Low Normal Levels of Factor V Enhance Thrombin Generation in Hemophilia a in a Tissue Factor Pathway Inhibitor Independent Manner

Background: Low normal levels (50%-75%) of factor V (FV) enhance thrombin generation in hemophilia A (Link, JTH, 2020). A mathematical model of coagulation predicts this observation that can be explained by substrate competition for FXa between FV and FVIII. Alternatively, this observation could be explained by reduced inhibition of TF:FVIIa by TFPIα bound to FV-short.

Aims: The objective of this study was to determine whether FXa substrate competition between FV and FVIII explains enhanced thrombin generation with decreasing FV levels independent of TFPIα.

Methods: FV and FVIII were immunodepleted to <1% normal levels from human pooled plasma (Affinity Biologics). Calibrated Automated Thrombogram (Stago, 1 pM TF or 100 nM FXa) was used to measure thrombin generation in FV/FVIII double deficient plasma spiked with human FV (2-12 µg/mL, Prolytix), recombinant FVIII (0.05, 1.0 U/mL Kogenate, Bayer), and full length recombinant TFPIα (12.5 µg/mL, Novo Nordisk). In some experiments, concizumab (4 µg/mL, NovoNordisk) and/or anti-A2 and anti-C2 FVIII function blocking antibodies (GMA-8015, GMA-8006, Green Mountain Antibodies) were added to the plasma. TFPIα levels were measured by ELISA. An in house generated pooled plasma was used for normalization of thrombin generation studies.

Results: FV/FVIII depleted plasma was also depleted of TFPIα (below ELISA detection limit) reflecting its high affinity interaction with FV-short. To inhibit any trace amounts of TFPIα, concizumab, an anti-TFPIα antibody, was added to the plasma at a saturating concentration. Anti-A2 and anti-C2 domain blocking antibodies were added in some experiments to block any residual FVIII activity. In TFPIα depleted and concizumab treated plasma, the TF initiated thrombin generation curve showed a right shift (increased lag time) and more thrombin (higher peak thrombin and total thrombin produced) with decreasing FV levels for both low and normal FVIII levels (0.05 U/mL, 1 U/mL). In plasma with <1% FVIII treated with anti-FVIII function blocking antibodies, the same trends in enhanced thrombin generation were even more pronounced than in samples with exogenous FVIII added. When TFPIα was added into the FV/FVIII deficient plasma at normal circulating concentrations, the same trends were observed albeit with lower total thrombin generation. Surprisingly, FXa initiated thrombin generation in the presence of TFPIα showed the opposite trend, thrombin generation was attenuated with decreasing FV concentrations.

Conclusions: These data support the FXa substrate competition mechanism independent of TFPIα. As FVIII levels are reduced, the effect size of lowering FV levels increases in TF-initiated thrombin generation. Based on these results we hypothesize that lower levels of FVIII yield faster activation of FV during the initiation of coagulation. Results from FXa initiated thrombin generation suggest this effect occurs upstream of the common pathway. FVa does inhibit activation of FX by TF:FVIIa (Al Dieri, JTH, 2013), thus lower FV may result in reduced inhibition of the initiation of coagulation, a phenomenon that appears to become more prominent in hemophilia A plasma.

Manco-Johnson:Genentech/Roche: Honoraria; NovoNordisk: Honoraria; Spark/Pfizer: Honoraria; BioMarin: Honoraria; CSL Behring: Honoraria. Neeves:Novo Nordisk: Research Funding.