Colubrid clotcraft: Divergent Factor Va cofactor utilisation underpins differential antivenom cross-reactivities for Boomslang (Dispholidus typus) and Eastern Twig Snake (Thelotornis mossambicanus) venoms.

Rear-fanged African colubrids are increasingly recognised as capable of causing severe haemotoxic envenomation, yet the mechanistic basis of their procoagulant venom activity and the efficacy of available therapeutics remain incompletely resolved. We investigated the coagulotoxic mechanisms of Boomslang (Dispholidus typus) and Eastern Twig Snake (Thelotornis mossambicanus) venoms using plasma coagulation assays, fluorometric clotting factor activation assays, Factor V cleavage studies, antivenom neutralisation experiments, and small-molecule inhibitor testing. Both venoms induced rapid clotting of human plasma, with T. mossambicanus producing substantially faster clotting than D. typus, indicating greater overall procoagulant potency. The SAIMR Boomslang monovalent antivenom demonstrated markedly greater efficacy than VINS Boomsven monovalent antivenom against both venoms, including superior cross-neutralisation of T. mossambicanus (albeit still at low levels of limited therapeutic usefulness), while Boomsven showed negligible effectiveness against the latter species. Metalloproteinase inhibitors neutralised both venoms, supporting metalloproteinases as key mediators of coagulation disturbance in both species. Fluorometric assays demonstrated activation of FVII, FX, and prothrombin (with a rank order of FVII>FX > prothrombin), but not FXI or FXII, implicating extrinsic and common pathway mechanisms rather than intrinsic pathway activation. Both venoms used exogenous FVa to potentiate prothrombin activation, particularly T. mossambicanus, but neither venom-generated FV cleavage product functioned equivalently to thrombin-generated FVa. SDS-PAGE analyses confirmed proteolytic cleavage of FV into FVa-like fragments, revealing mechanistically distinct forms of venom-mediated FV activation. These findings provide new insight into the convergent evolution of cofactor-dependent procoagulant venom systems, highlight clinically important differences in antivenom cross-reactivity between closely related rear-fanged snakes, and support metalloproteinase inhibitors as promising therapeutic candidates for African colubrid envenomation.