Impaired APC cofactor activity of factor V plays a major role in the APC resistance associated with the factor V Leiden (R506Q) and R2 (H1299R) mutations

Abstract

Activated protein C (APC) resistance is a major risk factor for venous thrombosis. Factor V (FV) gene mutations like FV Leiden (R506Q) and FV R2 (H1299R) may cause APC resistance either by reducing the susceptibility of FVa to APC-mediated inactivation or by interfering with the cofactor activity of FV in APC-catalyzed FVIIIa inactivation. We quantified the APC cofactor activity expressed by FV Leiden and FV R2 and determined the relative contributions of reduced susceptibility and impaired APC cofactor activity to the APC resistance associated with these mutations. Plasmas containing varying concentrations of normal FV, FV Leiden , or FV R2 were assayed with an APC resistance assay that specifically measures the APC cofactor activity of FV in FVIIIa inactivation, and with the activated partial thromboplastin time (aPTT)-based assay, which probes both the susceptibility and APC cofactor components. FV R2 expressed 73% of the APC cofactor activity of normal FV, whereas FV Leiden exhibited no cofactor activity in FVIIIa inactivation. Poor susceptibility to APC and impaired APC cofactor activity contributed equally to FV Leiden -associated APC resistance, whereas FV R2 -associated APC resistance was entirely due to the reduced APC cofactor activity of FV R2 . Thrombin generation assays confirmed the importance of the anticoagulant activity of FV and indicated that FV Leiden IntroductionThe protein C pathway is a major anticoagulant mechanism that down-regulates the prothrombin-and intrinsic factor X (FX)-activating complexes via inactivation of their respective cofactors, activated factors V (FVa) and VIII (FVIIIa). 1 Cofactor inactivation occurs by limited proteolysis at amino acid positions 306, 506, and 679 in FVa 2 and 336, 562, and 740 in FVIIIa. 3 These reactions are catalyzed by the serine protease activated protein C (APC) and stimulated by the APC cofactor protein S. In vitro experiments using purified proteins have shown that FV also stimulates the APC-mediated inactivation of FVIIIa. [4][5][6] To express full APC cofactor activity, FV must retain (part of) the B domain 4,6,7 and be cleaved by APC at Arg506. 8 Although this anticoagulant function of FV is still poorly characterized, a recent report suggests that it plays a critical antithrombotic role in vivo. 9 Functional defects of the protein C pathway, due to inherited or acquired conditions, determine a plasma phenotype known as APC resistance, 10 which is a prevalent and important risk factor for venous thrombosis. 11-13 A plasma is termed "APC resistant" when the addition of exogenous APC fails to prolong its clotting time in an activated partial thromboplastin time (aPTT) assay. 10 Since the discovery of APC resistance, several other methods have been developed to detect this condition and to study the underlying molecular mechanism.To date, a few FV gene mutations have been identified in association with APC resistance. In principle, these mutations may cause APC resistance either by reducing the susceptibility of FVa to APC-mediated inactivat...

Factor V Leiden Inhibits Fibrinolysis In Vivo

“…fV Leiden can potentially modulate thrombin production and fibrinolysis by different mechanisms, including enhanced fV procoagulant activity and diminished capacity to serve as a cofactor for the inactivation of fVIIIa by APC. 21,22 Differences in the relative impact of these processes in a static, in vitro system versus the pulmonary vasculature of a living mouse may contribute to the differences between our in vitro and in vivo assays in regard to the fibrinolytic defect produced by heterozygosity for fV Leiden . Effects of sample size must also be considered.…”

Factor VLeiden

“…Of these, one of the more significant is FV R2 (H1299R) which is tightly linked to several other polymorphisms and collectively named the R2 haplotype [29]. FV R2 exhibits APC resistance entirely due to reduced APC cofactor activity and leads to an increased thrombotic risk when present in compound heterozygotes with FV Leiden [29,33]. In the absence of FV Leiden , mild APC resistance has been found to be present [34,35] or absent [35,36] with homozygous or heterozygous FV R2 ; these studies used assays either with [34] or without [35,36] dilution into FV-deficient plasma.…”

An underestimated combination of opposites resulting in enhanced thrombotic tendency

“…11 The endogenous thrombin potential (ETP) was calculated using the Thrombinoscope software (Synapse BV, Maastricht, The Netherlands), and mean values were compared with the Mann-Whitney Wilcoxon test using SPSS 11.5 software. (SPSS, Chicago, IL).…”