Functional Properties of Factor V and Factor Va Encoded by the R2-gene

Lico Hoekema; Elisabetta Castoldi; Guido Tans; Domenico Girelli; Donato Gemmati; Francesco Bernardi; Jan Rosing

doi:10.1055/s-0037-1612907

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2001; 85(01): 75-81
DOI: 10.1055/s-0037-1612907

Review Article

Schattauer GmbH

Functional Properties of Factor V and Factor Va Encoded by the R2-gene

Authors

Lico Hoekema

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
Elisabetta Castoldi

²Department of Biochemistry and Molecular Biology, Ferrara University, Ferrara, Italy
Guido Tans

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands
Domenico Girelli

³Institute of Medical Pathology, Chair of Internal Medicine, Verona University, Italy
Donato Gemmati

⁴Center for the Study of Hemostasis and Thrombosis, Ferrara University, Italy
Francesco Bernardi

²Department of Biochemistry and Molecular Biology, Ferrara University, Ferrara, Italy
Jan Rosing

¹Department of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, The Netherlands

Abstract

Summary

Carriership of the factor V (FV) gene marked by the R2-haplotype, a series of linked polymorphisms encoding several amino acid changes in FV, is associated with mild resistance to activated protein C (APC) and with an increased risk of thrombosis. We compared the functional properties of normal FV(a) and R2-FV(a) in model systems and in plasma. FV and R2-FV were equally well activated by thrombin and expressed identical cofactor activities in prothrombin activation. Rate constants of APC-catalyzed inactivation of FVa and R2-FVa were similar both with and without protein S. However, significant differences were observed between haemostatic parameters determined in plasma from homozygous carriers of the R2-gene (n = 5) and age-matched non-carriers (n = 19). Plasma from R2-carriers contained significantly lower FV levels and the ratio of the two FV isoforms (FV₁ and FV₂) was shifted in favor of FV₁. The FV₂/FV₁ ratio was 1.4 (95% CI = 1.3-1.5) in homozygous carriers of R2 and 2.8 (95% CI = 2.5-3.1) in controls (p <0.00001). In an APC resistance test which quantifies the cofactor activity of FV in APC-catalyzed FVIII(a) inactivation, homozygous R2-carriers had significantly lower (p <0.00001) APC sensitivity ratios (APCsr = 1.54, 95% CI = 1.48-1.60) than controls (APCsr = 2.17, 95% CI = 2.05-2.28). This indicates that R2-FV has reduced cofactor activity in APC-catalyzed FVIII(a) inactivation. The changes of the relative amounts of FV₁ and FV₂ in carriers of the R2-gene will result in increased thrombin formation in the presence of APC and may provide a mechanistic explanation for the increased thrombotic risk associated with the R2-haplotype.

Keywords

Factor V isoforms - R2-haplotype - APC-resistance

Full Text

References

References
1 Rosing J, Tans G. Coagulation factor V: an old star shines again. Thromb Haemost 1997; 78: 427-33.
2 Fay PH, Smudzin TM, Walker FJ. Activated protein C-catalyzed inactivation of human factor VIII and factor Villa. J Biol Chem 1991; 266: 20139-45.
3 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Deficiency of protein C in congenital thrombotic disease. J Clin Invest 1981; 68: 1370-3.
4 Seligsohn U, Berger A, Abend M, Rubin L, Attias D, Zivelin A, Rapaport SI. Homozygous protein C deficiency manifested by massive thrombosis in the newborn. N Engl J Med 1984; 310: 559-62.
5 Schwarz HP, Fischer M, Hopmeier P, Batard MA, Griffin JH. Plasma protein S deficiency in familial thrombotic disease. Blood 1984; 64: 1297-300.
6 Comp PC, Nixon RR, Cooper MR, Esmon CT. Familial protein S deficiency is associated with recurrent thrombosis. J Clin Invest 1984; 74: 2082-8.
7 Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to a previously unrecognized mechanism characterized by poor anticoagulant response to activated protein C: prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-8.
8 Zöller B, Dahlbäck B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 1994; 343: 1536-8.
9 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ, De Ronde H, Van der Velden PA, Reitsma PH. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-7.
10 Koster T, Rosendaal FR, de Ronde H, Briët E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden Thrombophilia Study. Lancet 1993; 342: 1503-6.
11 Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood 1995; 85: 1504-8.
12 Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg506Gln mutation in factor Va. Lancet 1994; 343: 1361-2.
13 Voorberg J, Roelse JC, Koopman R, Büller HR, Berends F, Ten Cate JW, Mertens K, Van Mourik JA. Association of idiopathic venous thromboembolism with single point-mutation at Arg506 of factor V. Lancet 1994; 343: 1535-6.
14 Kalafatis M, Bertina RM, Rand MD, Mann KG. Characterization of the molecular defect in factor VR506Q. J Biol Chem 1995; 270: 4053-7.
15 Nicolaes GAF, Tans G, Thomassen MCLGD, Hemker HC, Pabinger I, Varadi K, Schwarz HP, Rosing J. Peptide bond cleavages and loss of functional activity during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 21158-66.
16 Rosing J, Hoekema L, Nicolaes GAF, Thomassen MCLGD, Hemker HC, Varadi K, Schwarz HP, Tans G. Effects of protein S and factor Xa on peptide bond cleavages during inactivation of factor Va and factor VaR506Q by activated protein C. J Biol Chem 1995; 270: 27852-8.
17 Shen L, Dahlbäck B. Factor V and protein S as synergistic cofactors to activated protein C in degradation of factor VIIIa. J Biol Chem 1994; 269: 18735-8.
18 Varadi K, Rosing J, Tans G, Schwarz HP. Influence of Factor V and Factor Va on APC-Induced Cleavage of Human Factor VIII. Thromb Haemost 1995; 73: 730-1.
19 Varadi K, Rosing J, Tans G, Pabinger I, Keil B, Schwarz HP. Factor V enhances the cofactor function of protein S in the APC-mediated inactivation of factor VIII: influence of the Factor VR506Q mutation. Thromb Haemost 1996; 76: 208-14.
20 Thorelli E, Kaufman RJ, Dahlbäck B. The C-terminal region of the factor V B-domain is crucial for the anticoagulant activity of factor V. Journal of Biological Chemistry 1998; 273: 16140-5.
21 Castoldi E, Rosing J, Girelli D, Hoekema L, Lunghi B, Mingozzi F, Ferraresi P, Friso S, Corrocher R, Tans G, Bernardi F. Mutations in the R2-FV gene affect the ratio between the FV isoforms in plasma. Thromb Haemost 2000; 83: 362-5.
22 de Visser MC, Guasch JF, Kamphuisen PW, Vos HL, Rosendaal FR, Bertina RM. The HR2 haplotype of factor V: effects on factor V levels, normalized activated protein C sensitivity ratios and the risk of venous thrombosis. Thromb Haemost 2000; 83: 577-82.
23 Bernardi F, Faioni EM, Castoldi E, Lunghi B, Castaman G, Sacchi E, Mannucci PM. A Factor V Genetic Component Differing from Factor V R506Q Contributes to the Activated Protein C Resistance Phenotype. Blood 1997; 90: 1552-7.
24 Lunghi B, Iacoviello L, Gemmati D, Dilasio MG, Castoldi E, Pinotti M, Castaman G, Redaelli R, Mariani G, Marchetti G, Bernardi F. Detection of new polymorphic markers in the factor V gene: Association with factor V levels in plasma. Thromb Haemost 1996; 75: 45-8.
25 Alhenc-Gelas M, Nicaud V, Gandrille S, Van Dreden P, Amiral J, Aubry M-L, Fiessinger J-N, Emmerich J, Aiach M. The Factor V Gene A4070G Mutation and the Risk of Venous Thrombosis. Thromb Haemost 1999; 82: 193-7.
26 Faioni EM, Franchi F, Bucciarelli P, Margaglione M, De Stefano V, Castaman G, Finazzi G, Mannucci PM. Coinheritance of the HR2 haplotype in the factor V gene confers an increased risk of venous thromboembolism to carriers of factor V R506Q (Factor V Leiden). Blood 1999; 94: 3062-6.
27 Castoldi E, Rosing J, Lunghi B, Hoekema L, Girelli D, Mingozzi F, Ferraresi P, Friso S, Corrocher R, Tans G, Bernardi F. Factor V gene mutations (R2 gene) are associated with coronary artery disease in elderly people. Thromb Haemost 1999; suppl 684.
28 Luddington R, Jackson A, Pannerselvam S, Brown K, Baglin T. The factor V R2 allele: risk of venous thromboembolism, factor V levels and resistance to activated protein C. Thromb Haemost 2000; 83: 204-8.
29 Rosing J, Bakker HM, Thomassen MCLGD, Hemker HC, Tans G. Characterization of two forms of human factor Va with different cofactor activities. J Biol Chem 1993; 268: 21130-6.
30 DiScipio RG, Davie EW. Characterization of protein S, a gamma-carboxyglutamic acid containing protein from bovine and human plasma. Biochemistry 1979; 18: 899-904.
31 Pletcher CH, Nelsestuen GL. The rate-determining step of the heparin-catalyzed antithrombin/thrombin reaction is independent of thrombin. J Biol Chem 1982; 257: 5342-5.
32 Bock PE, Craig PA, Olson ST, Singh P. Isolation of human blood coagulation alpha-factor Xa by soybean trypsin inhibitor-sepharose chromatography and its active-site titration with fluorescein mono-pguanidinobenzoate. Arch Biochem Biophys 1989; 273: 375-88.
33 Tans G, Nicolaes GA, Thomassen MCLGD, Hemker HC, van Zonneveld AJ, Pannekoek H, Rosing J. Activation of human factor V by meizothrombin. J Biol Chem 1994; 269: 15969-72.
34 Rosing J, Tans G, Govers-Riemslag JWP, Zwaal RFA, Hemker HC. The role of phospholipids and factor Va in the prothrombinase complex. J Biol Chem 1980; 255: 274-83.
35 Tans G, Rosing J, Thomassen MCLGD, Heeb MJ, Zwaal RF, Griffin JH. Comparison of anticoagulant and procoagulant activities of stimulated platelets and platelet-derived microparticles. Blood 1991; 77: 2641-8.
36 de Ronde H, Bertina RM. Laboratory Diagnosis of APC-Resistance: A Critical Evaluation of the Test and the Development of Diagnostic Criteria. Thromb Haemost 1994; 72: 880-6.
37 Varadi K, Moritz B, Lang H, Bauer K, Preston E, Peake I, Rivard GE, Keil B, Schwarz HP. A chromogenic assay for activated protein C resistance. Br J Haematol 1995; 90: 884-91.
38 Hoekema L, Rosing J, Tans G. An assay to quantify the two plasma isoforms of factor V. Thromb Haemost 2000; 84: 1066-71.
39 Monkovic D, Tracy P. Activation of human factor V by factor Xa and thrombin. Biochemistry 1990; 29: 1118-28.
40 Lindhout T, Govers-Riemslag JWP, van de Waart P, Hemker HC, Rosing J. Factor Va- factor Xa interaction. Effects of phospholipid vesicles of varying composition. Biochemistry 1982; 21: 5494-502.
41 Rijn van JLML, Govers-Riemslag JWP, Zwaal RFA, Rosing J. Kinetic studies of prothrombin activation: Effect of factor Va and phospholipids on the formation of the enzyme-substrate complex. Biochemistry 1984; 23: 4557-63.
42 van de Waart P, Hemker HC, Lindhout T. Interaction of prothrombin with factor Va-phospholipid complexes. Biochemistry 1984; 23: 2838-42.
43 Gedde-Dahl TW, Jeremic M, Weisert O. Factor V (proaccelerin) concentration in 1016 blood donors. The effects of age, sex, and ABO blood groups. Scand J Clin Lab Invest 1975; 35: 25-30.
44 Brozovic M, Chakrabarti R, Stirling Y, Fenton S, North WR, Meade TW. Factor V in an industrial population. Br J Haematol 1976; 33: 543-50.
45 Ortel TL, Devore Carter D, Quinn-Allen MA, Kane WH. Deletion analysis of recombinant human factor V. Evidence for a phosphatidylserine binding site in the second C-type domain. J Biol Chem 1992; 267: 4189-98.
46 Ortel TL, Yoo L, Quinn-Allen MA, Kane WH. Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V is the structural basis of the light chain doublet. Blood 1994; 84: 387a.
47 Nicolaes GAF, Villoutreix BO, Dahlback B. Partial glycosylation of Asn(2181) in human factor V as a cause of molecular and functional heterogeneity. Modulation of glycosylation efficiency by mutagenesis of the consensus sequence for N-linked glycosylation. Biochemistry 1999; 38: 13584-91.
48 Hoekema L, Nicolaes GA, Hemker HC, Tans G, Rosing J. Human Factor Va1 and Factor Va2: Properties in the Procoagulant and Anticoagulant Pathways. Biochemistry 1997; 36: 3331-5.