PB1155 - Using zebrafish and fish-consuming snake species to identify venom proteins that affect hemostasis

Background: Snake venoms are complex protein mixtures used to incapacitate prey and often target blood coagulation. Related snake species consume divergent diets, including aquatic and terrestrial. Deconstructing the functional effects of venom variation and components provides an unparalleled opportunity to leverage natural selection to identify novel protein functions. Conservation of the coagulation cascade enables the use of zebrafish to study hemostasis in the context of intact vasculature, clotting factors, and blood cells, in which many aspects of biological function can be directly observed.

Aims: Determine the molecular basis of species-specific venom targets on coagulation.

Methods: We infused the venoms of related snakes (fish-, amphibian-, or mammal-consuming) into zebrafish larvae and evaluated effects on heart rate, blood flow, and thrombocytes (zebrafish platelets). Venom was spiked into human and trout whole blood and plasma followed by observation of clotting times. Infusion of fractionated venom serine proteases into zebrafish larvae was followed by measurement of time to venous occlusion after laser-mediated endothelial injury.

Results: We found that Agkistrodon piscivorus (fish-eating) venom was most lethal to zebrafish, causing thrombocyte aggregation and adhesion to the endothelium in zebrafish larvae, with circulating emboli. This resulted in cessation of blood flow in larvae. A. piscivorus and Sisturus miliarius (amphibian eating) venoms caused trout whole blood to clot, however venoms from mammal-consuming snakes only clotted human, but not trout whole blood. Fractionated serine proteases from A. piscivorus venom prevented venous occlusion in larvae, but the same was not true for mammal-consuming snake species, suggesting evolved targeting of fish coagulation factors.

Conclusion(s): Using zebrafish as a model, we have discovered functional specificity of uncharacterized serine proteases specifically from venom of fish-eating snakes. Feeding on deeply divergent prey such as fish versus rodents exerts divergent natural selection, generating endless functional variations in venoms observed among species. These venom proteins have potential as new coagulation therapeutics.