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...
Elevated plasma prothrombin levels, due to the prothrombin 20210 G/A mutation or to acquired causes, are a risk factor for venous thrombosis, partly because of prothrombin-mediated inhibition of the protein C anticoagulant pathway and consequent activated protein C (APC) resistance. We determined the effect of plasma prothrombin concentration on the APC resistance phenotype and evaluated the role of protein S levels as a modulating variable. The effect of prothrombin and protein S levels on APC resistance was investigated in reconstituted plasma systems and in a population of healthy individuals using both the aPTT-based and the thrombin generation-based APC resistance tests. In reconstituted plasma, APC resistance increased at increasing prothrombin concentration in both assays. Enhanced APC resistance was caused by the effect of prothrombin on the clotting time in the absence of APC in the aPTT-based test, and on thrombin formation in the presence of APC in the thrombin generation-based test. In plasma from healthy individuals prothrombin levels were highly correlated to protein S levels. Since prothrombin and protein S had opposite effects on the APC resistance phenotype, the prothrombin/protein S ratio was a better predictor of APC resistance than the levels of either protein alone. Prothrombin titrations in plasmas containing different amounts of protein S confirmed that protein S levels modulate the ability of prothrombin to induce APC resistance. These findings suggest that carriers of the prothrombin 20210 G/A mutation, who have a high prothrombin/protein S ratio, may experience a higher thrombosis risk than non-carriers with comparable prothrombin levels.
Coagulation factor V (FV) is a large plasma glycoprotein with functions in both the pro- and anticoagulant pathways. In carriers of the so-called R2-FV haplotype, the FV D2194G mutation, in the C2 membrane-binding domain, is associated with low expression levels, suggesting a potential folding/stability problem. To analyze the molecular mechanisms potentially responsible for this in vitro phenotype, we used molecular dynamics (MD) and continuum electrostatic calculations. Implicit solvent simulations were performed on the x-ray structure of the wild-type C2 domain and on a model of the D2194G mutant. Because D2194 is located next to a disulfide bond (S-S bond), MD calculations were also performed on S-S bond depleted structures. D2194 is part of a salt-bridge network and investigations of the stabilizing/destabilizing role of these ionic interactions were carried out. Five mutant FV molecules were created and the expression levels measured with the aim of assessing the tolerance to amino acid changes in this region of molecule. Analysis of the MD trajectories indicated increased flexibility in some areas and energetic comparisons suggested overall destabilization of the structure due to the D2194G mutation. This substitution causes electrostatic destabilization of the domain by approximately 3 kcal/mol. Together these effects likely explain the lowered expression levels in R2-FV carriers.
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