBG promoter activity 2-fold, requiring promoter-proximal Site I and Site II; the promoter-distal Site III had no effect on dexamethasone potentiation of IL-6-induced promoter activity. Notably the propensity for Stat3 binding to human gammaFBG IL-6REs was low compared with Stat3 binding to the alpha2-macroglobulin IL-6RE. Together these data suggest that Stat3 transactivation via IL-6REs on FBG promoters likely involves participation of additional transcription factors and/or coactivators to achieve optimal coordinated up-regulation during an APR.

    Funded by: NHLBI NIH HHS: P01-HL30616, R01-HL50615

    The Journal of biological chemistry 2003;278;42;41270-81

  • Fibrinogen gamma-chain splice variant gamma' alters fibrin formation and structure.

    Cooper AV, Standeven KF and Ariëns RA

    Academic Unit of Molecular Vascular Medicine, University of Leeds, Leeds General Infirmary, United Kingdom.

    Fibrinogen gammaA/gamma' results from alternative splicing of mRNA. This variant, which constitutes approximately 8% to 15% of plasma fibrinogen, contains FXIII and thrombin binding sites. Our objective was to investigate whether gammaA/gamma' differs in fibrin formation and structure from the more common variant gammaA/gammaA. Both variants were separated and purified by anion-exchange chromatography. Fibrin formation and clot structure of the variants and unfractionated fibrinogen were investigated by turbidity and scanning electron microscopy (SEM). Thrombin cleavage of fibrinopeptides was analyzed by high-performance liquid chromatography (HPLC). Turbidity analysis showed significantly altered polymerization rates and overall fiber thickness in gammaA/gamma' clots compared with gammaA/gammaA and unfractionated fibrinogen. This finding was consistent with a range of thrombin concentrations. HPLC demonstrated reduced rates of fibrinopeptide B (FpB) release from gammaA/gamma' fibrinogen compared with gammaA/gammaA. Delayed FpB release was associated with delayed lateral aggregation of protofibrils and significant differences were found on SEM, with gammaA/gamma' clots consisting of smaller diameter fibers and increased numbers of branch points compared with both gammaA/gammaA and unfractionated fibrinogen. These results demonstrate that the gammaA/gamma' splice variant of fibrinogen directly alters fibrin formation and structure, which may help to explain the increased thrombotic risk associated with this variant.

    Blood 2003;102;2;535-40

  • Fibrinogen gamma chain functions.

    Mosesson MW

    The Blood Research Institute of the Blood Center of Southeastern Wisconsin, PO Box 2178, Milwaukee, Wisconsin 53201-2178, USA. mwmosesson@bcsew.edu

    This review covers the functional features of the fibrinogen gamma chains including their participation in fibrin polymerization and cross-linking, their role in the initiation of fibrinolysis, their binding and regulation of factor XIII activity, their interactions with platelets and other cells, and their role in mediating thrombin binding to fibrin, a thrombin inhibitory function termed 'antithrombin I'.

    Funded by: NHLBI NIH HHS: R01 HL59507, R01 HL6888

    Journal of thrombosis and haemostasis : JTH 2003;1;2;231-8

  • Identification of a novel human angiopoietin-like gene expressed mainly in heart.

    Zeng L, Dai J, Ying K, Zhao E, Jin W, Ye Y, Dai J, Xu J, Xie Y and Mao Y

    State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai 200433, PR China.

    The angiopoietins are an important family of growth factors specific for vascular endothelium. Most of them bind to the TIE2 receptor and are related to regulation of angiogenesis. During large-scale DNA sequencing of the human fetal brain cDNA library, we cloned a novel human angiopoietin-like cDNA and termed it human angiopoietin-like 5 ( ANGPTL5). Like other members of the angiopoietin family, ANGPTL5-deduced protein also has an N-terminal cleavable signal peptide, a predicted coiled-coil domain, and a fibrinogen-like domain. The search against the human genome database indicated that ANGPTL5 maps to 11q22. Expression analysis of ANGPTL5 shows that it is mainly expressed in adult human heart.

    Journal of human genetics 2003;48;3;159-62

  • The crystal structure of fragment double-D from cross-linked lamprey fibrin reveals isopeptide linkages across an unexpected D-D interface.

    Yang Z, Pandi L and Doolittle RF

    Center for Molecular Genetics, University of California at San Diego, La Jolla, CA 92093-0634, USA.

    The crystal structure of fragment double-D from factor XIII-cross-linked lamprey fibrin has been determined at 2.9 A resolution. The 180 kDa covalent dimer was cocrystallized with the peptide Gly-His-Arg-Pro-amide, which in many fibrinogens, but not that of lamprey, corresponds to the B-knob exposed by thrombin. The structure was determined by molecular replacement, a recently determined structure of lamprey fragment D being used as a search model. GHRPam was found in both the gamma- and beta-chain holes. Unlike the situation with fragment D, the crystal packing of the cross-linked double-D structure exhibits two different D-D interfaces, each gamma-chain facing gamma-chains on two other molecules. One of these (interface I) involves the asymmetric interface observed in all other D fragments and related structures. The other (interface II) encompasses a completely different set of residues. The two abutments differ in that interface I results in an "in line" arrangement of abutting molecules and the interface II in a "zigzag" arrangement. So far as can be determined (the electron density could only be traced on one side of the cross-links), it is the gamma-chains of the newly observed zigzag units (interface II) that are joined by the reciprocal epsilon-amino-gamma-glutamyl cross-links. Auspiciously, the same novel D-D interface was observed in two lower-resolution crystal structures of human double-D preparations that had been crystallized under unusual circumstances. These observations show that double-D structures are linked in a way that is sufficiently flexible to accommodate different D-D interfaces under different circumstances.

    Funded by: NHLBI NIH HHS: HL 26873

    Biochemistry 2002;41;52;15610-7

  • 2.8 A crystal structures of recombinant fibrinogen fragment D with and without two peptide ligands: GHRP binding to the "b" site disrupts its nearby calcium-binding site.

    Kostelansky MS, Betts L, Gorkun OV and Lord ST

    Department of Chemistry, University of North Carolina, Chapel Hill, NC 27599, USA.

    We report two crystal structures, each at a resolution of 2.8 A, of recombinant human fibrinogen fragment D (rfD) in the absence and presence of peptide ligands. The bound ligands, Gly-Pro-Arg-Pro-amide and Gly-His-Arg-Pro-amide, mimic the interactions of the thrombin exposed polymerization sites, "A" and "B", respectively. This report is the first to describe the structure of fragment D in the presence of both peptide ligands. The structures reveal that recombinant fibrinogen is nearly identical to the plasma protein but with minor changes, like the addition of a proximal fucose to the carbohydrate linked to residue betaGln364, and slightly different relative positions of the beta- and gamma-modules. Of major interest in our structures is that a previously identified calcium site in plasma fibrinogen is absent when Gly-His-Arg-Pro-amide is bound. The peptide-dependent loss of this calcium site may have significant biological implications that are further discussed. These structures provide a foundation for the detailed structural analysis of variant recombinant fibrinogens that were used to identify critical functional residues within fragment D.

    Funded by: NHLBI NIH HHS: HL 31048

    Biochemistry 2002;41;40;12124-32

  • New insights into fibrin (ogen) structure and function.

    Everse SJ

    Dept. of Biochemistry, University of Vermont, Burlinton, VT, USA. stephen.everse@uvm.edu

    Vox sanguinis 2002;83 Suppl 1;375-82

  • Analysis of fibrinogen gamma-chain truncations shows the C-terminus, particularly gammaIle387, is essential for assembly and secretion of this multichain protein.

    Okumura N, Terasawa F, Tanaka H, Hirota M, Ota H, Kitano K, Kiyosawa K and Lord ST

    Laboratory of Clinical Chemistry, Department of Medical Technology, School of Allied Medical Sciences, Shinshu University, the Second Department of Internal Medicine, Shinshu University School of Medicine, Shinshu University Hospital, Matsumoto, Japan.

    To examine the role of the fibrinogen gamma chain in the assembly and secretion of this multichain protein, we synthesized a series of fibrinogen variants with truncated gamma chains, terminating between residues gamma379 and the C-terminus, gamma411. The variant fibrinogens were synthesized from altered gamma-chain complementary DNAs in cultured Chinese hamster ovary cells. Immunoassays of the culture media demonstrated that only those variants with gamma chain longer than 386 residues were secreted and that the concentration of fibrinogen decreased with the length of the gamma chain, from 1.4 microg/mL for normal fibrinogen to 0.39 microg/mL for gamma 387 fibrinogen. Immunoassays of cell lysates showed that all variant gamma chains were synthesized, although the levels varied significantly. For variants longer than 386 residues, levels decreased with length but remained near normal. In contrast, expression of the 4 variants with 386 residues or less was about 20-fold reduced. Quantitative reverse transcription-polymerase chain reaction demonstrated that the gamma-chain messenger RNA level was independent from chain length. Western blot analyses showed that lysates expressing variants with 387 residues or more contained species comparable to the known intermediates in fibrinogen assembly, including half-molecules. For shorter variants, these intermediates were not evident. We conclude that residues near the C-terminus of the gamma chain are essential for fibrinogen assembly, and more specifically, that gamma387 is critical. We propose that the loss of residue gamma387 destabilized the structure of gamma chain, preventing assembly of alphagamma and betagamma dimers, essential intermediates in the assembly of normal fibrinogen.

    Funded by: NHLBI NIH HHS: HL31048

    Blood 2002;99;10;3654-60

  • Fibrinogen Hillsborough: a novel gammaGly309Asp dysfibrinogen with impaired clotting.

    Mullin JL, Brennan SO, Ganly PS and George PM

    Molecular Pathology Laboratory, Canterbury Health Laboratories, Christchurch Hospital, Christchurch, New Zealand. jennifer.mullin@chmeds.ac.nz

    We present a novel gamma-chain dysfibrinogen that was discovered in a 32-year-old asymptomatic man admitted to the hospital after a car accident. He presented with a low fibrinogen concentration, 0.5 mg/mL, and a prolonged thrombin clotting time, 58 seconds. Analysis of purified fibrinogen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a gamma-chain variant with an apparently higher molecular weight. Isoelectric focusing (IEF) demonstrated an anodal shift in the banding pattern of the chains and electrospray ionization mass spectrometry (ESIMS) showed a 27-Da increase in the average mass of the unresolved variant and normal gamma chains. DNA sequence analysis showed a heterozygous mutation of GGC (Gly)-->GAC (Asp) at codon 309 of the gamma chain gene. This Gly--> Asp substitution was consistent with the charge change shown by IEF as well as the mass change identified by ESIMS. Functional analysis revealed that thrombin-catalyzed polymerization occurred with a longer lag time, lower rate of lateral aggregation, and similar final turbidity compared to normal and that factor XIII cross-linking was normal. The polymerization results suggest that residue gamma309 is necessary for proper alignment of fibrinogen molecules, specifically in protofibril formation and D:D interactions. gammaGly309 is highly conserved and x-ray structures support the conclusion that the lack of a side chain at this position helps facilitate the close contact between abutting gammaD domains of condensing fibrin monomers during polymerization.

    Funded by: NHLBI NIH HHS: F32 HL10436-01

    Blood 2002;99;10;3597-601

  • Incorporation of vitronectin into fibrin clots. Evidence for a binding interaction between vitronectin and gamma A/gamma' fibrinogen.

    Podor TJ, Campbell S, Chindemi P, Foulon DM, Farrell DH, Walton PD, Weitz JI and Peterson CB

    Department of Pathology and Molecular Medicine, McMaster University and the Hamilton Civic Hospitals Research Centre, 711 Concession Street, Hamilton, Ontario L8V 1C3, Canada. podort@mcmaster.ca

    Vitronectin is an abundant plasma protein that regulates coagulation, fibrinolysis, complement activation, and cell adhesion. Recently, we demonstrated that plasma vitronectin inhibits fibrinolysis by mediating the interaction of type 1 plasminogen activator inhibitor with fibrin (Podor, T. J., Peterson, C. B., Lawrence, D. A., Stefansson, S., Shaughnessy, S. G., Foulon, D. M., Butcher, M., and Weitz, J. I. (2000) J. Biol. Chem. 275, 19788-19794). The current studies were undertaken to further examine the interactions between vitronectin and fibrin(ogen). Comparison of vitronectin levels in plasma with those in serum indicates that approximately 20% of plasma vitronectin is incorporated into the clot. When the time course of biotinylated-vitronectin incorporation into clots formed from (125)I-fibrinogen is monitored, vitronectin incorporation into the clot parallels that of fibrinogen in the absence or presence of activated factor XIII. Vitronectin binds specifically to fibrin matrices with an estimated K(d) of approximately 0.6 microm. Additional vitronectin subunits are assembled on fibrin-bound vitronectin multimers through self-association. Confocal microscopy of fibrin clots reveals the globular vitronectin aggregates anchored at intervals along the fibrin fibrils. This periodicity raised the possibility that vitronectin interacts with the gamma A/gamma' variant of fibrin(ogen) that represents about 10% of total fibrinogen. In support of this concept, the vitronectin which contaminates fibrinogen preparations co-purifies with the gamma A/gamma' fibrinogen fraction, and clots formed from gamma A/gamma' fibrinogen preferentially bind vitronectin. These studies reveal that vitronectin associates with fibrin during coagulation, and may thereby modulate hemostasis and inflammation.

    The Journal of biological chemistry 2002;277;9;7520-8

  • Genetic variation in coagulation and fibrinolytic proteins and their relation with acute myocardial infarction: a systematic review.

    Boekholdt SM, Bijsterveld NR, Moons AH, Levi M, Büller HR and Peters RJ

    Department of Cardiology, Academic Medical Center, Amsterdam, Netherlands.

    Background: It is pathophysiologically conceivable that genetic variations in coagulation and fibrinolytic proteins are associated with the risk of myocardial infarction. Methods and Results- We performed a literature search to identify published case-control studies correlating the factor V Leiden or prothrombin G20210A mutations or fibrinogen G-455A or plasminogen activator inhibitor-1 (PAI-1) 4G/5G polymorphisms with the risk of myocardial infarction. Studies were included only if they used solid diagnostic criteria and complied with published methodological criteria. A common OR with corresponding 95% CI was calculated for the risk of myocardial infarction in a fixed-effect model according to Mantel-Haenszel. The factor V Leiden and prothrombin G20201A mutations did not significantly correlate with myocardial infarction (OR 1.26, 95% CI 0.94 to 1.67, P=0.12 and OR 0.89, 95% CI 0.59 to 1.35, P=0.6, respectively). Inclusion of the studies that investigated young patients (<55 years) made the association significant for factor V Leiden (OR 1.29, 95% CI 1.03 to 1.61, P=0.02). Homozygosity for the fibrinogen -455A allele was significantly associated with a decreased risk of myocardial infarction (OR 0.66, 95% CI 0.44 to 0.99, P=0.04), whereas the PAI-1 4G4G genotype was significantly associated with increased risk (OR 1.20, 95% CI 1.04 to 1.39, P=0.04).

    Conclusions: Associations between these genetic variations and myocardial infarction were weak or absent. In the absence of clinical implications, our results indicate that screening of patients with myocardial infarction for these genetic variations is not warranted.

    Circulation 2001;104;25;3063-8

  • Identification of four novel polymorphisms in the Aalpha and gamma fibrinogen genes and analysis of association with plasma levels of the protein.

    Menegatti M, Asselta R, Duga S, Malcovati M, Bucciarelli P, Mannucci PM and Tenchini ML

    Angelo Bianchi Bonomi Hemophilia and Thrombosis Center and Fondazione Luigi Villa, Department of Internal Medicine, University of Milan, Milan, Italy. marzia.menegati@unimi.it

    Four novel polymorphisms were identified in the fibrinogen gene cluster. Three of them were localized in the promoter regions of the Aalpha-chain (alpha -128 C/G, alpha -58 G/A) or the gamma-chain (gamma -239 A/G) gene, while the remaining one was identified in intron 9 of the gamma-chain gene (gamma 7792 C/T). Genotype distributions for these polymorphisms were analyzed in 200 healthy Italian individuals and were in Hardy-Weinberg equilibrium. Since high levels of plasma fibrinogen have been associated with an increased risk of cardiovascular disease and genetic variations have been evaluated as thrombotic risk predictors, we analyzed their role in determining the plasma levels of this protein. Owing to the low frequency of the rare allele of alpha -128 C/G and gamma -239 A/G polymorphisms, association with plasma fibrinogen levels was investigated for only alpha -58 G/A and gamma 7792 C/T. We also investigated in the same population two previously identified polymorphisms in the fibrinogen gene cluster (alpha TaqI and beta -455 G/A) chosen for their widely studied association with plasma fibrinogen levels. In the multivariate linear regression analysis, no statistically significant association with plasma fibrinogen levels was found.

    Thrombosis research 2001;103;4;299-307

  • Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aalpha R16C and gamma G165R.

    Bolliger-Stucki B, Lord ST and Furlan M

    Central Hematology Laboratory, Inselspital, University Hospital, Bern, Switzerland. bettina_bollinger@med.unc.edu

    Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: Aalpha R16C and gamma G165R. As seen previously with other heterozygous Aalpha R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to Aalpha 16 C. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the alpha-helix content in the variant D3 fragment. It is concluded that the Aalpha-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the gamma-chain induced a conformational change in the D3 fragment.

    Funded by: NHLBI NIH HHS: R01 HL 31048

    Blood 2001;98;2;351-7

  • The amino acid sequence in fibrin responsible for high affinity thrombin binding.

    Meh DA, Siebenlist KR, Brennan SO, Holyst T and Mosesson MW

    Blood Research Institute, The Blood Center of Southeastern Wisconsin, Milwaukee 53201-2178, USA. dmeh@bcsew.edu

    Human fibrin has a low affinity thrombin binding site in its E domain and a high affinity binding site in the carboxy-terminal region of its variant gamma' chain (gamma'408-427). Comparison of the gamma' amino acid sequence (VRPEHPAETEYDSLYPEDDL) with other protein sequences known to bind to thrombin exosites such as those in GPIbalpha, the platelet thrombin receptor, thrombomodulin, and hirudin suggests no homology or consensus sequences, but Glu and Asp enrichment are common to all. Tyrosine sulfation in these sequences enhances thrombin exosite binding, but this has not been uniformly investigated. The fibrinogen gamma' chain mass determined by electrospray ionization mass spectrometry, was 50,549 Da, a value 151 Da greater than predicted from its amino acid/carbohydrate sequence. Since each sulfate group increases mass by 80 Da, this indicates that both tyrosines at 418 and 422 are sulfated. A series of overlapping gamma' peptides was prepared for evaluation of their inhibition of 125I-labeled PPACK-thrombin binding to fibrin. gamma'414-427 was as effective an inhibitor as gamma'408-427 and its binding affinity was dependent on all carboxy-terminal residues. Mono Tyr-sulfated peptides were prepared by substituting non-sulfatable Phe for Tyr at gamma'418 or 422. Sulfation at either Tyr residue increased binding competition compared with non-sulfated peptides, but was less effective than doubly sulfated peptides, which had 4 to 8-fold greater affinity. The reverse gamma' peptide or the forward sequence with repositioned Tyr residues did not compete well for thrombin binding, indicating that the positions of charged residues are important for thrombin binding affinity.

    Funded by: NHLBI NIH HHS: HL59507

    Thrombosis and haemostasis 2001;85;3;470-4

  • AlphaMbeta2 (CD11b/CD18, Mac-1) integrin activation by a unique monoclonal antibody to alphaM I domain that is divalent cation-sensitive.

    Orchekowski RP, Plescia J, Altieri DC and Bajt ML

    Cell and Molecular Biology, Pharmacia Corporation, Kalamazoo, Michigan, USA.

    The beta2 (CD18) leukocyte integrins play a key role in normal and inflammatory immune responses. In resting leukocytes, these receptors do not bind ligands. However, when leukocytes are exposed to an appropriate agonist, high-affinity ligand binding is achieved, presumably as a result of conformational changes in the integrin. In this study, we describe a novel monoclonal antibody, mAb 6C1, directed against the alphaM subunit, which directly induces adhesion of alphaMbeta2-transfected CHO cells to fibrinogen, ICAM-1, and iC3b. Induction of binding could also be accomplished by monovalent Fab fragments of mAb 6C1 at concentrations similar to that observed with intact IgG, demonstrating stimulation of adhesion was not because of receptor cross-linking at the cell surface. The binding of mAb 6C1 induces conformational changes in the receptor, as evidenced by the expression of an "activation reporter" epitope recognized by mAb 24. The binding of mAb 6C1 is modulated by divalent cations. Mn2+ promoted high levels of 6C1 binding, and Mg2+ supported low levels of binding, however Ca2+ failed to support binding. A unique distinction of mAb 6C1 is localization of its epitope to the alphaM I domain. The alphaM I domain is essential for ligand binding, can directly bind divalent cations, and participates in the regulation of alphaMbeta2 ligand-binding affinity. Thus, these studies have identified a novel alphaM I domain activation epitope of alphaMbeta2 and support the idea that the I domain modulates the activational state of the beta2 integrins.

    Journal of leukocyte biology 2000;68;5;641-9

  • Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation.

    Asselta R, Duga S, Simonic T, Malcovati M, Santagostino E, Giangrande PL, Mannucci PM and Tenchini ML

    Department of Biology and Genetics for Medical Sciences, University of Milan; Institute of Veterinary Physiology and Biochemistry, University of Milan, Italy.

    Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of plasma fibrinogen and by a bleeding tendency ranging from mild to moderately severe. Beside a deletion of the almost entire Aalpha-chain gene, only 2 missense mutations in the C-terminal domain of the Bbeta-chain have been very recently described as being associated with afibrinogenemia. We studied a Pakistani patient with unmeasurable plasma levels of functional and immunoreactive fibrinogen. Sequencing of the fibrinogen genes revealed a homozygous G-->A transition at position +5 of intron 1 of the gamma-chain gene. The predicted mutant fibrinogen gamma-chain would contain the signal peptide, followed by a short stretch of aberrant amino acids, preceding a premature stop codon. To demonstrate the causal role of the identified mutation, we prepared expression vectors containing a region of the fibrinogen gamma-chain gene spanning from exon 1 to intron 4 and carrying either a G or an A at position +5 of intron 1. Transient transfection of the mutated plasmid in HeLa cells, followed by RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, allowed us to demonstrate the production of an erroneously spliced messenger RNA (mRNA), retaining intron 1, as shown by direct sequencing. A normal splicing occurred in HeLa cells transfected with the wild-type plasmid. This is the first report of a mutation in the fibrinogen gamma-chain gene causing afibrinogenemia and indicates that, in addition to the Aalpha and Bbeta-chain genes, the gamma-chain gene must also be considered in mutation screening for afibrinogenemia.

    Blood 2000;96;7;2496-500

  • Interactions of human fibrinogens with factor XIII: roles of calcium and the gamma' peptide.

    Moaddel M, Farrell DH, Daugherty MA and Fried MG

    Department of Biochemistry and Molecular Biology, Penn State University College of Medicine, 500 University Drive, Hershey, Pennsylvania 17033, USA.

    Plasma factor XIII is the zymogen of the transglutaminase factor XIIIa. This enzyme catalyzes the formation of isopeptide cross-links between fibrin molecules in nascent blood clots that greatly increase the mechanical stability of clots and their resistance to thrombolytic enzymes. We have characterized the solution interactions of factor XIII with two variants of fibrinogen, the soluble precursor of fibrin. Both the predominant fibrinogen gamma(A)/gamma(A) and the major variant gamma(A)/gamma' form complexes with a 2 fibrinogen:1 factor XIII ratio. The absence of detectable concentrations of 1:1 complexes in equilibrium mixtures containing free factor XIII and 2:1 complexes suggests that this interaction is cooperative. Factor XIII binds fibrinogen gamma(A)/gamma' approximately 20-fold more tightly than fibrinogen gamma(A)/gamma(A), and the interaction with fibrinogen gamma(A)/gamma' (but not fibrinogen gamma(A)/gamma(A)) is accompanied by a significant release of Ca(2+). Taken together, these results suggest that the strikingly anionic gamma' C-terminal sequence contains features that are important for factor XIII binding. Consistent with this notion, a synthetic 20-residue polypeptide containing the gamma' sequence was found to associate with factor XIII in a 2:1 molar ratio and act as an efficient competitor for fibrinogen gamma(A)/gamma' binding.

    Funded by: NHLBI NIH HHS: R29HL53997

    Biochemistry 2000;39;22;6698-705

  • Identification of amino acid sequences in fibrinogen gamma -chain and tenascin C C-terminal domains critical for binding to integrin alpha vbeta 3.

    Yokoyama K, Erickson HP, Ikeda Y and Takada Y

    Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.

    Integrin alpha(v)beta(3) recognizes fibrinogen gamma and alpha(E) chain C-terminal domains (gammaC and alpha(E)C) but does not require the gammaC dodecapeptide sequence HHLGGAKQAGDV(400-411) for binding to gammaC. We have localized the alpha(v)beta(3) binding sites in gammaC using gammaC-derived synthetic peptides. We found that two peptides GWTVFQKRLDGSV(190-202) and GVYYQGGTYSKAS(346-358) block the alpha(v)beta(3) binding to gammaC or alpha(E)C, block the alpha(v)beta(3)-mediated clot retraction, and induce the ligand-induced binding site 2 (LIBS2) epitope in alpha(v)beta(3). Neither peptide affects fibrinogen binding to alpha(IIb)beta(3). Scrambled or inverted peptides were not effective. These results suggest that the two gammaC-derived peptides directly interact with alpha(v)beta(3) and specifically block alpha(v)beta(3)-gammaC or alpha(E)C interaction. The two sequences are located next to each other in the gammaC crystal structure, although they are separate in the primary structure. Asp-199, Ser-201, Gln-350, Thr-353, Lys-356, Ala-357, and Ser-358 residues are exposed to the surface. This suggests that the two sequences are part of alpha(v)beta(3) binding sites in fibrinogen gammaC domain. We also found that tenascin C C-terminal fibrinogen-like domain specifically binds to alpha(v)beta(3). Notably, a peptide WYRNCHRVNLMGRYGDNNHSQGVNWFHWKG from this domain that includes the sequence corresponding to gammaC GVYYQGGTYSKAS(346-358) specifically binds to alpha(v)beta(3), suggesting that fibrinogen and tenascin C C-terminal domains interact with alpha(v)beta(3) in a similar manner.

    Funded by: NIGMS NIH HHS: GM47157, GM49899

    The Journal of biological chemistry 2000;275;22;16891-8

  • Signaling through platelet integrin alpha IIb beta 3: inside-out, outside-in, and sideways.

    Shattil SJ

    Department of Vascular Biology, Scripps Research Institute, La Jolla, CA 92037, USA. shattil@scripps.edu

    Thrombosis and haemostasis 1999;82;2;318-25

  • Characterization of single-nucleotide polymorphisms in coding regions of human genes.

    Cargill M, Altshuler D, Ireland J, Sklar P, Ardlie K, Patil N, Shaw N, Lane CR, Lim EP, Kalyanaraman N, Nemesh J, Ziaugra L, Friedland L, Rolfe A, Warrington J, Lipshutz R, Daley GQ and Lander ES

    Whitehead Institute/MIT Center for Genome Research, Cambridge, Massachusetts 02139, USA. lander@genome.wi.mit.edu

    A major goal in human genetics is to understand the role of common genetic variants in susceptibility to common diseases. This will require characterizing the nature of gene variation in human populations, assembling an extensive catalogue of single-nucleotide polymorphisms (SNPs) in candidate genes and performing association studies for particular diseases. At present, our knowledge of human gene variation remains rudimentary. Here we describe a systematic survey of SNPs in the coding regions of human genes. We identified SNPs in 106 genes relevant to cardiovascular disease, endocrinology and neuropsychiatry by screening an average of 114 independent alleles using 2 independent screening methods. To ensure high accuracy, all reported SNPs were confirmed by DNA sequencing. We identified 560 SNPs, including 392 coding-region SNPs (cSNPs) divided roughly equally between those causing synonymous and non-synonymous changes. We observed different rates of polymorphism among classes of sites within genes (non-coding, degenerate and non-degenerate) as well as between genes. The cSNPs most likely to influence disease, those that alter the amino acid sequence of the encoded protein, are found at a lower rate and with lower allele frequencies than silent substitutions. This likely reflects selection acting against deleterious alleles during human evolution. The lower allele frequency of missense cSNPs has implications for the compilation of a comprehensive catalogue, as well as for the subsequent application to disease association.

    Nature genetics 1999;22;3;231-8

  • Fibrinogen.

    Herrick S, Blanc-Brude O, Gray A and Laurent G

    PfizerCentral Research, Sandwich, Kent, UK. s.herrick@ucl.ac.uk

    Fibrinogen is a blood-borne glycoprotein comprised of three pairs of nonidentical polypeptide chains. Following vascular injury, fibrinogen is cleaved by thrombin to form fibrin which is the most abundant component of blood clots. As well as controlling blood loss at sites of tissue damage, other properties of fibrinogen have recently been discovered. For example, various cleavage products of fibrinogen and fibrin, released during coagulation and fibrinolysis, respectively, regulate cell adhesion and spreading, display vasoconstrictor and chemotactic activities, and are mitogens for several cell types including fibroblasts, endothelial and smooth muscle cells. Current research aims to define the bioactive fibrinogen molecule moieties and cellular receptors involved in these processes. Future studies may provide us with new opportunities to develop agents which are useful in promoting tissue repair or conversely in inhibiting fibrosis in inflammatory and fibroproliferative diseases where endothelial cell damage or chronic leakage of blood proteins is a feature.

    The international journal of biochemistry & cell biology 1999;31;7;741-6

  • Conformational changes in fragments D and double-D from human fibrin(ogen) upon binding the peptide ligand Gly-His-Arg-Pro-amide.

    Everse SJ, Spraggon G, Veerapandian L and Doolittle RF

    Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0634, USA.

    The structure of fragment double-D from human fibrin has been solved in the presence and absence of the peptide ligands that simulate the two knobs exposed by the removal of fibrinopeptides A and B, respectively. All told, six crystal structures have been determined, three of which are reported here for the first time: namely, fragments D and double-D with the peptide GHRPam alone and double-D in the absence of any peptide ligand. Comparison of the structures has revealed a series of conformational changes that are brought about by the various knob-hole interactions. Of greatest interest is a moveable "flap" of two negatively charged amino acids (Glubeta397 and Aspbeta398) whose side chains are pinned back to the coiled coil with a calcium atom bridge until GHRPam occupies the beta-chain pocket. Additionally, in the absence of the peptide ligand GPRPam, GHRPam binds to the gamma-chain pocket, a new calcium-binding site being formed concomitantly.

    Funded by: NHLBI NIH HHS: HL-26873

    Biochemistry 1999;38;10;2941-6

  • Identification of a novel recognition sequence for integrin alphaM beta2 within the gamma-chain of fibrinogen.

    Ugarova TP, Solovjov DA, Zhang L, Loukinov DI, Yee VC, Medved LV and Plow EF

    Joseph J. Jacobs Center for Thrombosis and Vascular Biology, Department of Molecular Cardiology, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.

    The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.

    Funded by: NHLBI NIH HHS: HL-54924, HL-56051, R01 HL056051

    The Journal of biological chemistry 1998;273;35;22519-27

  • Crystal structure of fragment double-D from human fibrin with two different bound ligands.

    Everse SJ, Spraggon G, Veerapandian L, Riley M and Doolittle RF

    Center for Molecular Genetics, University of California, San Diego, La Jolla, California 92093-0634, USA.

    Factor XIII-cross-linked fragment D (double-D) from human fibrin was crystallized in the presence of two different peptide ligands and the X-ray structure determined at 2.3 A. The peptide Gly-Pro-Arg-Pro-amide, which is an analogue of the knob exposed by the thrombin-catalyzed removal of fibrinopeptide A, was found to reside in the gamma-chain holes, and the peptide Gly-His-Arg-Pro-amide, which corresponds to the beta-chain knob, was found in the homologous beta-chain holes. The structure shows for the first time that the beta-chain knob does indeed bind to a homologous hole on the beta-chain. The gamma- and beta-chain holes are structurally very similar, and it is remarkable they are able to distinguish between these two peptides that differ by a single amino acid. Additionally, we have found that the beta-chain domain, like its gamma-chain counterpart, binds calcium.

    Funded by: NHLBI NIH HHS: HL-26873

    Biochemistry 1998;37;24;8637-42

  • Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin.

    Spraggon G, Everse SJ and Doolittle RF

    Center for Molecular Genetics, University of California, San Diego, La Jolla 92093-0634, USA.

    In blood coagulation, units of the protein fibrinogen pack together to form a fibrin clot, but a crystal structure for fibrinogen is needed to understand how this is achieved. The structure of a core fragment (fragment D) from human fibrinogen has now been determined to 2.9 A resolution. The 86K three-chained structure consists of a coiled-coil region and two homologous globular entitles oriented at approximately 130 degrees to each other. Additionally, the covalently bound dimer of fragment D, known as 'double-D', was isolated from human fibrin, crystallized in the presence of a Gly-Pro-Arg-Pro-amide peptide ligand, which simulates the donor polymerization site, and its structure solved by molecular replacement with the model of fragment D.

    Nature 1997;389;6650;455-62

  • The primary fibrin polymerization pocket: three-dimensional structure of a 30-kDa C-terminal gamma chain fragment complexed with the peptide Gly-Pro-Arg-Pro.

    Pratt KP, Côté HC, Chung DW, Stenkamp RE and Davie EW

    Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. kpratt@u.washington.edu

    After vascular injury, a cascade of serine protease activations leads to the conversion of the soluble fibrinogen molecule into fibrin. The fibrin monomers then polymerize spontaneously and noncovalently to form a fibrin gel. The primary interaction of this polymerization reaction is between the newly exposed N-terminal Gly-Pro-Arg sequence of the alpha chain of one fibrin molecule and the C-terminal region of a gamma chain of an adjacent fibrin(ogen) molecule. In this report, the polymerization pocket has been identified by determining the crystal structure of a 30-kDa C-terminal fragment of the fibrin(ogen) gamma chain complexed with the peptide Gly-Pro-Arg-Pro. This peptide mimics the N terminus of the alpha chain of fibrin. The conformational change in the protein upon binding the peptide is subtle, with electrostatic interactions primarily mediating the association. This is consistent with biophysical experiments carried out over the last 50 years on this fundamental polymerization reaction.

    Funded by: NHLBI NIH HHS: HL16919, HL50355, R01 HL016919

    Proceedings of the National Academy of Sciences of the United States of America 1997;94;14;7176-81

  • Induction of fibrinogen biosynthesis and secretion from cultured pulmonary epithelial cells.

    Haidaris PJ

    Department of Medicine/Hematology Unit, University of Rochester School of Medicine and Dentistry, NY 14642, USA.

    Although the liver is the primary site of fibrinogen (FBG) synthesis, epithelial cells from diverse tissues have been shown to express one or more of the FBG A alpha, B beta, and gamma chain genes. In contrast, marrow megakaryocytes, which store FBG in the alpha-granules, are thought not to express the FBG genes. Our earlier studies have shown that epithelial cells in a variety of extrahepatic tissues express the gamma chain gene ubiquitously, while the mRNAs for the A alpha and B beta chain genes are essentially undetectable. During systemic inflammation, the liver secretes increased levels of FBG into the circulation, and lung epithelium responds to local inflammation during pulmonary infection by increased transcription of the gamma-FBG gene. Therefore, to determine whether extrahepatic epithelial cells express the A alpha, B beta, and gamma chain genes in response to proinflammatory mediators, cultured lung epithelial cells were treated with interleukin-6 (IL-6) and dexamethasone (DEX). Northern blot analysis demonstrated that the levels of gamma-FBG mRNA in cultured lung (A549) and liver (HepG2) epithelial cells increased 2- to 10-fold in response to treatment. Reverse-transcriptase-polymerase chain reaction amplification demonstrated increased accumulation of steady state levels of FBG A alpha, B beta, and gamma chain mRNAs in lung epithelial cells after treatment. The basal level of lung cell gamma-FBG gene transcription was not accompanied by appreciable levels of A alpha and B beta chain gene transcription; however, nuclear run-on analysis suggested that the increase in lung cell FBG mRNAs in response to DEX +/- IL-6 was due to new transcription. Furthermore, stimulation of lung epithelial cells with IL-6 + DEX resulted in maximal secretion of intact FBG (340 kD) composed of the characteristic A alpha, B beta, and gamma chain polypeptides. The data suggest that basal expression of the gamma-FBG gene in extrahepatic tissue occurs ubiquitously in the absence of detectable levels of A alpha- and B beta-FBG gene expression, which are then upregulated on induction of an inflammatory response. This would result in local synthesis and secretion of FBG in the injured tissue, supporting the hypothesis that production of FBG by extrahepatic epithelial cells in response to inflammation plays a role in wound repair.

    Funded by: NHLBI NIH HHS: HL 30616, HL 50615

    Blood 1997;89;3;873-82

  • Crystal structure of a 30 kDa C-terminal fragment from the gamma chain of human fibrinogen.

    Yee VC, Pratt KP, Côté HC, Trong IL, Chung DW, Davie EW, Stenkamp RE and Teller DC

    Department of Biochemistry, Biomolecular Structure Center, University of Washington, Seattle, WA 98195, USA.

    Background: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, (alphabetagamma)2, held together by 29 disulfide bonds. The globular C terminus of the gamma chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation.

    Results: The X-ray crystallographic structure of the 30 kDa globular C terminus of the gamma chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible.

    Conclusions: The polymerization domain in the gamma chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.

    Funded by: NHLBI NIH HHS: HL16919, HL50355

    Structure (London, England : 1993) 1997;5;1;125-38

  • Hierarchies in the binding of human factor XIII, factor XIIIa, and endothelial cell transglutaminase to human plasma fibrinogen, fibrin, and fibronectin.

    Achyuthan KE, Rowland TC, Birckbichler PJ, Lee KN, Bishop PD and Achyuthan AM

    ZymeTx, Inc., Oklahoma Medical Research Foundation, Oklahoma City 73104, USA.

    The affinities of Factor XIII (FXIII), Factor XIIIa (FXIIIa), and cellular transglutaminase (Tg) for fibrinogen (Fgn), fibrin (Fbn), and fibronectin (Fn) were compared using a solid-phase binding assay. Initial rates of binding were as follows: FXIII bound Fbn 3-fold more than Fgn. FXIII did not bind Fn till 20 min. Increasing the ligands concentrations and binding time, resulted in weak binding of FXIII to Fn. FXIIIa bound Fbn 2-fold more than Fgn and 28-fold more than Fn. Tg bound Fn approximately 130-fold more than either Fgn or Fbn. At equilibrium, the extent of binding was determined to be as follows: FXIII bound Fbn 3-15-fold more than Fgn and 8-fold more than Fn. FXIIIa bound Fgn and Fbn equally and 12-25-fold more than Fn. FXIIIa bound Fgn or Fbn 2-fold and 25-fold greater than FXIII-Fbn and FXIII-Fgn interactions, respectively. Tg bound about equally to Fgn and Fbn and 10-20-fold less than Fn. The Kds' for FXIIIa binding to Fn, Fgn, and Fbn were 100, 23, and 19 nM, respectively. The Kd for Tg binding to Fn was 6.5 nM. The binding hierarchies are: [Tg-Fn] > [FXIIIa-Fgn] = [FXIIIa-Fbn] > [FXIII-Fbn] > [FXIII-Fgn] = [FXIIIa-Fn] > [Tg-Fbn] = [Tg-Fgn] > [FXIII-Fn]. Such hierarchies could regulate the cross-linkings by FXIIIa and Tg during hemostasis, wound healing, and cell adhesion.

    Molecular and cellular biochemistry 1996;162;1;43-9

  • Plasma factor XIII binds specifically to fibrinogen molecules containing gamma chains.

    Siebenlist KR, Meh DA and Mosesson MW

    Department of Basic Health Sciences, School of Dentistry, Marquette University, Milwaukee, Wisconsin 53233, USA.

    The difference between peak 1 and peak 2 fibrinogen lies in their gamma chains. Peak 1 molecules contain 2 gamma A chains; peak 2 molecules contain 1 gamma A and 1 gamma chain, the latter of which contains a 20 amino acid extension (gamma 408-427) replacing the carboxyl-terminal 4 amino acids of the gamma A chain (gamma A 408-411). While the existence of gamma chains in plasma fibrinogen molecules has been known for many years, their function remains unknown. When fibrinogen is purified from plasma, the factor XIII zymogen (A2B2) copurifies with it and is found only in the peak 2 fibrinogen when this fraction is separated from peak 1 fibrinogen by ion-exchange chromatography on DEAE-cellulose. Factor XIII alone applied to the same DEAE column elutes at a position between peak 1 and peak 2. When mixtures of peak 1 fibrinogen plus factor XIII or peak 2 fibrinogen plus factor XIII are applied to DEAE columns, the peak 1/factor XIII mixture elutes in two peaks, whereas the peak 2/factor XIII mixture elutes in the peak 2 fibrinogen position. Gel sieving on Superose 6 of peak 1/factor XIII mixtures results in two protein peaks, the first of which contains the fibrinogen. Most factor XIII activity elutes in the second peak with a small amount of activity emerging with the trailing end of the fibrinogen peak. Gel sieving of mixtures of peak 2 and factor XIII results in a single protein peak with all factor XIII activity emerging with the leading edge of the fibrinogen peak. The interaction between peak 2 fibrinogen and plasma factor XIII appears to be through binding to the B subunit of factor XIII since placental or platelet factor XIII (A2), which does not contain B subunits, elutes independently from peak 2 fibrinogen on DEAE-cellulose chromatography. The results indicate that peak 2 fibrinogen gamma chains have a physiologically significant affinity for the B subunits of plasma factor XIII and that through this interaction fibrinogen serves as a carrier for the plasma zymogen in circulating blood.

    Funded by: NHLBI NIH HHS: HL-47000

    Biochemistry 1996;35;32;10448-53

  • Cloning and characterization of a lung-specific cDNA corresponding to the gamma chain of hepatic fibrinogen.

    Simpson-Haidaris PJ, Wright TW, Earnest BJ, Hui Z, Neroni LA and Courtney MA

    Department of Medicine, Microbiology and Pathology, University of Rochester School of Medicine and Dentistry, NY 14642, USA.

    The gene (gamma FBG) encoding the fibrinogen gamma chain (gamma FBG) has been shown to exhibit both tissue-specific and ubiquitous expression. To confirm the identity of the gamma FBG transcripts expressed in extrahepatic tissue, lung tissue was chosen as a model of extrahepatic gamma FBG gene expression. A ferret lung cDNA clone bank was constructed in lambda gt11 and several positive plaques were isolated using cross-species hybridization with the rat gamma FBG cDNA. Sequence data of the longest clone, designated pFLG gamma 3, was compared at the nucleotide and deduced amino acid (aa) levels with sequences of gamma FBG from other species. The results indicated that the identity of the ferret lung-specific gamma FBG cDNA to pig, rat, bovine and human gamma FBG cDNAs ranged from 78-88%; the similarity of the ferret lung-specific gamma FBG deduced aa sequence ranged from 84-88% across species. Cysteine aa involved in intra- and inter-chain disulfide-bonded secondary and tertiary structure are absolutely conserved in ferret gamma FBG. The putative cell-cell adhesion sites for both platelet alpha IIb beta 3 and intercellular adhesion molecule-1 receptor binding to ferret gamma FBG are > 90% similar to the corresponding sites in the human gamma FBG. The results of Northern hybridization indicated that the ferret lung gamma FBG mRNA was equivalent in size to the liver gamma FBG mRNA; Southern hybridization suggested that ferret gamma FBG is a single-copy gene, as is the gamma FBG of other species. Lung-specific gamma FBG expression was localized to epithelial cells of the large and small airways and chondrocytes by in situ RNA:RNA hybridization. The functional significance of gamma FBG expression in lung is not presently known. Since expression of FBG is up-regulated 2-10-fold in the liver during an inflammatory event, it is possible that lung-specific gamma FBG expression occurs predominantly during lung disease or injury.

    Funded by: NHLBI NIH HHS: HL-07152, HL-49610, HL-50615; ...

    Gene 1995;167;1-2;273-8

  • Regulation of leukocyte-endothelium interaction and leukocyte transendothelial migration by intercellular adhesion molecule 1-fibrinogen recognition.

    Languino LR, Duperray A, Joganic KJ, Fornaro M, Thornton GB and Altieri DC

    Department of Vascular Biology, Scripps Research Institute, La Jolla, CA 92037.

    Although primarily recognized for its role in hemostasis, fibrinogen is also required for competent inflammatory reactions in vivo. It is now shown that fibrinogen promotes adhesion to and migration across an endothelial monolayer of terminally differentiated myelomonocytic cells. This process does not require chemotactic/haptotactic gradients or cytokine stimulation of the endothelium and is specific for the association of fibrinogen with intercellular adhesion molecule 1 (ICAM-1) on endothelium. Among other adhesive plasma proteins, fibronectin fails to increase the binding of leukocytes to endothelium, or transendothelial migration, whereas vitronectin promotes the binding but not the migration. The fibrinogen-mediated leukocyte adhesion and transendothelial migration could be inhibited by a peptide from the fibrinogen gamma-chain sequence N117NQKIVNL-KEKVAQLEA133, which blocks the binding of fibrinogen to ICAM-1. This interaction could also be inhibited by new anti-ICAM-1 monoclonal antibodies that did not affect the ICAM-1-CD11a/CD18 recognition, thus suggesting that the fibrinogen binding site on ICAM-1 may be structurally distinct from regions previously implicated in leukocyte-endothelium interaction. Therefore, binding of fibrinogen to vascular cell receptors is sufficient to initiate (i) increased leukocyte adhesion to endothelium and (ii) leukocyte transendothelial migration. These two processes are the earliest events of immune inflammatory responses and may also contribute to atherosclerosis.

    Funded by: NHLBI NIH HHS: HL-54131, R01 HL-43773

    Proceedings of the National Academy of Sciences of the United States of America 1995;92;5;1505-9

  • Structural recognition of a novel fibrinogen gamma chain sequence (117-133) by intercellular adhesion molecule-1 mediates leukocyte-endothelium interaction.

    Altieri DC, Duperray A, Plescia J, Thornton GB and Languino LR

    Boyer Center for Molecular Medicine, Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06536.

    In addition to its role in hemostasis, fibrinogen is obligatorily required to mount competent inflammatory responses in vivo. A molecular prerequisite of fibrinogen-dependent inflammation may reside in its ability to associate with intercellular adhesion molecule-1 (ICAM-1), and enhance monocyte adhesion to endothelium by bridging the two cell types. Structure-function characterization of the novel ICAM-1 recognition of fibrinogen was carried out by synthetic peptidyl mimicry of the fibrinogen gamma chain. A novel peptide sequence, N117NQ-KIVNLKEKVAQLEA133, designated gamma 3, dose-dependently inhibited (IC50 approximately 20-40 micrograms/ml) binding of 125I-fibrinogen to endothelial cells or ICAM-1-expressing B lymphoblastoid Daudi cells. In contrast, none of the previously identified vascular cell fibrinogen interacting sequences was effective. Increasing concentrations of gamma 3 completely inhibited fibrinogen-mediated adhesion of peripheral blood mononuclear cells or vitamin D3-differentiated monocytic HL-60 cells to endothelium, but did not affect leukocyte-endothelium interaction in the absence of fibrinogen. 125I-Labeled gamma 3 bound specifically and saturably to genetically engineered ICAM-1 transfectants, but not to control non-transfected cells, and associated with ICAM-1 on cytokine-activated endothelium with a Kd of 34 microM. Consistent with functional recognition of ICAM-1, immobilized gamma 3 supported adhesion of JY lymphoblasts in a dose-dependent reaction inhibited by monoclonal antibodies to ICAM-1. We conclude that a novel fibrinogen gamma 3 sequence N117NQKIVNLKEKVAQLEA133 binds to ICAM-1 and modulates ICAM-1-dependent adhesion. These findings define the structural basis of fibrinogen:ICAM-1 recognition and provide a potential selective target for inhibiting fibrinogen-dependent inflammatory responses.

    Funded by: NHLBI NIH HHS: HL-54131, R01 HL-43773

    The Journal of biological chemistry 1995;270;2;696-9

  • A new substitution, gamma 358 Ser-->Cys, in fibrinogen Milano VII causes defective fibrin polymerization.

    Steinmann C, Bögli C, Jungo M, Lämmle B, Heinemann G, Wermuth B, Redaelli R, Baudo F and Furlan M

    Central Hematology Laboratory, Inselspital, Bern, Switzerland.

    Fibrinogen Milano VII is a hereditary fibrinogen variant detected in a woman with no clinical symptoms of bleeding or thrombosis. Thrombin and reptilase clotting times were prolonged in six family members from three generations. Release of fibrinopeptides A and B was normal. Fibrin polymerization was strongly delayed both in the presence and in the absence of calcium. The structural defect was determined by sequence analysis of a 290-bp fragment of genomic DNA amplified by polymerase chain reaction and cloned in M13mp19. The triplet TCT coding for the amino acid residue gamma 358 was found to be replaced by TGT, resulting in the substitution gamma 358 Ser-->Cys. Immunoblot analysis demonstrated the presence of covalently linked fibrinogen albumin and fibrinogen (albumin)2 complexes. Albumin was released from fibrinogen Milano VII by limited reduction with 2-mercaptoethanol. Fibrin polymerization was not normalized after removal of albumin from fibrinogen Milano VII, suggesting that the delayed clot formation is not due to steric hindrance caused by bound albumin but by substitution of gamma 358 Ser by Cys itself. Our results indicate that the residue gamma 358 Ser is essential for normal expression of the carboxy terminal polymerization site on the fibrinogen gamma-chain.

    Blood 1994;84;6;1874-80

  • Fibrinogen Milano V: a congenital dysfibrinogenaemia with a gamma 275 Arg-->Cys substitution.

    Steinmann C, Bögli C, Jungo M, Lämmle B, Heinemann G, Wermuth B, Redaelli R, Baudo F and Furlan M

    Central Haematology Laboratory, Inselspital, University Hospital of Bern, Switzerland.

    An abnormal fibrinogen was discovered in a clinically asymptomatic woman from Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times and a discrepancy between the plasma fibrinogen levels determined by the clotting assay and electroimmunoassay. Release of fibrinopeptides A and B from fibrinogen Milano V by thrombin was normal. Fibrin polymerization was strongly delayed in the presence of EDTA and was partially corrected at physiological calcium concentration. Normal migration of mercaptolysed polypeptide chains was observed in polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Moreover, there was no apparent abnormality in the charge of the reduced chains of the variant fibrinogen, as judged by two-dimensional gel electrophoresis. A fragment of the gamma-chain gene coding for the amino acids 259-350 was amplified and cloned. The amino acid gamma 275 arginine was found to be substituted by cysteine. Immunoblotting analysis with a rabbit antiserum against human serum albumin indicated that albumin was not linked to the odd sulphydryl group of fibrinogen Milano V. Treatment of fibrinogen Milano V with cysteamine, that is surmised to convert the mutant cysteine to a positively charged lysine analogue, did not improve the clotting properties of fibrinogen Milano V.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 1994;5;4;463-71

  • Fibrinogen Bern I: substitution gamma 337 Asn-->Lys is responsible for defective fibrin monomer polymerization.

    Steinmann C, Reber P, Jungo M, Lämmle B, Heinemann G, Wermuth B and Furlan M

    Central Hematology Laboratory, Inselspital, University of Bern, Switzerland.

    An inherited fibrinogen variant, fibrinogen Bern I, was isolated from plasma of an asymptomatic woman. Routine coagulation studies showed prolonged thrombin and reptilase clotting times. Fibrinogen concentration was diminished when determined by a functional assay, but was normal by the heat precipitation method. The release of fibrinopeptides A and B was not delayed. Two-dimensional gel electrophoresis of mercaptolyzed fragments D of fibrinogen, obtained by digestion with plasmin, showed an abnormal electrophoretic mobility in the gamma-chain remnants of fragments D1 and D2 from fibrinogen Bern I, whereas conversion of D2 to D3 by plasmin resulted in the loss of the abnormal charge, suggesting that the structural abnormality in this variant is located in the region gamma 303 through 356. The molecular defect in fibrinogen Bern I was identified by sequence analysis of genomic DNA amplified by polymerase chain reaction and cloned in M13mp19. The triplet AAC coding for asparagine at position gamma 337 was found to be substituted by AAA coding for lysine. We conclude that the substitution gamma 337 Asn-->Lys in fibrinogen Bern I is responsible for defective polymerization of fibrin monomers and for impaired protection by calcium against plasmic degradation.

    Blood 1993;82;7;2104-8

  • Extrahepatic expression of fibrinogen by granulosa cells: potential role in ovulation.

    Parrott JA, Whaley PD and Skinner MK

    Reproductive Endocrinology Center, University of California, San Francisco 94143-0556.

    Granulosa cells from ovarian follicles were shown to express and secrete fibrinogen under the control of FSH. Conditioned medium was collected from granulosa cell cultures and found to contain FSH-dependent 50-kilodalton (kDa) and 93- to 95-kDa proteins. N-Terminal microsequence analysis identified these proteins as fibrinogen beta- and gamma-chains, respectively. Proteins migrating at 93 and 95 kDa contain identical gamma-chain sequences at the N-terminal, suggesting differential processing of fibrinogen. These fibrinogen chains were specifically detected with antifibrinogen antibodies in immunoblot and immuno-precipitation analysis. Fibrinogen gamma-chain mRNA was detected in granulosa cells by polymerase chain reaction analysis, confirming fibrinogen gene expression by these cells. Fibrinogen secretion by granulosa cells was measured by a competitive enzyme-linked immunosorbent assay. Granulosa cells treated with FSH (100 ng/ml) secreted 2-3 times more fibrinogen than untreated cells. These data show that fibrinogen, a major product of the liver, is also a secretory product of granulosa cells. This provides a novel extrahepatic site of fibrinogen expression. As hepatic parenchymal cells normally maintain high circulating levels of fibrinogen, the local production of fibrinogen in the ovary is anticipated to have specialized functions. Locally produced fibrinogen may be important in the clotting process following tissue rupture at ovulation. In addition, fibrinogen fragments may be involved in the mechanism of ovulation by increasing the activity of tissue-type plasminogen activator to control the proteolytic activity required for ovulation.

    Funded by: NICHD NIH HHS: HD-20922

    Endocrinology 1993;133;4;1645-9

  • Paris I dysfibrinogenemia: a point mutation in intron 8 results in insertion of a 15 amino acid sequence in the fibrinogen gamma-chain.

    Rosenberg JB, Newman PJ, Mosesson MW, Guillin MC and Amrani DL

    Dept. of Health Sciences, University of Wisconsin-Milwaukee.

    Paris I dysfibrinogenemia results in the production of a fibrinogen molecule containing a functionally abnormal gamma-chain. We determined the basis of the molecular defect using polymerase chain reaction (PCR) to amplify the gamma-chain region of the Paris I subject's genomic DNA. Comparative sequence analysis of cloned PCR segments of normal and Paris I genomic DNA revealed only an A-->G point mutation occurring at nucleotide position 6588 within intron 8 of the Paris I gamma-chain gene. We examined six normal individuals and found only normal sequence in this region, indicating that this change is not likely to represent a normal polymorphism. This nucleotide change leads to a 45 bp fragment being inserted between exons 8 and 9 in the mature gamma Paris I chain mRNA, and encodes a 15 amino acid insert after gamma 350 [M-C-G-E-A-L-P-M-L-K-D-P-C-Y]. Alternative splicing of this region from intron 8 into the mature Paris I gamma-chain mRNA also results after translation into a substitution of S for G at position gamma 351. Biochemical studies of 14C-iodoacetamide incorporation into disulfide-reduced Paris I and normal fibrinogen corroborated the molecular biologic predictions that two additional cysteine residues exist within the gamma Paris I chain. We conclude that the insertion of this amino acid sequence leads to a conformationally-altered, and dysfunctional gamma-chain in Paris I fibrinogen.

    Funded by: NHLBI NIH HHS: HL28444, HL44612, HL47000

    Thrombosis and haemostasis 1993;69;3;217-20

  • Binding of urinary protein C inhibitor to fibrin(ogen) and its binding mechanism.

    Hayashi S and Yamada K

    Thrombosis Chemical Institute, Tokyo, Japan.

    The region and mechanism of urinary protein C inhibitor (PCI) binding to fibrin(ogen) were examined using fibrin(ogen)-Sepharose and ligand blotting. Urinary PCI bound to fibrin(ogen)-Sepharose in a heparin-dependent manner at a level about 1.6-fold higher to fibrin-Sepharose than to fibrinogen-Sepharose. Scatchard analysis of the binding between urinary PCI and fibrin(ogen)-Sepharose showed that the Kd for fibrin-Sepharose and fibrinogen-Sepharose were 4.0 nM and 5.7 nM respectively. Ligand blotting using urinary PCI and an enzyme-linked immunoassay showed that urinary PCI bound to fibrinogen, fibrinogen degradation products (X, Y, D and E) and the A alpha-, B beta- and gamma-chains of fibrinogen. Binding between urinary PCI and fibrin(ogen)-Sepharose was slightly suppressed (16%) by alpha-methylmannose and largely suppressed (42%) by EACA, which indicates that the carbohydrate chain and the lysine binding site participate in the binding. These findings suggest that urinary PCI binds to fibrin(ogen) via the A alpha-, B beta- and gamma-chains and its binding is partly mediated by carbohydrate and the lysine binding site.

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 1993;4;1;153-8

  • Assembly and secretion of fibrinogen. Degradation of individual chains.

    Roy S, Yu S, Banerjee D, Overton O, Mukhopadhyay G, Oddoux C, Grieninger G and Redman C

    Lindsley F. Kimball Research Institute, New York Blood Center, New York 10021.

    Hep G2 cells produce surplus A alpha and gamma fibrinogen chains. These excess chains, which are not secreted, exist primarily as free gamma chains and as an A alpha-gamma complex. We have determined the intracellular location and the degradative fate of these polypeptides by treatment with endoglycosidase-H and by inhibiting lysosomal enzyme activity, using NH4Cl, chloroquine, and leupeptin. Free gamma chain and the gamma component of A alpha-gamma are both cleaved by endoglycosidase-H, indicating that the gamma chains accumulate in a pre-Golgi compartment. Lysosomal enzyme inhibitors did not affect the disappearance of free gamma chains but inhibited A alpha-gamma by 50%, suggesting that A alpha-gamma is degraded in lysosomes. The degradative fate of individual chains was determined in transfected COS cells which express but do not secrete single chains. Leupeptin did not affect B beta chain degradation, had very little affect on gamma chain, but markedly inhibited A alpha chain degradation. Antibody to immunoglobulin heavy chain-binding protein (GRP 78) co-immunoprecipitated B beta but not A alpha or gamma chains. Preferential binding of heavy chain-binding protein to B beta was also noted in Hep G2 cells and in chicken hepatocytes. Taken together these studies indicate that B beta and gamma chains are degraded in the endoplasmic reticulum, but only B beta is bound to BiP. By contrast A alpha chains and the A alpha-gamma complex undergo lysosomal degradation.

    Funded by: NHLBI NIH HHS: HL37457

    The Journal of biological chemistry 1992;267;32;23151-8

  • Heterozygous abnormal fibrinogen Osaka III with the replacement of gamma arginine-275 by histidine has an apparently higher molecular weight gamma-chain variant.

    Yoshida N, Imaoka S, Hirata H, Matsuda M and Asakura S

    Institute of Hematology, Jichi Medical School, Tochigi, Japan.

    Congenitally abnormal fibrinogen Osaka III with the replacement of gamma Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of alpha- and gamma-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal gamma-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 gamma remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

    Thrombosis and haemostasis 1992;68;5;534-8

  • Gene analyses of abnormal fibrinogens with a mutation in the gamma chain.

    Mimuro J, Muramatsu S, Maekawa H, Sakata Y, Kaneko M, Yoshitake S, Okuma M, Ito Y, Takeda Y and Matsuda M

    Division of Hemostasis and Thrombosis Research, Jichi Medical School, Tochigi-Ken, Japan.

    In four abnormal fibrinogens with a point mutation in the gamma chain, all characterized by impaired fibrin polymerization, we identified single base exchanges in the respective mutant gamma chain genes by polymerase chain reaction followed by DNA sequence analysis. These base exchanges accounted for the amino acid substitutions previously reported from our laboratory. They were exchanges of C to T (CGC for gamma Arg-275 to TGC for Cys) in fibrinogen Osaka II, T to G (AAT for gamma Asn-308 to AAG for Lys) in fibrinogen Kyoto I, T to C (ATG for gamma Met-310 to ACG for Thr) in fibrinogen Asahi, and G to T (GAT for gamma Asp-330 to TAT for Tyr) in fibrinogen Kyoto III. These base exchanges were found to reside in exon VIII of the gamma chain gene. Since many abnormal molecules are associated with polymerization defects, unless associated with the impaired release of fibrinopeptides A and/or B, exon VIII of the gamma chain gene may deserve careful study to define the structural alterations.

    International journal of hematology 1992;56;2;129-34

  • Characterization of an abnormal fibrinogen Osaka V with the replacement of gamma-arginine 375 by glycine. The lack of high affinity calcium binding to D-domains and the lack of protective effect of calcium on fibrinolysis.

    Yoshida N, Hirata H, Morigami Y, Imaoka S, Matsuda M, Yamazumi K and Asakura S

    Institute of Hematology, Jichi Medical School, Tochigi, Japan.

    Prolonged thrombin time was completely corrected by the addition of millimolar concentrations of calcium in a new abnormal fibrinogen, Osaka V. Analysis of lysyl endopeptidase digests of A alpha-, B beta-, or gamma-chains by high performance liquid chromatography, and the following amino acid sequence analysis of relevant peptides revealed that about 50% of the gamma-chain has a replacement of gamma-arginine 375 by glycine. When fibrinogen was digested with plasmin in the presence of millimolar concentration of calcium, the amount of fragment D1 was about 50% of the normal control, and the rest was further cleaved to fragment D2, D3, or D62 with an apparent Mr of 62,000. Plasmic digestion of cross-linked fibrin in the presence of calcium resulted in the appearance of an abnormal fragment with an apparent Mr of 123,000 as well as fragments D2, D3, and D62, concomitant with the decrease of D dimer. The gamma-remnant of the abnormal fragment proved to be a cross-linked complex of the normal D1 gamma-remnant and residues 374-406/411 of the abnormal gamma-chain. The number of high affinity Ca(2+)-binding sites for the normal fibrinogen and fibrinogen Osaka V obtained by equilibrium dialysis was 2.88 (about 3) and 1.85, respectively, and that for the abnormal molecules was calculated as 0.9 (about 1) from their relative amounts in the samples, suggesting the lack of two Ca(2+)-binding sites in the D-domains. These data suggest that the normal structure of the COOH-terminal portion of the gamma-chain including residue 375 is required for the full expression of high affinity calcium binding to D-domains, the ability to be protected by calcium against plasmic digestion, and fibrin polymerization. During these studies, we found that the NH2-terminal amino acid of the gamma-remnant in fragments D or D dimer which were obtained after prolonged digestion with plasmin is gamma-Met89.

    The Journal of biological chemistry 1992;267;4;2753-9

  • Endogenous platelet fibrinogen surface expression on activated platelets.

    Gralnick HR, Williams S, McKeown L, Connaghan G, Shafer B, Hansmann K, Vail M and Fenton J

    Hematology Service, Clinical Center, National Institutes of Health, Bethesda, MD 20892.

    Intracellular platelet fibrinogen surface expression was studied in arabinogalactan-purified, resting, and thrombin-stimulated platelets. Platelet fibrinogen is derived from endocytosis of plasma fibrinogen by megakaryocytes. Like a variety of other adhesive proteins, it is stored in the platelet alpha-granule. Platelet fibrinogen surface expression was studied by using the antigen-binding fragments of a murine monoclonal antibody to platelet fibrinogen, F26, and an immunopurified polyclonal antifibrinogen antibody. Studies correlating platelet fibrinogen surface expression with the presence of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex showed that in the presence of ethylene glycol tetraacetic acid (EGTA) at 37 degrees C, neither the GPIIb-IIIa complex nor platelet fibrinogen was expressed on the surface of thrombin-activated platelets. Similar experiments performed in the presence of EGTA and calcium showed proportional expression of the GPIIb-IIIa complex and platelet fibrinogen. The addition of Arg-Gly-Asp-Ser-containing peptides, the pentadecapeptide of the fibrinogen gamma-chain carboxy terminus, or the monoclonal antibody 10E5, when directed against the GPIIb-IIIa complex before thrombin activation, inhibited 65% to 94% of the platelet fibrinogen expression, as determined with the polyclonal and monoclonal antigen-binding fragments. When these same inhibitory agents were added immediately after or 5 minutes after thrombin, the amount of inhibition decreased significantly. Similar studies with a washed platelet system revealed that when the inhibitors of platelet fibrinogen expression were added before thrombin stimulation, the degree of inhibition observed was only 24% to 38%. This suggests that the major portion of platelet fibrinogen expression involves the release of platelet fibrinogen and its subsequent binding to GPIIb-IIIa. This binding may occur within the open canalicular system or on the platelet surface; in either case, wherever the site of released platelet fibrinogen binding occurs, it can be markedly inhibited by the RGD-containing peptides and the gamma-chain fibrinogen peptides. Approximately 10% to 30% of platelet fibrinogen may be expressed prebound to a platelet receptor, or else it is released and binds to a platelet receptor other than the GPIIb-IIIa complex.

    Funded by: NHLBI NIH HHS: HL-13160

    The Journal of laboratory and clinical medicine 1991;118;6;604-13

  • Recombinant human fibrinogen and sulfation of the gamma' chain.

    Farrell DH, Mulvihill ER, Huang SM, Chung DW and Davie EW

    Department of Biochemistry, University of Washington, Seattle 98195.

    Human fibrinogen and the homodimeric gamma'-chain-containing variant have been expressed in BHK cells using cDNAs coding for the alpha, beta, and gamma (or gamma') chains. The fibrinogens were secreted at levels greater than 4 micrograms (mg of total cell protein)-1 day-1 and were biologically active in clotting assays. Recombinant fibrinogen containing the gamma' chain incorporated 35SO4 into its chains during biosynthesis, while no incorporation occurred in the protein containing the gamma chain. The identity of the sulfated gamma' chain was verified by its ability to form dimers during clotting. In addition, carboxypeptidase Y digestion of the recombinant fibrinogen containing the gamma' chain released 96% of the 35S label from the sulfated chain, and the radioactive material was identified as tyrosine O-sulfate. These results clarify previous findings of the sulfation of tyrosine in human fibrinogen.

    Funded by: NHLBI NIH HHS: HL 16919

    Biochemistry 1991;30;39;9414-20

  • A congenitally abnormal fibrinogen (Vlissingen) with a 6-base deletion in the gamma-chain gene, causing defective calcium binding and impaired fibrin polymerization.

    Koopman J, Haverkate F, Briët E and Lord ST

    Gaubius Institute TNO, Leiden, The Netherlands.

    A congenitally abnormal fibrinogen (Vlissingen) was isolated from the blood of a young woman suffering from massive pulmonary embolism. Fibrinogen Vlissingen showed an abnormal clotting time with both thrombin and Reptilase. The release of the fibrino-peptides A and B by thrombin was normal, but fibrin polymerization was impaired both in the presence and absence of Ca2+ ions. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed according to Laemmli the gamma-chain of fibrinogen Vlissingen showed two bands, one normal and one having an apparently lower molecular mass of about 1,500 daltons. The previously described protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain of normal fibrinogen was only partially detectable in fibrinogen Vlissingen. In addition the binding of Ca2+ ions was decreased. Fibrinogen Vlissingen bound 2.4 Ca2+ ions per fibrinogen molecule at pH 7.4, whereas normal fibrinogen bound 3.1 Ca2+ ions. At pH 5.8 fibrinogen Vlissingen bound 1.1 Ca2+ ions, whereas normal fibrinogen bound 2.0 Ca2+ ions per molecule fibrinogen in the D-domains, again indicating a structural change in the carboxyl terminus of fibrinogen. The structural defect was determined by sequence analysis of DNA amplified by use of the polymerase chain reaction. Exons VIII, IX, and X of the gamma-chain gene were amplified and the DNA sequence of the amplified fragments was determined. A 6-base deletion was found in 50% of the fragments corresponding to exon VIII, indicating that the patient was heterozygous for the mutation. This deletion codes for amino acids Asn-319 and Asp-320 in the normal fibrinogen gamma-chain. The data indicate that Asn-319 and Asp-320 are crucial for maintaining the integrity of the carboxyl-terminal polymerization sites, the protective effect of Ca2+ ions on plasmin degradation of the carboxyl terminus of the gamma-chain, and the calcium binding domain at the carboxyl terminus of fibrinogen.

    Funded by: NHLBI NIH HHS: HL-31048

    The Journal of biological chemistry 1991;266;20;13456-61

  • Polymorphism of the human gamma chain fibrinogen gene.

    Marchetti L, Zanelli T, Malcovati M and Tenchini ML

    Dipartimento di Biologia e Genetica per le Scienze mediche, Milano, Italy.

    Nucleotide sequence comparisons of the published cDNAs and genomic sequences coding for the gamma-chain of human fibrinogen revealed several differences. We isolated two independent human cDNA clones, coding for part of this protein and compared their sequences, which are identical with the published relevant data. Our sequence allowed us to solve a conflict for aminoacid 88. All the remaining differences resulting from this comparison occurred in the third position of a codon and did not change codon properties or restriction sites. The level of polymorphism of this gene is discussed, taking into account also the nucleotide differences among all the published relevant data.

    DNA sequence : the journal of DNA sequencing and mapping 1991;1;6;419-22

  • Fibrinogen Baltimore I: polymerization defect associated with a gamma 292Gly----Val (GGC----GTC) mutation.

    Bantia S, Mane SM, Bell WR and Dang CV

    Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205.

    Fibrinogen Baltimore I is one of the very first congenital abnormal fibrinogens reported over several decades ago; however, the molecular defect of this dysfibrinogen has eluded identification. In fact, several reports misidentified the functional defect of Baltimore I, which has impaired fibrin monomer polymerization. Reversed-phase high-performance liquid chromatography analysis of lysyl endopeptidase digest of the purified Baltimore I gamma-chain showed an abnormal peptide not found in the co-existing normal gamma-chain of this heterozygote. Amino acid sequencing of this peptide indicated that gamma-chain Gly292 is replaced by valine. This observation was confirmed, and the genetic defect was determined by direct nucleotide sequencing of a polymerase chain reaction product containing codon gamma 292, which is mutated: GGC----GTC. The molecular defect of Fibrinogen Baltimore I lies in a region of the gamma-chain required for fibrin polymerization, suggesting that the integrity of gamma Gly292 is critical for fibrin assembly.

    Funded by: NHLBI NIH HHS: HL07525, HL36260

    Blood 1990;76;11;2279-83

  • Tissue-specific and ubiquitous expression of fibrinogen gamma-chain mRNA.

    Haidaris PJ and Courtney MA

    University of Rochester School of Medicine and Dentistry, Department of Medicine, Rochester, NY 14642.

    Fibrinogen gamma-chains are derived from differential mRNA splicing resulting in polypeptide that differ in their carboxyterminal sequences as well as tissue distribution. We examined the expression of the rat fibrinogen gamma-chain mRNAs to determine the nature of the differential tissue expression of the gamma-fibrinogens. Here we demonstrate that the expression of the rat gamma B mRNA is tissue-specific. Northern blot analysis indicated that gamma A mRNA was expressed in liver, lung, brain and marrow, while the alternatively spliced gamma B mRNA was expressed only in liver. Three distinct full-length species of gamma B mRNA were identified in liver, indicative of multiple sites for polyadenylation that is suggestive of regulation at the level of 3' RNA processing. The restricted expression of gamma B mRNA in liver was contrasted by the ubiquitous expression of the gamma-chain promoter in brain and lung tissues that are not known for production of plasma coagulation proteins. The results of in situ hybridization showed that only gamma A mRNA was found in lung, localized to bronchiolar epithelial cells and chondrocytes but not smooth muscle or endothelial cells. Tissue-specific regulation of the gamma-chain gene results in compartmentalization of gamma B-fibrinogen to the circulation.

    Funded by: NCRR NIH HHS: S7RR05403-25; NHLBI NIH HHS: HL-07152, HL-30616

    Blood coagulation & fibrinolysis : an international journal in haemostasis and thrombosis 1990;1;4-5;433-7

  • Polymerization defect of fibrinogen Baltimore III due to a gamma Asn308----Ile mutation.

    Bantia S, Bell WR and Dang CV

    Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD.

    Fibrinogen Baltimore III, a congenital abnormal fibrinogen with impaired fibrin monomer polymerization, displays a normal gamma-chain and a gamma-variant that has an apparently lower relative molecular weight (mol wt) than normal on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Reverse phase high-performance liquid chromatography (HPLC) analysis of the lysyl endopeptidase digest of the purified gamma-chains of fibrinogen Baltimore III revealed the presence of a peptide that is not found in the digest of the normal fibrinogen gamma-chain. Amino acid sequence analysis of this peptide indicated that the gamma-chain residue 308, asparagine, is replaced by isoleucine. Concanavalin A bound both normal and variant gamma-chains of fibrinogen Baltimore III, indicating that the carbohydrate moiety is not altered and is not responsible for the increase in electrophoretic mobility of the Baltimore III gamma-chain. This study suggests that the integrity of gamma Asn308 is critical for fibrin monomer polymerization, since alteration to either a basic (fibrinogen Kyoto I, Asn----Lys) or hydrophobic (Asn----Ile) residue results in significantly delayed polymerization of fibrinogen to fibrin.

    Funded by: NHLBI NIH HHS: HL07525, HL36260

    Blood 1990;75;8;1659-63

  • Fibrinogen Kyoto III: a congenital dysfibrinogen with a gamma aspartic acid-330 to tyrosine substitution manifesting impaired fibrin monomer polymerization.

    Terukina S, Yamazumi K, Okamoto K, Yamashita H, Ito Y and Matsuda M

    Institute of Hematology, Jichi Medical School, Tochigi-Ken, Japan.

    A congenital dysfibrinogen characterized by impaired fibrin monomer polymerization was found in an asymptomatic 50-year-old woman and her two sons. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) run according to the method of Laemmli, we noticed two gamma chain species in fibrinogen and its plasmic fragments D1 and D2, consisting of a normal species and an apparently lower molecular weight (mol wt) variant in respective fractions. However, in fragment D3 only a single gamma chain remnant was observed. By chromatofocusing of the plasmic-CaCl2 digests of the abnormal fibrinogen, we separately isolated the normal and abnormal D1 species, the latter having been eluted in a slightly higher pH range. As expected, the abnormal D1 species failed to interfere with thrombin clotting of normal fibrinogen and normal fibrin monomer polymerization, whereas the normal D1 species inhibited them markedly. By analyzing the lysyl endopeptidase digests of the isolated gamma chain, we identified a replacement of aspartic acid by tyrosine at position 330 of the mutant gamma chain. The point mutation from an aspartic acid (pK for the beta-carboxyl = 3.86) to a tyrosine (pK for the aromatic hydroxyl = 10.07) may have perturbed the folding gamma chain structure in the D domain of fibrinogen specifically required for polymerization.

    Blood 1989;74;8;2681-7

  • Megakaryocyte and hepatocyte origins of human fibrinogen biosynthesis exhibit hepatocyte-specific expression of gamma chain-variant polypeptides.

    Haidaris PJ, Francis CW, Sporn LA, Arvan DS, Collichio FA and Marder VJ

    Department of Medicine, University of Rochester School of Medicine and Dentistry, NY.

    The gamma chain of human fibrinogen is heterogeneous in length at the C-terminus due to differential RNA processing of the gamma chain-gene primary transcript. We have produced two specific monoclonal antibodies (MoAbs) against the gamma-chain epitopes generated by this alternative processing event: anti-gamma 57.5(408-416) (L2B), which reacts with gamma 57.5 and gamma 55 chains, and anti-gamma 50(337-411) (H9B7), which reacts preferentially with gamma 50 chains. Using these MoAbs we have studied the expression of gamma-chain polypeptides by immunofluorescence microscopy in the tissues of fibrinogen biosynthesis and have determined that gamma 57.5 polypeptide is expressed in hepatocytes but is absent or present in significantly reduced amounts in megakaryocytes. Therefore the gamma 50 chain is found in plasma, platelet, and megakaryocyte fibrinogens, but the gamma 57.5 chain is found only in plasma fibrinogen. The C-terminal amino acid sequence of gamma 55 includes the L2B epitope 57.5(408-416). Using MoAb L2B we have determined that gamma 55, which is a post-translationally modified gamma 57.5 chain, is found only in plasma fibrinogen and is absent or present in markedly reduced amounts in platelet or megakaryocyte fibrinogen. In addition, the conformation of the L2B epitope is preserved in gamma 55, as determined by Western blot analysis. The hepatocyte-specific expression of the gamma 57.5-chain polypeptide and the post-translational modification to gamma 55 result in a compartmentalization of gamma-chain polypeptide expression. This is suggestive of different mechanisms regulating human fibrinogen gamma-chain gene expression in hepatocytes v megakaryocytes that may operate in a tissue-specific manner at the level of 3' RNA processing events.

    Funded by: NCRR NIH HHS: S7RR05403-25; NHLBI NIH HHS: HL-30616

    Blood 1989;74;2;743-50

  • A gamma methionine-310 to threonine substitution and consequent N-glycosylation at gamma asparagine-308 identified in a congenital dysfibrinogenemia associated with posttraumatic bleeding, fibrinogen Asahi.

    Yamazumi K, Shimura K, Terukina S, Takahashi N and Matsuda M

    Institute of Hematology, Jichi Medical School, Tochigi, Japan.

    In an abnormal fibrinogen with severely impaired polymerization of fibrin monomers, we identified a methionine-to-threonine substitution at position 310 of the gamma chain. Furthermore, asparagine at position 308 was found to be N-glycosylated due to a newly formed consensus sequence, asparagine(308)-glycine(309)-threonine(310). The two structural defects in the mutant gamma chain may well perturb the conformation required for fibrin monomer polymerization that is specifically assigned to the D domain of fibrinogen. This alteration also seems to affect the intermolecular gamma chain cross-linking of fibrin and fibrinogen, although the amine acceptor gamma glutamine-398 was found to function normally. These functional abnormalities may well be related to posttraumatic hemorrhage as observed in a 33-yr-old man with moderate hemorrhagic diathesis related to injuries since his early adolescence. The structure of the extra carbohydrate moiety attached to asparagine-308 was found to be identical with those derived from the normal B beta and gamma chains as evidenced by HPLC.

    The Journal of clinical investigation 1989;83;5;1590-7

  • High-resolution NMR studies of fibrinogen-like peptides in solution: interaction of thrombin with residues 1-23 of the A alpha chain of human fibrinogen.

    Ni F, Konishi Y, Frazier RB, Scheraga HA and Lord ST

    Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301.

    The interaction of the following human fibrinogen-like peptides with bovine thrombin was studied by use of one- and two-dimensional NMR techniques in aqueous solution: Ala(1)-Asp-Ser-Gly-Glu-Gly-Asp-Phe(8)-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg(16 )- Gly(17)-Pro-Arg(19)-Val(20)-Val-Glu-Arg (F10), residues 1-16 of F10 (fibrinopeptide A), residues 17-23 of F10 (F12), residues 1-20 of F10 (F13), residues 6-20 of F10 with Arg(16) replaced by a Gly residue (F14), and residues 6-19 of F10 with Arg(16) replaced by a Leu residue (F15). At pH 5.3 and 25 degrees C, the Arg(16)-Gly(17) peptide bonds of both peptides F10 and F13 were cleaved instantaneously in the presence of 0.6 mM thrombin, whereas the cleavage of the Arg(19)-Val(20) peptide bonds in peptides F12, F13, and F14 took over 1 h for completion. On the basis of observations of line broadening, fibrinopeptide A was found to bind to thrombin. While resonances from residues Ala(1)-Glu(5) were little affected, binding of fibrinopeptide A to thrombin caused significant line broadening of NH and side-chain proton resonances within residues Asp(7)-Arg(16). There is a chain reversal within residues Asp(7)-Arg(16) such that Phe(8) is brought close to the Arg(16)-Gly(17) peptide bond in the thrombin-peptide complex, as indicated by transferred NOEs between the aromatic ring protons of Phe(8) and the C alpha H protons of Gly(14) and the C gamma H protons of Val(15). A similar chain reversal was obtained in the isolated peptide F10 at a subzero temperature of -8 degrees C. The titration behavior of Asp(7) in peptide F13 does not deviate from that of the reference peptide, N-acetyl-Asp-NHMe at both 25 and -8 degrees C, indicating that no strong interaction exists between Asp(7) and Arg(16) or Arg(19). Peptides with Arg(16) replaced by Gly and Leu, respectively, i.e., F14 and F15, were also found to bind to thrombin but with a different conformation, as indicated by the absence of the long-range NOEs observed with fibrinopeptide A. Residues Asp(7)-Arg(16) constitute an essential structural element in the interaction of thrombin with fibrinogen.

    Funded by: NHLBI NIH HHS: HL-30616, HL-31048; NIGMS NIH HHS: GM-24893; ...

    Biochemistry 1989;28;7;3082-94

  • Fibrinogen Nagoya, a replacement of glutamine-329 by arginine in the gamma-chain that impairs the polymerization of fibrin monomer.

    Miyata T, Furukawa K, Iwanaga S, Takamatsu J and Saito H

    Department of Biology, Faculty of Science, Kyushu University, Fukuoka.

    Structural studies on a hereditary abnormal fibrinogen, fibrinogen Nagoya (Takamatsu, J., Ogata, K., Kamiya, T., Koie, K., Takagi, T., & Iwanaga, S. (1979) Thromb. Haemost. 42, 78), were performed to identify the abnormality responsible for the impaired polymerization of fibrin monomer. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, fibrinogen Nagoya showed the presence of an extra protein band with an apparent molecular weight of 49,500 in addition to the normal three subunit chains. Amino acid sequence analysis of a peptide isolated from a lysyl endopeptidase digest of one of the CNBr fragments derived from fibrinogen Nagoya indicated that Gln-329 in the gamma-chain had been replaced by Arg. This substitution can be explained by a single nucleotide change in the codon for Gln-329 (CAG----CGG). We conclude that Gln-329 in the gamma-chain is indispensable for the normal polymerization of fibrin monomer.

    Journal of biochemistry 1989;105;1;10-4

  • Normal plasmic cleavage of the gamma-chain variant of "fibrinogen Saga" with an Arg-275 to His substitution.

    Yamazumi K, Terukina S, Onohara S and Matsuda M

    Division of Hemostasis and Thrombosis Research, Jichi Medical School, Tochigi-Ken, Japan.

    We have identified a gamma-Arg-275 to His substitution in an abnormal fibrinogen designated as "fibrinogen Saga" characterized by impaired fibrin monomer polymerization. By chromatofocusing chromatography, we isolated normal and abnormal fragment D1 populations separately from the plasmic-calcium digests of fibrinogen derived from the propositus, a heterozygote for the abnormality. We found that both normal and abnormal fragment D1's were similarly protected from digestion by plasmin in the presence of calcium ions and further degraded to fragments D2 and D3 due to cleavage of the gamma-chain remnant when calcium ions were replaced by chelating agents. Abnormal fragment D1 failed to inhibit both thrombin-clotting of normal fibrinogen and polymerization of normal fibrin monomer, while normal D1 exhibited marked inhibitory activities. In an aberrant peptide comprising residues gamma-274-302 isolated by HPLC from the lysyl endopeptidase-digests of abnormal fragment D1, we identified a His substituting for an Arg at position 2, which corresponds to position 275 of the mutant gamma-chain.

    Thrombosis and haemostasis 1988;60;3;476-80

  • Characterization of an apparently lower molecular weight gamma-chain variant in fibrinogen Kyoto I. The replacement of gamma-asparagine 308 by lysine which causes accelerated cleavage of fragment D1 by plasmin and the generation of a new plasmin cleavage site.

    Yoshida N, Terukina S, Okuma M, Moroi M, Aoki N and Matsuda M

    Institute of Hematology, Jichi Medical School, Tochigi, Japan.

    Congenitally abnormal fibrinogen Kyoto I with impaired fibrin monomer polymerization contains a normal gamma-chain and a gamma-chain variant (gamma Kyoto I) that has an apparently lower Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the Laemmli system (Laemmli, U. K. (1970) Nature 227, 680-685) but migrates with apparently normal Mr in the Weber and Osborn system (Weber, K., and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412). Reverse-phase high performance liquid chromatographic analyses of the cyanogen bromide or lysyl endopeptidase cleavage fragments of the purified gamma-chains of fibrinogen Kyoto I showed the presence of peptides not seen from normal fibrinogen. Amino acid sequence analysis of these peptides indicated that gamma Asn308 of the gamma-chain variant is replaced by lysine. Purified fragment D1 of fibrinogen Kyoto I also contains two types of D1 gamma-remnants: normal and apparently lower Mr types. Abnormal fragment D1 is cleaved faster to fragments D2 and D3 by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) than normal fragment D1, as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by immunoblotting using anti-gamma-chain monoclonal antibody. Analysis of peptides released from fragment D1 by plasmin in the presence of EGTA demonstrated the cleavage of the gamma Lys308-Gly309 bond. Fragment D1 of fibrinogen Kyoto I has normal calcium binding properties. The data suggest that a region or conformation containing gamma Asn308 affects the polymerization of fibrin monomers and that the gamma Asn308----Lys replacement causes a conformational change in the gamma-chain which results in the accelerated cleavage of gamma Lys356-Ala357 and gamma Lys302-Phe303 bonds by plasmin and also results in the generation of a new plasmin cleavage site between Lys308 and Gly309 in the presence of EGTA. During these studies, we found that part of the gamma Lys212-Glu213 bond in fragment D1 is cleaved by plasmin in the presence of EGTA.

    The Journal of biological chemistry 1988;263;27;13848-56

  • Substitution of gamma Arg-275 by Cys in an abnormal fibrinogen, "fibrinogen Osaka II". Evidence for a unique solitary cystine structure at the mutation site.

    Terukina S, Matsuda M, Hirata H, Takeda Y, Miyata T, Takao T and Shimonishi Y

    Division of Hemostasis and Thrombosis Research, Jichi Medical School, Tochigi-Ken, Japan.

    In an abnormal fibrinogen with impaired fibrin monomer polymerization designed as fibrinogen Osaka II, we have identified substitution of Arg by Cys at position 275 of the gamma chain. This Cys is linked to a free cysteine molecule by a disulfide link as evidenced by fast atom bombardment mass spectrometry. This finding was supported by identification of a single cysteine released from isolated abnormal fragment D1 upon reduction. This unique cystine structure at the mutation site has not been reported heretofore in any abnormal protein including fibrinogen. The substitution may well perturb the structure required for fibrin monomer polymerization, specifically that assigned to the carboxyl-terminal D domain of fibrinogen. Indeed, isolated fragment D1 with the Cys substitution failed to inhibit thrombin-mediated clotting of normal fibrinogen and normal fibrin monomer polymerization, while normal fragment D1 inhibited them markedly. Our data seem to provide supporting evidence that the putative polymerization site(s) assigned to the D domain of fibrinogen may be structure-dependent, including the carboxyl-terminal segment of the gamma chain as well as a contiguous region that contains the gamma 275 residue.

    The Journal of biological chemistry 1988;263;27;13579-87

  • An apparently higher molecular weight gamma-chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of gamma arginine-275 by cysteine.

    Yoshida N, Ota K, Moroi M and Matsuda M

    Institute of Hematology, Jichi Medical School, Tochigi, Japan.

    A gamma-chain variant with an apparently higher molecular weight than the normal gamma-chain was detected in a new congenital abnormal fibrinogen with impaired polymerization of the fibrin monomer and with normal release of fibrinopeptides A and B in a 51-year-old male. Purified fibrinogen analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under the reduced condition in the system of Laemmli contained two protein bands in the gamma-chain region (molecular weight, 50,500 as compared with 50,000 for the normal), both with normal crosslinking ability. The presence of two types of gamma-chains was more clearly detected when reduced and carboxymethylated fibrinogen was analyzed by SDS-PAGE or when reduced fragment D2 was analyzed on SDS-PAGE followed by Western blotting, and identified by positive staining for anti gamma-chain monoclonal antibody. Cyanogen bromide- or lysylendopeptidase-cleavage of purified gamma-chains analyzed on reverse-phase high performance liquid chromatography showed the decrease of one peptide compared with the normal and the appearance of an abnormal peptide peak. Amino acid sequence analysis demonstrated that the gamma arginine-275 of gamma-chain variant was replaced by a cysteine. These data suggest that some regions or conformations containing gamma 275 will affect the polymerization of fibrin monomers. The propositus' two daughters had the same abnormal fibrinogen. This unique inherited abnormal fibrinogen was designated as fibrinogen Tochigi, and the gamma-chain variant as gamma Tochigi.

    Blood 1988;71;2;480-7

  • Three abnormal fibrinogen variants with the same amino acid substitution (gamma 275 Arg----His): fibrinogens Bergamo II, Essen and Perugia.

    Reber P, Furlan M, Henschen A, Kaudewitz H, Barbui T, Hilgard P, Nenci GG, Berrettini M and Beck EA

    We report on three unrelated individuals with the same uncommon type of dysfibrinogenemia, originating from Bergamo, Essen and Perugia. None of them showed bleeding symptoms while the Bergamo patient and members of her family presented with a thrombotic tendency. The presence of a defective fibrinogen was suggested by prolonged thrombin and reptilase times. Furthermore, fibrinogen concentrations of less than 0.28 g/L were determined by the functional assay whereas values of 1.5-2.4 g/L were measured by heat precipitation or electroimmunoassay. Fibrinogen was isolated by affinity chromatography on insoluble fibrin monomer. The rate of fibrinopeptide release by thrombin was normal while the fibrin polymerization reaction was strongly delayed. An abnormal peptide (gamma 265-310) was isolated by high-performance liquid chromatography after cyanogen bromide cleavage of the purified gamma-chain of fibrinogen Bergamo II and Essen. The same peptide was also isolated following cyanogen bromide treatment of the intact fibrinogen Perugia. Sequence analyses of these peptides demonstrated the same amino acid exchange in all three fibrinogens: gamma 275 arginine----histidine. The described fibrinogen variants appear to possess a molecular defect which has thus far only been observed in fibrinogen Haifa.

    Thrombosis and haemostasis 1986;56;3;401-6

  • Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization.

    Reber P, Furlan M, Rupp C, Kehl M, Henschen A, Mannucci PM and Beck EA

    An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called "fibrinogen Milano l." The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303-356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.

    Blood 1986;67;6;1751-6

  • Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence.

    Dang CV, Ebert RF and Bell WR

    Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.

    Funded by: NHLBI NIH HHS: HL 07377, HL 24898, HL 29067

    The Journal of biological chemistry 1985;260;17;9713-9

  • Nucleotide sequence of the gene for the gamma chain of human fibrinogen.

    Rixon MW, Chung DW and Davie EW

    A human genomic DNA library was screened for the gene coding for the gamma chain of fibrinogen by using a human cDNA for the gamma chain as a hybridization probe. The gene was identified in three overlapping recombinant lambda bacteriophage, and its sequence, including the immediate 5' and 3' flanking regions, was determined. The DNA sequence analysis revealed the presence of 10 exons coding for 411 amino acids present in the mature protein and a signal sequence of 26 amino acids. Two 30 base pair (bp) direct repeats of 93% identity were found 468 bp upstream from the transcription initiation site. The DNA sequence of the gene for the gamma chain of human fibrinogen showed considerable sequence homology with a partial sequence reported for the gene for the gamma chain of rat fibrinogen.

    Funded by: NHLBI NIH HHS: HL 16919, HL 28598

    Biochemistry 1985;24;8;2077-86

  • Structure of the human gamma-fibrinogen gene. Alternate mRNA splicing near the 3' end of the gene produces gamma A and gamma B forms of gamma-fibrinogen.

    Fornace AJ, Cummings DE, Comeau CM, Kant JA and Crabtree GR

    The gamma chain of human fibrinogen exists in 2 nonallelic forms, gamma A and gamma B, which differ only in their carboxyl termini. We have found that only one genomic locus exists for gamma-fibrinogen and that the gamma A and gamma B chains arise by alternate mRNA splicing near the 3' end of this gene. In contrast to the rat gamma B mRNA which is produced by alternate splicing with identical polyadenylation sites, human gamma B is produced when the eighth intervening sequence remains unspliced and a polyadenylation signal within this intron is used. The new carboxyl terminus is 16 amino acids longer than the gamma A protein, and although there is only minimal homology between the rat and human carboxyl termini they share a very high proportion of acidic amino acids.

    The Journal of biological chemistry 1984;259;20;12826-30

  • Localization of a fibrin gamma-chain polymerization site within segment Thr-374 to Glu-396 of human fibrinogen.

    Horwitz BH, Váradi A and Scheraga HA

    Fibrinogen fragment D1 was converted to fragment D3 by plasmic digestion. This conversion eliminates the ability of the fragment to interact with thrombin-exposed sites on fibrin monomer. Peptides released during this plasmic digestion were assayed for the presence of a polymerization site by affinity chromatography on fibrin monomer-Sepharose. We found that a 33-residue peptide, corresponding to gamma-chain Thr-374 to Lys-406, binds to immobilized fibrin monomer. This peptide is a shorter variant of a previously isolated 38-residue peptide (gamma-chain Thr-374 to Val-411) that contains a polymerization site [Olexa, S. A. & Budzynski, A. Z. (1981) J. Biol. Chem. 256, 3544-3549]. The peptide mixture derived from fragment D1 was digested further with Staphylococcus aureus protease V8, and a 23-residue peptide, gamma-chain Thr-374 to Glu-396, carrying a polymerization site, was isolated by affinity chromatography. This 23-residue peptide inhibits the polymerization of desA-fibrinogen. We conclude that a polymerization site complementary to the site exposed by removal of fibrinopeptide A is present in this segment. The localization of the polymerization site within the gamma-chain segment 374-396 implies that the polymerization site does not overlap with segments of the gamma-chain that are responsible for platelet aggregation and for Staphylococcus clumping (residues 400-411 and 397-411, respectively) or with the residues involved in factor XIIIa-catalyzed fibrin crosslinking (Gln-398 and Lys-406).

    Funded by: NHLBI NIH HHS: HL-30616

    Proceedings of the National Academy of Sciences of the United States of America 1984;81;19;5980-4

  • gamma and gamma' chains of human fibrinogen are produced by alternative mRNA processing.

    Chung DW and Davie EW

    cDNAs and the genomic DNA coding for the gamma and gamma' chains of human fibrinogen have been isolated and characterized by sequence analysis. The cDNAs coding for the gamma and gamma' chains share a common nucleotide sequence coding for the first 407 amino acid residues in each polypeptide chain. The predominant gamma chain contains an additional four amino acids on its carboxyl-terminal end (residues 408-411). These four amino acids, together with the 3' noncoding sequences, are encoded by the tenth exon. Removal of the ninth intervening sequence following the processing and polyadenylation reactions yields a mature mRNA coding for the predominant gamma chain. The less prevalent gamma' chain contains 20 amino acids at its carboxyl-terminal end (residues 408-417). These 20 amino acids are encoded by the immediate 5' end of the ninth intervening sequence. This results from an occasional processing and polyadenylation reaction that occurs within the region normally constituting the ninth intervening sequence. Accordingly, the gene for the gamma chain of human fibrinogen gives rise to two mRNAs that differ in sequence on their 3' ends. These mRNAs code for polypeptide chains with different carboxyl-terminal sequences. Both of these polypeptides are incorporated into the fibrinogen molecule present in plasma.

    Funded by: NHLBI NIH HHS: HL 16919, HL 28598

    Biochemistry 1984;23;18;4232-6

  • Evidence that three adhesive proteins interact with a common recognition site on activated platelets.

    Plow EF, Srouji AH, Meyer D, Marguerie G and Ginsberg MH

    Synthetic peptides corresponding to the extreme COOH terminus of the gamma chain of fibrinogen gamma 400-411, (400)HHLGGAKQAGDV(411), have been used to analyze recognition specificities of the platelet-binding sites for fibrinogen, fibronectin, and von Willebrand factor. gamma 403-411 did not inhibit 125I-fibrinogen binding to platelets. In contrast, gamma 402-411 produced dose-dependent inhibition of fibrinogen binding to ADP and thrombin-stimulated living or fixed cells and was a competitive antagonist. Inhibitory activity was not modified by addition of one (gamma 401-411) or two (gamma 400-411) histidinyl residues to the NH2 terminus, but peptides with a trifluoroacetyl group on the epsilon-amino group of lysine 406 were inactive. 125I-Fibronectin and 125I-von Willebrand factor binding to thrombin-stimulated living or fixed cells was inhibited in the same dose range by the same set of peptides which inhibited fibrinogen binding and not by gamma 403-411 or trifluoroacetate-blocked peptides. The capacity of the peptides to inhibit binding to cells with an expressed receptor, i.e. fixed cells, excludes an effect on receptor induction. Thus, these results suggest that the three adhesive glycoproteins share a common site on thrombin-activated platelets, and a 10-amino acid peptide, corresponding to gamma 402-411, defines the recognition specificity of this site.

    Funded by: NHLBI NIH HHS: HL 16411, HL 28235; NIADDK NIH HHS: AM 00720

    The Journal of biological chemistry 1984;259;9;5388-91

  • Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the gamma chain.

    Kloczewiak M, Timmons S, Lukas TJ and Hawiger J

    Binding of fibrinogen to human platelets depends on the interaction of the gamma-chain carboxy-terminal segment with specific receptors exposed by different agonists such as ADP, epinephrine, and thrombin. The functions of a series of synthetic peptides encompassing the sequence of the 15 carboxy-terminal residues of the gamma chain were investigated in this study. Both pentadecapeptide (gamma 397-411) and dodecapeptide (gamma 400-411) inhibited binding of 125I-fibrinogen to ADP-treated platelets, with the concentration causing 50% inhibition (IC50) being 28 microM. In comparison, decapeptide (gamma 402-411) was almost 4 times less active (IC50 = 106 microM), thus suggesting that the two histidine residues (gamma 400-401) are required for a full inhibitory effect. A heptapeptide (gamma 405-411) had a similar effect (IC50 = 102 microM) whereas a pentapeptide (gamma 407-411) was even less inhibitory (IC50 = 190 microM), indicating that the lack of lysine (gamma 406) further diminishes the reactivity of the platelet recognition site on the gamma chain of human fibrinogen. The heptapeptide (gamma 400-406) containing two histidine residues and derived from the dodecapeptide by proteolytic degradation with trypsin had very low inhibitory activity. The synthetic peptides inhibited fibrinogen-supported platelet aggregation in the same order of decreasing reactivity: pentadecapeptide = dodecapeptide greater than decapeptide = heptapeptide greater than pentapeptide. Modified synthetic pentadecapeptides bearing tyrosine or cysteinyltyrosine at the amino terminal were prepared to provide a means for radiolabeling and for formation of molecules of higher valency.(ABSTRACT TRUNCATED AT 250 WORDS)

    Funded by: NHLBI NIH HHS: HL-30648, HL-30649; NIGMS NIH HHS: GM 30861

    Biochemistry 1984;23;8;1767-74

  • Fibrinogen and fibrin.

    Doolittle RF

    Funded by: NHLBI NIH HHS: HL 18,576

    Annual review of biochemistry 1984;53;195-229

  • Isolation and characterisation of cDNA clones for the A alpha- and gamma-chains of human fibrinogen.

    Imam AM, Eaton MA, Williamson R and Humphries S

    cDNA clones coding for the A alpha- and gamma-chains of human fibrinogen have been isolated from an adult liver cDNA library. Clones were identified by hybridisation with mixtures of synthetic oligonucleotides 17 bases long, predicted using amino acid sequence data for each chain. The cDNA insert sizes are 1,950bp for A alpha-fibrinogen and 950bp for gamma-fibrinogen. The clones do not show any cross-hybridisation. Each cDNA hybridises to a unique sequence in the human genome. In adult human liver, Northern blots give an estimated messenger RNA size of 2.6kb for A alpha-fibrinogen and 1.8kb for gamma-fibrinogen.

    Nucleic acids research 1983;11;21;7427-34

  • Partial mRNA sequences for human A alpha, B beta, and gamma fibrinogen chains: evolutionary and functional implications.

    Kant JA, Lord ST and Crabtree GR

    Using rat cDNA and genomic probes to screen a human liver cDNA library, we have isolated clone of 2,274, 855, and 736 base pairs (bp) coding for the A alpha, B beta and gamma chains of human fibrinogen. Sequence analysis reveals a hitherto unrecognized extension of 15 amino acids at the carboxyl terminus of the A alpha chain, the terminal residue of which is proline. This brings the known length of the human A alpha chain to 625 amino acids. The 13-amino-acid repeated region in the midportion of the A alpha chain clearly has arisen through an 8-fold duplication of a 39-bp genetic element, which itself appears to have been constructed from smaller 6-bp repeating units. Greater than 50% sequence homology between B beta- and gamma-chain coding regions confirms postulates that these genes have arisen by duplication and subsequent divergence of an ancestral gene. A comparison of human and rat gamma-chain cDNAs shows more than 88% sequence homology over the carboxyl-terminal 162 amino acids, implying strong selective pressures on these portions of the gamma-chain gene.

    Proceedings of the National Academy of Sciences of the United States of America 1983;80;13;3953-7

  • Covalent structure of fibrinogen.

    Henschen A, Lottspeich F, Kehl M and Southan C

    Annals of the New York Academy of Sciences 1983;408;28-43

  • Characterization of a complementary deoxyribonucleic acid coding for the gamma chain of human fibrinogen.

    Chung DW, Chan WY and Davie EW

    A number of cDNAs coding for the gamma chain of human fibrinogen have been isolated from a liver cDNA library by employing a synthetic nucleotide mixture as a probe. One of the positive clones was then employed to screen the entire cDNA library of 18000 recombinants, yielding 320 positive clones for the gamma chain. The largest cDNA was 1638 base pairs in length and contained 10 base pairs of poly(G) at the 5'-end followed by 71 base pairs of noncoding nucleotides. The next 78 base pairs coded for a leader sequence that was 26 amino acids in length and included a methionine start signal and a typical hydrophobic core. The following 1233 base pairs coded for 411 amino acids that are present in the mature protein followed by a stop codon of TAA, 207 base pairs of noncoding nucleotides, a poly(A) track of 15 base pairs, and 22 base pairs of poly(C). Specific regions of the cDNA of the gamma chain were then compared with the cDNAs for the alpha and beta chains of human fibrinogen.

    Funded by: NHLBI NIH HHS: HL 16919, HL 28598

    Biochemistry 1983;22;13;3250-6

  • Dimeric half-molecules of human fibrinogen are joined through disulfide bonds in an antiparallel orientation.

    Hoeprich PD and Doolittle RF

    Human fibrinogen is a dimer composed of two identical halves. Each dimeric half contains three peptide chains (alpha, beta, and gamma) linked by disulfide bonds. The two half-molecules are joined by three disulfide bonds, one between the two alpha-chains (residue alpha-28) and two between the two gamma-chains (residues gamma-8 and gamma-9). In the absence of any difinitive experimental evidence, it has been presumed that the joined halves were aligned in a parallel orientation similar to the situation found in immunoglobulins. We have now determined that the two gamma-chains--hence, the dimeric halves--are connected in an antiparallel manner. A tryptic peptide containing gamma-chain residues 6-14 was isolated as a disulfide-linked dimer from CNBr-treated fragment E. Synthetic peptides corresponding to this sequence were prepared, from which parallel and antiparallel dimers were constructed. During the syntheses, cysteine thiol groups were protected as p-methoxybenzyl and acetamidomethyl sulfides; the peptides were dimerized by selective deprotection and disulfide bond formation. First, the p-methoxybenzyl groups were removed by liquid hydrogen fluoride and the newly exposed thiols oxidized in the presence of potassium ferricyanide. Then the monocystine compound was converted to the double-cystine product by iodolytic cleavage of the acetamidomethyl group with concomitant disulfide bond formation. This selectivity was used to prepare peptide dimers which modeled both parallel and antiparallel arrangements. The antiparallel-oriented synthetic peptide was indistinguishable from the native tryptic peptide as judged by elution from reverse-phase high-performance liquid chromatography and circular dichroism spectroscopy. The parallel-oriented synthetic peptide differed from the native material by both criteria.

    Funded by: NHLBI NIH HHS: HL 18576

    Biochemistry 1983;22;9;2049-55

  • gamma and alpha chains of human fibrinogen possess sites reactive with human platelet receptors.

    Hawiger J, Timmons S, Kloczewiak M, Strong DD and Doolittle RF

    Fibrinogen, a clottable plasma protein, agglutinates both prokaryotic cells (e.g., staphylococci) and eukaryotic cell fragments (e.g., platelets) through interaction with specific receptors. To identify the region of the fibrinogen molecule responsible for its interaction with human platelets, we prepared polypeptide chain subunits (alpha, beta, and gamma) of human fibrinogen by reduction and carboxymethylation. A mixture of the chains induced aggregation (clumping) of human platelets separated from plasma proteins and treated with ADP. When individual chains of fibrinogen were tested, gamma-chain multimers caused platelet aggregation at a molar concentration comparable with that of intact human fibrinogen. The beta chain remained inactive, and the alpha chain was 1/4th to 1/5th as reactive as the gamma chain. Monospecific antibody fragments against the gamma chain inhibited binding of 125I-labeled fibrinogen to the human platelet receptor and blocked aggregation of platelets induced by ADP in the presence of fibrinogen or gamma-chain multimers. These results indicate that the gamma chain of human fibrinogen bears the main site for interaction with the platelet receptor.

    Funded by: NHLBI NIH HHS: HL-08,338, HL-25,935, HL-25-107

    Proceedings of the National Academy of Sciences of the United States of America 1982;79;6;2068-71

  • Fibrinogen gamma chain locus is on chromosome 4 in man.

    Olaisen B, Teisberg P and Gedde-Dahl T

    A molecular fibrinogen variant has been detected by two-dimensional electrophoresis of human plasma samples. Fibrinogen is a complex molecule consisting of three different polypeptide chains A alpha, B beta, and gamma. The presently described variation resides in the gamma-chain, which in the variant is slightly more basic and heavier than the common form of this chain. In a family material it has been shown that the variant is genetically determined, and the segregation pattern shows autosomal codominant inheritance. The family material has been typed in approximately 30 marker systems, and linkage studies have shown close linkage between the gamma-chain locus (FGG) and MNSs. The MNSs loci are known to be located on chromosome 4 in humans and the fibrinogen gamma-chain locus is thus on this chromosome. The MNSs/FGG distance is approximately 8 centimorgans. Supplementing data suggest that FGG is distal to MNSs on the long arm of chromosome 4.

    Human genetics 1982;61;1;24-6

  • Carboxy-terminal amino acid sequence of a human fibrinogen gamma-chain variant (gamma').

    Wolfenstein-Todel C and Mosesson MW

    A normal human fibrinogen gamma-chain variant, termed gamma', is larger than the gamma chain (51 500 vs. 49 500) due to an extended COOH-terminal sequence. The extended COOH-terminal cyanogen bromide peptide (CNBr e') was isolated by high-pressure liquid chromatography, and its amino acid sequence was determined. Comparison with the corresponding COOH-terminal gamma-chain peptide (CNBr e) showed that the last four amino acids of the gamma chain were replaced in gamma' chains by a 20-residue fragment rich in aspartic and glutamic acids, having the sequence Val-Tyr-Pro-Glu-His-Pro-Ala-Glx-Thr-Glx-Tyr-Asx-Ser-Leu-Arg-Pro-Glx-Asx-Asx-Leu . Mutant gamma chains (gamma Paris I) from a congenitally dysfunctional fibrinogen molecule (fibrinogen Paris 1) express both gamma and gamma' features, suggesting that both gamma and gamma' chains are produced from a single gene. If this suggestion is correct, the observed differences in amino acid sequence could be explained by the existence of different mRNAs for gamma and gamma' chains, respectively, which are transcribed from one gene by differential RNA splicing.

    Funded by: NHLBI NIH HHS: HL-17419

    Biochemistry 1981;20;21;6146-9

  • Localization of a fibrin polymerization site.

    Olexa SA and Budzynski AZ

    The formation of a fibrin clot is initiated after the proteolytic cleavage of fibrinogen by thrombin. The enzyme removes fibrinopeptides A and B and generates fibrin monomer which spontaneously polymerizes. Polymerization appears to occur though the interaction of complementary binding sites on the NH2-terminal and COOH-terminal (Fragment D) regions of the molecule. A peptide has been isolated from the gamma chain remnant of fibrinogen Fragment D1 which has the ability to bind to the NH2-terminal region of fibrinogen as well as to inhibit fibrin monomer polymerization. The peptide reduces the maximum rate and extent of the polymerization of thrombin or batroxobin fibrin monomer and increases the lag time. The D1 peptide does not interact with disulfide knot, fibrinogen, or Fragment D1, but it binds to thrombin-treated disulfide knot with a Kd of 1.45 X 10(-6) M at approximately two binding sites per molecule of disulfide knot. Fibrin monomer formed either by thrombin or batroxobin binds approximately two molecules of D1 peptide per molecule of fibrin monomer, indicating that the complementary site is revealed by the loss of fibrinopeptide A. The NH2-terminal sequence (Thr-Arg-Trp) and COOH-terminal sequence (Ala-Gly-Asp-Val) of the D1 peptide were determined. Therefore the gamma 373-410 region of fibrinogen contains a polymerization site which is complementary to the thrombin-activated site on the NH2-terminal region of fibrinogen.

    Funded by: NHLBI NIH HHS: HL 14217

    The Journal of biological chemistry 1981;256;7;3544-9

  • Ca2+-mediated association of glycoprotein G (thrombinsensitive protein, thrombospondin) with human platelets.

    Phillips DR, Jennings LK and Prasanna HR

    Washed human platelets suspended in buffers containing either 1.8 mM Ca2+ and 0.49 mM Mg2+ or 1 mM EDTA were treated with human alpha-thrombin to induce secretion. Glycoprotein G, a major glycoprotein in alpha-granules, was quantitatively secreted from platelets activated in the EDTA-containing buffer but remained with the platelet in the presence of Ca2+ and Mg2+. Addition of Ca2+ to the platelets that were activated in the presence of EDTA caused glycoprotein G to bind to platelets. To determine if glycoprotein G is expressed on the membrane surface of the activated platelet, platelets were rapidly labeled by a method employing lactoperoxidase-catalyzed iodination. Although glycoprotein G was barely detected on the surface of unstimulated platelets, labveling 1 min after thrombin treatment showed that glycoprotein G rapidly became one of the prominent surface proteins. These findings show that an alpha-granule protein, glycoprotein G, is one of the major glycoproteins on the membrane surface of thrombin-activated platelets and that its binding is dependent on divalent cations.

    Funded by: NHLBI NIH HHS: HL 00080, HL 15616, HL 21487

    The Journal of biological chemistry 1980;255;24;11629-32

  • Human plasma fibrinogen heterogeneity: evidence for an extended carboxyl-terminal sequence in a normal gamma chain variant (gamma').

    Wolfenstein-Todel C and Mosesson MW

    Two types of normal human plasma fibrinogen--peak 1 and peak 2--are distinquishable by DEAE-cellulose gradient elution chromatography. The elution characteristics of peak 2 fibrinogen, which amounts to about 15% of the total, are attributable to the presence of a gamma chain variant, gamma', which is more negatively charged than gamma chains and makes up about half of all such chains in that peak [Mosesson M. W., Finlayson, J. S. & Umfleet, R. A. (1972), J. Biol. Chem. 247, 5223-5227]. Analyses of reduced S-carboxymethylated fibrin that had first been incubated in the presence of Factor XIIIa plus the fluorescent amine donor dansylcadaverine (DNScad) showed that the same amount of this compound could be incorporated covalently into either type of gamma chain. Furthermore, the DNScad-labeled COOH-terminal CNBr fragment (CNBr e) derived from the S-carboxymethylated gamma chain was smaller than the DNScad-labeled fragment (CNBr e') from the gamma' chain (Mr, 3200 and 4900) by about the same amount as the difference in size between the respective parent chains (Mr, 49,400 and 51,500). DNScad-CNBr e or DNScad-cNBR e' could be further cleaved by trypsin to yield a smaller fluorescent fragment corresponding to the penultimate tryptic gamma chain peptide containing the DNScad-glutamine acceptor and lysine donor crosslinking functions. The COOH-terminal amino acids of gamma and gamma' chains were valine and leucine, respectively. The rates of Factor XIIIa-catalyzed crosslinking of peak 1 and peak 2 fibrin were the same, but peak 1 fibrin gamma chains formed only one species of crosslinked dimer (gamma gamma) whereas peak 2 fibrin gamma chains yielded three (gamma gamma, gamma gamma', gamma'gamma'). We conclude that gamma' chains are functionally normal but have an extended COOH-terminal sequence accounting for their more negative charge and larger size relative to gamma chains.

    Funded by: NHLBI NIH HHS: HL-17419

    Proceedings of the National Academy of Sciences of the United States of America 1980;77;9;5069-73

  • Studies on synthetic peptides that bind to fibrinogen and prevent fibrin polymerization. Structural requirements, number of binding sites, and species differences.

    Laudano AP and Doolittle RF

    Biochemistry 1980;19;5;1013-9

  • Disulfide bridges in nh2 -terminal part of human fibrinogen.

    Blombäck B, Hessel B and Hogg D

    Thrombosis research 1976;8;5;639-58

  • Quantitative studies on the release of platelet fibrinogen by thrombin.

    Keenan JP and Solum NO

    British journal of haematology 1972;23;4;461-6

Gene lists (2)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
© G2C 2014. The Genes to Cognition Programme received funding from The Wellcome Trust and the EU FP7 Framework Programmes:
EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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