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DOI: 10.1055/s-0037-1614172
An Assay to Quantify the Two Plasma Isoforms of Factor V
Supported by Program Grant 900-526-192 from the Dutch Organisation for Scientific Research (N.W.O.).Publication History
Received
19 April 2000
Accepted after revision
29 June 2000
Publication Date:
13 December 2017 (online)
![](https://www.thieme-connect.de/media/10.1055-s-00035024/200006/lookinside/thumbnails/10-1055-s-0037-1614172-1.jpg)
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.
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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.