PB0214 - Snake venom-based hemocoagulase restores in vivo hemostasis and limits bleeding in murine model of hemophilia A

Background: Hemophilia A is a hereditary life-long bleeding disorder and FVIII replacement has been the mainstay of treatment. However, there is an unmet need for novel therapeutics to prevent and treat bleeding. We recently reported that slounase, a thrombin-like enzyme batroxobin from a snake venom containing activated Factor X, enhances platelet-fibrin clot formation in vivo in heparin-anticoagulated mice, suggesting slounase may restore hemostasis by bypassing coagulation.

Aims: To determine the effect of slounase treatment on hemostatic clot formation in FVIII-/- mice with and without inhibitors in vivo and in vitro and confirm slounase restores hemostasis in hemophilia.

Methods: The effect of slounase on hemostasis and bleeding was determined in FVIII-/- mice, with and without inhibitors, using intravital microscopy hemostatic models, thromboelastography and a tail-bleeding assay.

Results: FVIII-/- mice are unable to form hemostatic clots in vivo due to a severe defect in platelet accumulation and absence of fibrin formation at the site of vascular injury, as confirmed in micro and macro hemostatic models under real-time intravital microscopy. Prophylactic intravenous treatment of 1U/kg slounase in FVIII-/- mice significantly enhanced platelet activation, accumulation, and fibrin formation in response to vascular injury resulting in stable hemostatic clot formation in the cremaster artery and saphenous vein laser ablation hemostasis models. Platelet-fibrin hemostatic clots also formed following ferric chloride injury to the carotid artery in FVIII-/- mice pretreated with slounase without reaching vessel occlusion. Importantly, in vivo hemostatic enhancement persisted in FVIII-/- mice intravenously treated with anti-FVIII antibodies and slounase. Furthermore, the hemostatic effect of slounase was also confirmed by in vitro thromboelastography and in vivo tail-bleeding assays.

Conclusion(s): Our data indicates slounase is a novel bypassing agent that promotes platelet procoagulant activity and fibrin formation, to restore hemostasis and limit bleeding in hemophilia A, with and without inhibitors.

This is a collaborative research study funded by Lee’s Pharm.