Abstract
Background: Factor V (FV) has pro- and anticoagulant roles in the regulation of coagulation. This is reflected in the paradoxical observation that both low and high FV levels are correlated with risk of venous thromboembolism (Rietveld et al., RPTH, 2, 2018). Moreover, low normal levels (50-75%) of FV enhance thrombin generation in hemophilia A (Link et al., JTH, 18, 2020). Reducing FV levels may increase thrombin generation via its anticoagulant functions as a carrier for TFPIα, in the form of the splice variant FV-short, or as a co-factor in the activated protein C pathway. However, there is also evidence that FV and FVa can interact with the tissue factor-factor VIIa (TF:FVIIa) complex directly (Safa et al., Biochemistry, 38, 1999; Al Dieri et al., JTH, 11, 2013).
Aims: The objective of this study was to measure the effect of FV levels on thrombin generation and factor X (FX) activation by tissue factor-initiated coagulation.
Methods: FV and factor VIII (FVIII) were immunodepleted to <1% normal levels from human pooled plasma (Affinity Biologics). FVIII- and FIX-deficient plasma were from human donors (George King Biomedical). Calibrated Automated Thrombogram (Stago, 1 pM TF, 100 µg/mL kaolin) was used to measure thrombin generation in FV/FVIII depleted plasma supplemented with either human FV or FVa (1-40 nM, Prolytix) and, where indicated, recombinant FVIII (0.05, 1.0 U/mL Kogenate, Bayer). In some experiments, concizumab (4 µg/mL, NovoNordisk) was added to the plasma to block TFPIα or antibodies against FV's light and heavy chains (Prolytix, AHV-5101, AHV-5146). FX activation was measured with 1 nM FVIIa, 1 pM TF, 125 nM FX, 2-40 nM FV or FVa, 4 µM phospholipids, and 500 µM of a FXa substrate (RGR-XaChrom, Enzyme Research Laboratory). TFPIα levels were measured by ELISA.
Results: Increasing FV levels results in decreases in lag time, peak thrombin, and velocity index in FV/FVIII immunodepleted plasma in a FVIII-dependent manner in the presence of concizumab. TFPIα activity was undetectable in this mixture. The effect size for the reduction in thrombin generation is greater for low FVIII levels than normal levels. For example, increasing FV levels from 2 nM to 40 nM reduces peak thrombin approximately by 50% for <0.01 and 0.05 U/mL FVIII, and 20% for 1 U/mL FVIII. Conversely, increasing FVa levels has the opposite effect in a FVIII-independent manner; lag times, peak thrombin, and velocity index all increase up to 20 nM FV. Increasing FV levels only affects lag time in kaolin-initiated thrombin generation. FX activation by TF:FVIIa is significantly reduced by FV in a dose-dependent manner with plasma levels of 20 nM FV reducing the rate of FX activation by 4-fold, compared to 20 nM FVa which only reduced the activation rate by 20%. Inhibition of FV's light chain, and but not its heavy chain, increased the rate FX activation and enhances thrombin generation in FVIII- and FIX-deficient plasma, more than doubling peak thrombin and the velocity index in both types of plasma.
Conclusions: These data support a new anticoagulant role of FV wherein it inhibits FX activation by TF:FVIIa during the initiation of coagulation, independent of TFPIα. FV reduces the rate and extent of thrombin generation, an effect that is attenuated at normal FVIII levels, suggesting complementary FX activation by intrinsic tenase (FVIIIa:FIXa). Coagulation initiated by the contact pathway via kaolin does not show the same trends. FV is a potent inhibitor of FX activation by TF:FVIIa; normal plasma FV levels reduce the rate of normal plasma levels of FX by a factor of four. Inhibition of the FV light chain rescues FXa activation and enhances thrombin generation in plasma deficient in FVIII and FIX. The light chain includes the C1 and C2 domains of FV, which are responsible for its interaction with negatively charged phospholipids. Variants in these domains including FV Basançon and FV Nara, lead to recurrent venous thrombosis, by affecting APC-cofactor activity (Mohapatra, RPTH, 8, 2024), but these data suggest a potential added role for FV in regulating initial FX activation. These data could also explain, in part, the correlation between low FV levels and VTE and the observation that low FV levels enhance thrombin generation in hemophilia.
