Venom versatility: Dynamic anticoagulant and procoagulant variations between and within Bothrocophias (toad-head) and basal Bothrops (lance-head) pit vipers

Pinpointing the emergence of toxicological evolutionary novelties can be challenging. In American pit vipers, anticoagulant venoms are the paradigm, with a notable exception being the genus Bothrops, which are typically procoagulant. A recent study found that the basal Bothrops (B. pictus) is anticoagulant, raising two competing hypotheses: ancestral Bothrops were anticoagulant with procoagulant venom evolving later, or ancestral Bothrops were procoagulant with anticoagulant venom in B. pictus being a derived trait. To help resolve this, we tested venoms of the sister genus Bothrocophias for pathophysiological actions upon blood clotting. The Ecuadorian Bothrocophias venoms (B. campbelli, B. lojanus, and B. microphthalmus) were compared to Bothrops pictus. Both Bothrocophias lojanus and B. pictus inhibited various blood clotting enzymes, but B. pictus was more potently anticoagulant. Intriguingly, B. campbelli and B. microphthalmus were procoagulant. Both B. microphthalmus populations activated prothrombin, but Zamora Chinchipe locality also activated Factors X and VII. Bothrocophias campbelli showed a novel activity, using Factor Va in a calcium-dependent manner as a cofactor to activate prothrombin, the first time this has been shown for any viperid venom. Organismal phylogenetics failed to resolve the relative positions of B. campbelli and B. lojanus, thus we were unable to ascertain the ancestral trait. To resolve this, more phylogenetic research and venom testing with other Bothrocophias species is needed. Neutralisation tests revealed differential efficacy of PoliVal-ICP (Instituto Clodomiro Picado) and Soro Antibotrópico (Instituto Butantan) antivenoms. Together, these findings aid in designing evidence-based clinical-management strategies and provide foundational data for reconstructing venom evolution