Hemostatic hellfire: Rhabdophis helleri (Red-necked Keelback) snake venom pathophysiologically activates Factor VII and Factor X through novel uses of the clotting cofactors calcium, phospholipid, and Factor Va

Rhabdophis helleri envenomations may cause lethal coagulopathy, yet the venom mechanisms are poorly characterized. This study investigated the procoagulant mechanisms of R. helleri venom, with particular focus on coagulation factor activation and cofactor dependencies. We demonstrate that R. helleri venom potently activates Factor VII (FVII) and Factor X (FX) in a highly cofactor-dependent manner. FVII activation was minimal with venom alone but increased dramatically with the addition of the cofactor protein Factor V (FV) and reached maximal activity with the addition of the cofactor Factor Va (FVa). In addition to using endogenous thrombin produced FVa, the itself is able to convert FV into a useable FVa product. Similarly, FX activation was greatly amplified in the presence of FV or FVa, and showed similar cofactor dependence and the ability to use venom-activated FVa. Under comparable FV or FVa cofactor conditions, FX was activated at substantially lower magnitude than Factor VII, suggesting that Factor VII is the primary pathophysiological target. Systematic calcium and phospholipid requirement analysis revealed extremely high levels of dependence upon both cofactors. This is the first time for any venom that such a high level of phospholipid dependence has been noted. These results represent the first documentation of Factor Va-dependent zymogen activation in a non-front-fanged species, indicating convergent evolution of this mechanism across Natricidae, Elapidae, and Viperidae snake families. The determination of the biochemistry underpinning the rapid plasma clotting induced by the venom provides mechanistic insight into the consumption coagulopathy observed clinically in human envenomations and the stroke-inducing effect in prey. These toxicological findings advance understanding of rear-fanged snake venom evolution and inform development of treatment strategies for this understudied but medically important species.