Characterization of an immunologic polymorphism (D79H) in the heavy chain of factor V

Human blood coagulation factor V (FV)2 is as a single-chain glycoprotein that consists of a mosaic domain structure composed of three homologous A-type domains, two smaller C-type domains, and a large B-domain (A1-A2-B-A3-C1-C2) (1). FV, which has little or no intrinsic procoagulant cofactor activity, is activated by thrombin or factor Xa (FXa) through limited proteolysis (2). Proteolytic activation of FV results in the removal of the FV B-domain and the exposure of regions in the FV molecule that are important for the expression of its procoagulant activity (3, 4). Activated FV (FVa) is a heterodimer that consists of a heavy chain (A3-A2 domains) and a light chain (A1-C1-C2 domains) that are noncovalently associated in a calcium-dependent manner (5, 6). FVa is the essential nonenzymatic cofactor of the prothrombinase complex, which upon complex formation with FXa accelerates FXa-catalyzed prothrombin activation in the presence of calcium ions and a phospholipid membrane surface by several orders of magnitude (2, 7).Although FV can be activated by a number of proteases (8, 9), thrombin is the most potent physiological activator of FV. Activation of FV by thrombin is achieved through limited proteolysis at Arg 709 , Arg 1018 , and Arg 1545 , of which the cleavage after Arg 1545 is required for full expression of FVa activity (10). Thrombin-catalyzed activation of FV follows a kinetically preferred order of bond cleavage, in which cleavage at Arg 709 occurs first, followed closely by cleavage at Arg 1018 and slow cleavage at Arg 1545 , which results in formation of the FVa light chain (11,12).A characteristic feature of thrombin are two distinct electropositive surface regions, termed exosite I and exosite II, that contribute to the specificity of thrombin by mediating the recognition of its substrates, inhibitors and receptors (13,14). Although both exosites have been implicated in FV activation (15-18), their role in the recognition of the individual thrombin cleavage sites is still unclear. In this study, we have used FV cleavage site mutants in combination with specific thrombin exosite I and II inhibitors to elucidate the importance of each exosite for the individual FV activation cleavages.Furthermore, we have used FV B-domain deletion mutants to probe the structural requirements for thrombin exosite interactions located in the B-domain and compared them with the structural requirements for FV activation by RVV-V and LVV-V, which are snake venom proteases that are known to activate FV by a single cleavage at Arg 1545 (19 -21

show abstract

“…The FV construct lacking the B-domain region from residues 828 to 1499 (FV des 827-1498) was made as described (30).…”

Section: Methodsmentioning

confidence: 99%

The Role of Thrombin Exosites I and II in the Activation of Human Coagulation Factor V

Segers

Dahlbäck

Bock

et al. 2007

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The Asp79His change 36 recently reported to modulate FV levels in plasma 42 and, from expression and functional studies, to represent a neutral polymorphism 43 was detected in 5 subjects by sequencing of the exon 3. Screening for this polymorphism was extended to the whole population.…”

Section: Search Of New Determinants By Nucleotide Sequencingmentioning

confidence: 98%

Modulation of Factor V Levels in Plasma by Polymorphisms in the C2 Domain

Scanavini¹,

Girelli²,

Lunghi³

et al. 2004

ATVB

View full text Add to dashboard Cite

Objective-Functional polymorphisms contributing to coagulation factor levels are preferential markers for association studies aimed at identifying prothrombic genetic components. Methods and Results-Factor V (FV) microsatellite genotypes were found to be associated with FV levels (Pϭ0.003).Single nucleotide polymorphisms analysis and sequencing of the promoter and of coding regions identified two polymorphisms (Met2120Thr, Asp2194Gly) present in 20% of the population (nϭ1013) that are responsible for genotype-phenotype associations. The effect of the Met2120Thr polymorphism, both in plasma (mean reduction of FV level in the heterozygous condition: 25%) and in recombinant FV studies (34% reduction), was comparable to that of the Asp2194Gly change (20% and 34%, respectively). The study of 10 subjects with a rare genotype indicated that the Asp2194Gly substitution is the functional determinant of the reduced FV levels associated with the FVHR2 haplotype. Among Leiden carriers, the doubly heterozygous condition for FV2120Thr was found to be associated with a significantly increased activated protein-C resistance (APCR) (PϽ0.05), and the doubly heterozygous condition for FV2194Gly was found to be more frequent (Pϭ0.009) in symptomatic than in asymptomatic subjects. Key Words: factor V levels Ⅲ functional polymorphisms Ⅲ FVHR2 haplotype Ⅲ recombinant FV Ⅲ APC resistance P olymorphisms contributing to modulate factor levels might constitute preferential markers for association studies aimed at identifying prothrombic or protective genetic components. 1-3 Among the coagulation factors, factor V (FV) 4 plays a pivotal role at the crossroads of procoagulant and anticoagulant pathways, 5 acting as a cofactor in prothrombin activation and in activated factor VIII (FVIII) inactivation. 6 A complex relationship between plasmatic FV levels and thrombosis might be present; high FV coagulant levels have been found to be an independent risk factor for myocardial infarction, 7 whereas no significant association was found between FV antigen level and thrombotic risk. 8 Moreover, low FV levels are associated with a reduced activated protein-C (APC) cofactor activity in the inactivation of the FVIIIa, 9 -11 which could be responsible for an APC-resistant phenotype. In particular, low FV levels in carriers of FV Leiden mutation result in a pseudohomozygous state of APC resistance (APCR). 12 Although FV genetic components of APCR have been extensively investigated, [13][14][15][16][17][18] no systematic analysis of the relationship between FV gene variation 19 and FV levels 8 has been conducted. The His1299Arg polymorphism, 20 which marks a FV gene haplotype (FVHR2) predicting several amino acid substitutions 21,22 in the A2, B, A3, and C2 domains, 23,24 has been associated with reduced FV activity 20 and antigen 25 levels. Because the increased risk for venous thrombosis 26 conferred by the FVHR2 haplotype is still a matter of debate, 25,27-31 markers of this haplotype are currently investigated in several laboratories. Conclusions-Ex...

show abstract

“…Bossone et al [11] reported that it was associated with low FV levels and that it contributed to APC resistance, if FV Leiden was present on the other chromosome. Others did not observe decreased FV plasma levels [12,13] and could not find an associated risk of thrombosis [13]. Lunghi et al [14] observed a modulation of the effect of D79H on FV levels by the G/A polymorphism at position –426 in the promoter of the FV gene.…”

Section: Factor V Variations and Venous Thrombosismentioning

confidence: 99%