Abstract
Introduction and Objectives: Novel oral anticoagulants (NOACs), such as Factor (F) Xa inhibitors (Rivaroxaban and Apixaban) or the direct thrombin inhibitor (Dabigatran), are used for prevention of venous thromboembolism and ischemic strokes. However, conventional hemostatic reversal strategies in case of bleeding or surgery are ineffective, posing a major unmet clinical need. We recently demonstrated that superFVa, a novel FVa variant engineered with mutations of 3 activated protein C (APC) cleavage sites and a disulfide bond between the A2 and A3 domains, has enhanced biological activity and APC resistance. SuperFVa provided efficient normalization of hemostasis in hemophilia A mice and in animal models of APC-induced bleeding. Moreover, superFVa synergistically reduced bleeding in combination with recombinant human (rh) FVIIa in hemophilia mice. Thus, superFVa fits the criteria for a prohemostatic biologic. Therefore, the in vitro and in vivo effects of superFVa alone and in combination with other prohemostatic agents such as rhFVIIa or 4-Factor prothrombin complex concentrates (4F-PCC) as a novel hemostatic reversal strategy for NOAC-induced bleeding were determined.
Materials and Methods: In vitro procoagulant properties of superFVa alone and in combination with rhFVIIa or 4F-PCC were studied using thrombin generation assays in normal human plasma (NHP) in the presence of FXa inhibitors (Rivaroxaban, Apixaban) or the direct thrombin inhibitor (Dabigatran, active form). In vivo prevention of blood loss by superFVa after intravenous injection of Rivaroxaban, Apixaban, or Dabigatran was studied using the tail clip model in BalbC mice.
Results: Rivaroxaban and Apixaban each dose-dependently reduced thrombin generation in NHP and reduced the Endogenous Thrombin Potential (ETP) by ~60% at therapeutic concentrations (200 nM). Addition of either superFVa (50 nM) or rhFVIIa (40 nM, equivalent to a 90 µg/kg therapeutic dose in hemophilia patients), increased thrombin generation to some extent. However, ETP and thrombin peak height increased dose-dependently when increasing concentrations of superFVa (6.25 to 400 nM) were added with rhFVIIa (40 nM). superFVa effects appeared synergistic, with a plateau reached at 25-50 nM superFVa and ETP restored to ~93% of normal. Synergistic effects of superFVa were also present and even more pronounced in combination with 4F-PCC (1.35 U/ml; therapeutic plasma concentration).
In the presence of Dabigatran (1 µM), superFVa (0.1 nM-100 nM) in combination with rhFVIIa (40 nM) or 4F-PCC (1.35 U/ml) showed a concentration dependent reduction of the lag time of thrombin generation. However, in contrast to the FXa inhibitors, in the presence of Dabigatran, no effects on ETP and thrombin peak height were observed for the addition of superFVa or combinations of superFVa and rhFVIIa or 4F-PCC.
The in vivo efficacy of superFVa to reduce NOAC-induced bleeding was determined by blood loss after tail transection in BalbC mice injected i.v. with Rivaroxaban (40 mg/kg), Apixaban (20 mg/kg), or Dabigatran (0.4 mg/kg). Mean blood loss in mice injected with Rivaroxaban (16 µL/g), Apixaban (16.5 µL/g) or Dabigatran (14.5 µL/g) was significantly higher than baseline bleeding (4 µL/g, p<0.001). superFVa reduced blood loss after Rivaroxaban or Apixaban administration significantly in a dose dependent manner, e.g., superFVa at 40 U/kg significantly reduced bleeding to the baseline control level (5 µL/g). RhFVIIa (1 mg/kg) was able to reduce bleeding caused by Rivaroxaban but not by Apixaban. Neither superFVa nor rhFVIIa was able to reduce bleeding caused by Dabigatran.
Conclusion: superFVa alone or in combination with rhFVIIa significantly improved in vitro thrombin generation in the presence of FXa inhibitors and to some extent in the presence of a direct thrombin inhibitor. SuperFVa alone consistently reduced bleeding in mice treated with FXa inhibitors, whereas a mixed response dependent on the type of FXa inhibitor was obtained with rhFVIIa. None of the agents was able to decrease bleeding in mice induced by Dabigatran. Because superFVa exerts a potent class effect as a hemostatic reversal strategy for FXa inhibitor-induced bleeding, it deserves further consideration for potential development as a hemostatic agent.
Disclosures
Gale: University of California, San Diego: University of California, San Diego Patents & Royalties. Griffin:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties. Mosnier:The Scripps Research Institute: The Scripps Research Institute Patents & Royalties. von Drygalski:University of California, San Diego: University of California, San Diego Patents & Royalties.
Synergistic Effect in Bleed Reduction By superfva and Recombinant Human FVIIa in Vivo Suggests a Novel Bypassing Strategy for Hemophilia Patients with Inhibitors