Slounase, a Snake Venom Hemocoagulase Combined with FXa, Enhances Hemostasis and Limits Bleeding in Both Normal and Hypocoagulant Conditions
Authors: Adili Reheman , Dai Xiangrong , Li Benjamin Xiaoyi , Holinstat Michael 
Published: November 2023

Background

Uncontrolled post‐traumatic bleeding is the leading cause of potentially preventable death. Hemocoagulase, also known as botroxobin, a thrombin‐like serine protease from the venom of the snake Bothops atrox, is capable of converting fibrinogen to fibrin and thus reduces blood loss in hemorrhagic medical conditions. Slounase, a hemocoagulase combined with FXa, may have better hemostatic effect compared to botroxobin alone. However, the effects of this hemostatic reagent on hemostasis in vivo are not well characterized due to lack of reliable in vivo hemostasis models. Here, we compare the hemostatic effect of slounase and botroxobin on platelet function in vitro and in vivo.

Aim

To determine the in vivo hemostatic efficacy of slounase compared to botroxobin and FXa in normal and hypocoagulant conditions using real‐time intravital microscopy murine models of hemostasis.

Methods

Effect of slounase, botroxobin, and FXa on human platelet function was assessed in vitro by platelet aggregometry and flow cytometry. In vivo hemostatic effect of slounase was determined by laser ablation of the cremaster arteriole or a saphenous vein hemostasis model under real‐time intravital microscopy. Bleeding time was assessed in a liver puncture hemostasis model.

Results

Both Slounase and FXa, but not botroxobin, enhanced human platelet aggregation induced by a low dose of thrombin. Incubation of human platelets with slounase, FXa, or botroxobin did not induce platelet activation in the absence of a platelet agonist or vascular injury as determined by flow cytometry and in vivo intravital microscopy. Intravenous injection of these three agents into control wild type mice strongly enhanced hemostasis in the laser‐induced cremaster arteriole rupture hemostatic model. As expected intravenous injection of 25U of heparin into WT mice resulted in hypocoagulant status that prevented clot formation in vivo. FXa did not have a significant effect on hemostasis in heparin‐treated hypocoagulant mice as heparin is known to inhibit FXa. Botroxobin treatment in hypocoagulant mice resulted in detectable fibrin formation but no notable change in platelet recruitment in the clot. Interestingly, slounase treatment resulted in a significant enhancement of both fibrin and platelet content and restored the clot formation in hypocoagulant mice, which significantly shortened the red blood cell leakage time. Similar results were also observed in laser ablation saphenous vein hemostasis model in hypocoagulant mice. The bleeding time was shorter in slounase treated hypocoagulant mice as measured using liver pricking injury model.

Conclusions

These comprehensive in vivo studies clearly demonstrated slounase enhances hemostasis in normal mice and restores hemostasis in hypocoagulant conditions via both enhancing fibrin formation and platelet activation while botroxobin only enhances fibrin formation. Our data strongly indicates that slounase has a better hemostatic effect in the both normal and hypocoagulant states.

Support or Funding Information

This work is funded by LEE'S PHARM.