An Assay to Quantify the Two Plasma Isoforms of Factor V

Summary

Blood coagulation factor V (FV) circulates in the blood in two forms, designated FV₁ and FV₂. In model systems containing purified proteins FV₁ appears to be more thrombogenic than FV₂. Recently, we reported that in plasma from carriers of the R2 haplotype, a polymorphism which encodes several amino acid changes in FV and which is associated with an increased risk of thrombosis, the FV₁/FV₂ ratio is shifted in favor of the more thrombogenic form FV₁. Here we describe in detail the assay that enables quantification of the plasma levels of FV₁ and FV₂. FV present in highly diluted plasma samples was activated with thrombin and the FVa generated was subsequently quantified in two prothrombinase-based assay systems. In the first assay, which is performed at saturating amounts of FXa and phospholipid vesicles with a high mole fraction phosphatidylserine, FVa₁ and FVa₂ express the same cofactor activity in prothrombin activation. Hence, this assay quantifies the total FV level (FV₁ + FV₂) present in plasma. In the second assay, which is performed at suboptimal amounts of FXa and phospholipid vesicles with a low mole fraction phosphatidylserine, FVa₂ has approximately an 8-fold higher cofactor activity than FVa₁. Therefore, the response in this assay depends on the relative amounts of FV₁ and FV₂ in the plasma sample. Calibration curves made with samples containing known concentrations of purified FVa₁ and FVa₂ subsequently allowed calculation of the amounts of FV₁ and FV₂ present in plasma.

Key words

Blood coagulation - Factor V - thrombosis

• References

• 1 Rosing J, Tans G. Coagulation factor V: an old star shines again. Thromb Haemost 1997; 78: 427-33.
• 2 Dahlbäck B. Procoagulant and anticoagulant properties of coagulation factor V: factor V Leiden (APC resistance) causes hypercoagulability by dual mechanisms. J Lab Clin Med 1999; 133: 415-22.
• 3 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.
• 4 Váradi 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.
• 5 Lu D, Kalafatis M, Mann KG, Long GL. Comparison of activated protein C/ protein S-mediated inactivation of human factor VIII and factor V. Blood 1996; 87: 4708-17.
• 6 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.
• 7 Kim SW, Ortel TL, Quinn-Allen MA, Yoo L, Worfolk L, Zhai X, Lentz BR, Kane WH. Partial glycosylation at asparagine-2181 of the second C-type domain of human factor V modulates assembly of the prothrombinase complex. Biochemistry 1999; 38: 11448-54.
• 8 Nicolaes GAF, Villoutreix BO, Dahlbäck B. Partial Glycosylation of Asn2181 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.
• 9 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.
• 10 Váradi 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 (02) 208-14.
• 11 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.
• 12 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.
• 13 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.
• 14 Faioni EM, Franchi F, Bucciarelli P, Margaglione M, De Stefano V, Castaman C, 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.
• 15 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.
• 16 DiScipio RG, Davie EW. Characterization of protein S, a gamma-carboxyglutamic acid containing protein from bovine and human plasma. Biochemistry 1979; 18: 899-904.
• 17 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.
• 18 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-p-guanidinobenzoate. Arch Biochem Biophys 1989; 273 (02) 375-88.
• 19 Böttcher CJF, van Gent CM, Pries C. A Rapid and Sensitive Sub-micro Phosphorus Determination. Anal Chim Acta 1961; 24: 203-7.
• 20 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.
• 21 Tracy PB, Eide LL, Bowie EJ, Mann KG. Radioimmunoassay of factor V in human plasma and platelets. Blood 1982; 60: 59-63.
• 22 Tans G, Janssen TClaessen, Hemker HC, Zwaal RF, Rosing J. Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma. J Biol Chem 1991; 266: 21864-73.