Superior in Vivo Hemostatic Properties of an Engineered Factor Va Variant for Hemophilia Mice

Abstract 17 Approximately 1/3rd of hemophiliacs with inhibitors fail treatment with FVIIa-based bypassing agents. New intervention strategies are urgently needed. Activated FV (FVa) enhances thrombin formation by the prothrombinase complex, but is rapidly inactivated by activated protein C (APC). Mutation of the APC cleavage site, R506Q (FV Leiden ), is prothrombotic and improves hemostasis in humans and mice with hemophilia. We therefore engineered FV-derivatives with additional mutations of the APC cleavage sites (Arg506/306/679Gln) and introduced a disulfide bond (Cys609-Cys1691) connecting the A2 and A3 domains (FV(A2-SS-A3)). Introduction of the disulfide bond prevents the dissociation of the A2 domain as the final irreversible step of inactivation. All FV mutants were activated with thrombin prior to experimentation. Procoagulant properties of FVa mutants were studied in vitro and in hemophilia mouse models. Mutation of all three APC cleavage sites (Arg506/306/679) and addition of the disulfide bond yielded “ super FVa” that had 2- to 3-fold higher specific activity compared to recombinant human (rh)FVa, rhFVa(A2-SS-A3), rhFVa Leiden or rhFVa Leiden (A2-SS-A3) in purified prothrombinase assays and in thrombin generation assays in FV- and FVIII-deficient plasma. super FVa corrected the aPTT at 20-fold lower concentrations compared to rhFVa in FV-deficient plasma. super FVa was highly resistant to inactivation by APC in purified prothrombinase assays and in thrombin generation assays in FV-deficient plasma. While super FVa retained 90–100% activity in the presence of APC, FVa(A2-SS-A3) and FVa Leiden (A2-SS-A3) lost ∼90% and ∼40% of their activities, respectively. In FVIII-deficient mice, super FVa reduced bleeding significantly compared to rhFVa in a dose dependent fashion after tail clip (n=10–17 per group). Blood loss was determined separately for the first and second 10 min after tail clip to study the effects of FVa mutants on re-bleeding. FVa mutants were dosed at equal activities determined in prothrombinase assays (expressed in arbitrary units). At low doses of intravenous FVa mutants (40 units), mean blood loss during the first 10 min after clip in FVIII-deficient mice was significantly decreased following injection of super FVa compared to saline (6.5 μL/g vs. 15.5 μL/g; p=0.02), whereas blood loss following injection of rhFVa was not (16.7 μL/g). At high doses of FVa mutants (100 units), super FVa and rhFVa were effective in reducing blood loss in FVIII-deficient mice following intravenous injection. Mean blood loss with super FVa was further improved to 3.9 μl/g, and with rhFVa was 8.2 μl/g. Both results were significant compared to mean blood loss following saline injection (15.5 μL/g; p≤0.05). FVIII-deficient mice were considered protected at 10 min when blood loss was ≤ 9.9 μL/g, which is the mean plus 3 SDs derived from tail bleeding of wild-type BalbC mice. While only ∼45% of FVIII-deficient mice injected with saline or rhFVa (40 units) were protected, protection against bleeding increased to 83% with super FVa (40 units). When 100 units of FVa mutants were given, protection with both mutants ( super FVa or FVa) was ≥ 90%. Of note, during the second 10 min and therefore during the combined 20 min of bleeding, only super FVa at high dose (100 units) effectively reduced bleeding. Mean blood loss in FVIII-deficient mice following intravenous super FVa was 11.7 μL/g during the 2nd 10 minutes of bleeding, compared to 20.2 μL/g with saline (p=0.02). Our results indicate that molecular engineering of FV, which combined two alterations for improving thrombin generation (increased specific activity and APC resistance), provided a FVa-mutant, super FVa, with unique procoagulant properties. In vivo, super FVa not only decreased initial bleeding, but also significantly decreased re-bleeding, indicating that super FVa efficiently improved clot formation and also enhanced clot stabilization. These results warrant more in depth characterization of the potential therapeutic benefits of super FVa as a novel bypassing strategy in hemophilia patients with inhibitors. Disclosures: No relevant conflicts of interest to declare.