Snakebite is a global tropical disease that has long had huge implications for human health and well-being. Despite its long-standing medical importance, it has been the most neglected of tropical diseases. Reflective of this is that many aspects of the pathology have been underinvestigated. Snakebite by species in the Elapidae family is typically characterised by neurotoxic effects that result in flaccid paralysis. Thus, while clinically significant disturbances to the coagulation cascade have been reported, the bulk of the research to date has focused upon neurotoxins. In order to fill the knowledge gap regarding the coagulotoxic effects of elapid snake venoms, we screened 30 African and Asian venoms across eight genera using in vitro anticoagulant assays to determine the relative inhibition of the coagulation function of thrombin and the inhibition of the formation of the prothrombinase complex through competitive binding to a nonenzymatic site on Factor Xa (FXa), thereby preventing FXa from binding to Factor Va (FVa). It was revealed that African spitting cobras were the only species that were potent inhibitors of either clotting factor, but with Factor Xa inhibited at 12 times the levels of thrombin inhibition. This is consistent with at least one death on record due to hemorrhage following African spitting cobra envenomation. To determine the efficacy of antivenom in neutralising the anticoagulant venom effects, for the African spitting cobras we repeated the same 8-point dilution series with the addition of antivenom and observed the shift in the area under the curve, which revealed that the antivenom performed extremely poorly against the coagulotoxic venom effects of all species. However, additional tests with the phospholipase A2 inhibitor LY315920 (trade name: varespladib) demonstrated a powerful neutralisation action against the coagulotoxic actions of the African spitting cobra venoms. Our research has important implications for the clinical treatment of cobra snakebites and also sheds light on the molecular mechanisms involved in coagulotoxicity within Naja. As the most coagulotoxic species are also those that produce characteristic extreme local tissue damage, future research should investigate potential synergistic actions between anticoagulant toxins and cytotoxins.

The binding of compounds to nicotinic acetylcholine receptors is of great interest in biomedical research. However, progress in this area is hampered by the lack of a high-throughput, cost-effective, and taxonomically flexible platform. Current methods are low-throughput, consume large quantities of sample, or are taxonomically limited in which targets can be tested. We describe a novel assay which utilizes a label-free bio-layer interferometry technology, in combination with adapted mimotope peptides, in order to measure ligand binding to the orthosteric site of nicotinic acetylcholine receptor alpha-subunits of diverse organisms. We validated the method by testing the evolutionary patterns of a generalist feeding species (Acanthophis antarcticus), a fish specialist species (Aipysurus laevis), and a snake specialist species (Ophiophagus hannah) for comparative binding to the orthosteric site of fish, amphibian, lizard, snake, bird, marsupial, and rodent alpha-1 nicotinic acetylcholine receptors. Binding patterns corresponded with diet, with the Acanthophis antarcticus not showing bias towards any particular lineage, while Aipysurus laevis showed selectivity for fish, and Ophiophagus hannah a selectivity for snake. To validate the biodiscovery potential of this method, we screened Acanthophis antarcticus and Tropidolaemus wagleri venom for binding to human alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-6, alpha-7, alpha-9, and alpha-10. While A. antarcticus was broadly potent, T. wagleri showed very strong but selective binding, specifically to the alpha-1 target which would be evolutionarily selected for, as well as the alpha-5 target which is of major interest for drug design and development. Thus, we have shown that our novel method is broadly applicable for studies including evolutionary patterns of venom diversification, predicting potential neurotoxic effects in human envenomed patients, and searches for novel ligands of interest for laboratory tools and in drug design and development.

The genus Bitis comprises 17 snake species that inhabit Africa and the Arabian Peninsula. They are responsible for a significant proportion of snakebites in the region. The venoms of the two independent lineages of giant Bitis (B. arietans and again in the common ancestor of the clade consisting of B. gabonica, B. nasicornis, B. parviocula and B. rhinoceros) induce an array of debilitating effects including anticoagulation, hemorrhagic shock and cytotoxicity, whilst the dwarf species B. atropos is known to have strong neurotoxic effects. However, the venom effects of the other species within the genus have not been explored in detail. A series of coagulation assays were implemented to assess the coagulotoxic venom effects of fourteen species within the genus. This study identified procoagulant venom as the ancestral condition, retained only by the basal dwarf species B. worthingtoni, suggesting anticoagulant venom is a derived trait within the Bitis genus and has been secondarily amplified on at least four occasions. A wide range of anticoagulant mechanisms were identified, such as coagulant and destructive activities upon fibrinogen in both giant and dwarf Bitis and the action of inhibiting the prothrombinase complex, which is present in a clade of dwarf Bitis. Antivenom studies revealed that while the procoagulant effects of B. worthingtoni were poorly neutralized, and thus a cause for concern, the differential mechanisms of anticoagulation in other species were all well neutralized. Thus, this study concludes there is a wide range of coagulotoxic mechanisms which have evolved within the Bitis genus and that clinical management strategies are limited for the procoagulant effects of B. worthingtoni, but that anticoagulant effects of other species are readily treated by the South African polyvalent antivenom. These results therefore have direct, real-work implications for the treatment of envenomed patients.

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