An Assay to Quantify the Two Plasma Isoforms of Factor V

Lico Hoekema; Jan Rosing; Guido Tans

doi:10.1055/s-0037-1614172

Thrombosis and Haemostasis, Table of Contents

Thromb Haemost 2000; 84(06): 1066-1071
DOI: 10.1055/s-0037-1614172

Review Article

Schattauer GmbH

An Assay to Quantify the Two Plasma Isoforms of Factor V

Lico Hoekema

¹From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands

,

Jan Rosing

¹From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands

,

Guido Tans

¹From the Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, The Netherlands

› Author Affiliations

Abstract

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

Full Text

References

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