| Discovering novel pro-coagulant agents from snake venom Raymond Adili. |
Abstract
Novel Snake Venom Hemocoagulase Restores in Vivo Hemostasis and Limits Bleeding Associated with Coagulation Disorders
Reheman Adili M.D
Bloodworks Research Institute, Seattle, WA
Background: Activation of platelets and coagulation at vascular injury sites are crucial for hemostasis and limiting excessive bleeding. Coagulation defects can lead to severe bleeding, which requires urgent restoration of hemostasis. However, there is an unmet medical need for novel therapeutic agents that restore hemostasis in vivo. We recently reported slounase, a novel hemocoagulase containing a snake venom-derived batroxobin (a thrombin-like enzyme that converts fibrinogen to fibrin independent of thrombin) and a Factor X activator, restores hemostatic clot formation in heparin-anticoagulated mice suggesting slounase may circumvent FXa inhibition and restore coagulation.
Objectives: To investigate the hemostatic effect of slounase in severe bleeding disorders associated with hemophilia and treatment with Factor Xa Inhibitors.
Methods: The effect of slounase on hemostasis and bleeding was determined in FVIII-/- mice, with and without inhibitors, and wild-type mice that were pretreated with apixaban or rivaroxaban (via oral gavage), using intravital microscopy hemostatic models, and a tail-bleeding assay. The effect of slounase on clot formation in whole human blood was studied using reconstituted Factor FVIII-/- deficient blood or whole human blood pretreated with apixaban or rivaroxaban in vitro determined by thromboelastography (TEG).
Results: Hemostatic clot formation in response to vascular injury is severely defective in FVIII-/- mice and WT mice pretreated with FXa inhibitors examined under 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 and by ferric chloride injury to the carotid artery. Importantly, in vivo, hemostatic enhancement persisted in FVIII-/- mice intravenously treated with anti-FVIII antibodies and slounase in vivo. Slounase treatment dose-dependently restored clot formation in FVIII-/- mice whole blood and FVIII-/- deficient reconstituted human whole blood examined by TEG. Slounase intervention (1U/kg) reversed the anticoagulant effect of apixaban or rivaroxaban in vivo and significantly enhanced platelet-fibrin hemostatic clot formation in response to vascular injury. Rivaroxaban or apixaban treatment in human whole blood dose-dependently inhibited clot formation in vitro on TEG. Slounase treatment dose-dependently restored clot formation and clot strength to the normal range. Lastly, Slounase treatment significantly corrected prolonged tail bleeding time and decreased blood loss in FVIII-/- mice and WT mice treated with FXa inhibitors in tail clip assays.
Conclusion: Slounase treatment restores hemostasis and limits bleeding hemophilia A, with and without inhibitors. Slounase treatment reverses the anticoagulant effect of FXa inhibitors rivaroxaban and apixaban in vitro and in vivo. Our results demonstrate that slounase is a potential bypassing agent to restore hemostasis in bleeding associated with inherited coagulation disorders and as a rapid reversal agent for anticoagulation by FXa inhibitors to decrease bleeding and blood loss.
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