The overall goal of this proposal is to provide new information on the molecular mechanisms that regulate the cofactor function of blood coagulation factor V (FV). Shedding light on these processes may provide a framework for developing novel strategies for prevention and treatment in cases of disregulation of the blood coagulation response. This is a uniquely complex process that protects organisms from significant blood loss following vascular damage. A failure of this system to respond or to restrict its response can result in life-threatening bleeding disorders or thrombosis. Coagulation FV plays an important role in the clotting system as precursor of FVa, the cofactor for the serine protease FXa that converts prothrombin to thrombin, a key regulatory enzyme in the formation of a blood clot. Interaction of FXa with FVa dramatically enhances its catalytic rate, highlighting the biological significance of FV.Factor Va only interacts with FXa on a phospholipid surface, such as that of platelets or ruptured atherosclerotic plaques. The exact mechanism underlying the membrane-dependence of this interaction is, despite three decades of FV structure-function research, still poorly understood. In the current project, we aim to address this longstanding question using a novel strategy based on a naturally occurring FV variant found in the venom of the Australian snake P.textilis (pt-FV), which has evolved to circumvent the membrane-dependence in FXa binding. We will target several unique structural features of pt-FV that we speculate are involved in its membrane-independent FXa interaction, which will be a) removed from pt-FV and b) introduced into human FV. By characterizing this recombinant FV panel, we anticipate to gain critical insight into the FVa-FXa enzyme complex that is central to coagulation and also into the biology of other macromolecular coagulation complexes, which represents information vital to the engineering of improved therapeutic proteins.