Pseudonaja textilis venom factor Va circumvents natural regulatory mechanisms via a unique A2 domain region.

BACKGROUND: The coagulation factor V (FV) variant derived from the venom of the Australian snake Pseudonaja textilis (ptFV) has several unique procoagulant adaptations that circumvent the normal regulatory mechanisms. Notably, ptFV comprises a significantly extended A2 domain C-terminus (A2T), a region of which the role in human FV biology is poorly understood. OBJECTIVES: In this study, we generated chimeric FV variants to elucidate the functional relevance of this extended structural region. METHODS: The ptFV A2T was exchanged for the homologous human FV region and vice versa, thereby generating ptFV-hA2T and hFV-ptA2T. RESULTS: Surprisingly, our findings demonstrate that enhanced procoagulant activity of ptFV is mediated by the A2T. Using this structural element, ptFV bypasses the need for membrane binding by facilitating productive interactions with P. textilis venom FXa (ptFXa) and prothrombin in solution. Substitution of the human A2T for the corresponding region in ptFV enabled human FVa to function in the absence of membranes, in a similar fashion to ptFVa, although this required complex formation with ptFXa, suggesting the presence of an essential element in the venom protease. In addition, we observed that the ptFV A2T region significantly contributes to the functional activated protein C-resistance. Unlike in mammalian FVa, a ptFVa variant comprising the human FV A2T retained structural integrity despite loss of cofactor function. CONCLUSIONS: Taken together, the ptFV A2T represents an exceptional structural element driving the enhanced procoagulant functions that are at the basis of the snake venom's extreme toxicity.