he coagulation arm of hemostasis (the maintenance of vascular patency) is triggered in response to various stimuli and results in the explosive generation of thrombin. The co-factor proteins, factors V and VIII, act as receptors and activity modulators in assembling their respective serine protease partners into the enzymatic complexes (prothrombinase and intrinsic tenase, respectively) without which thrombin generation is negligible. This application proposes to elucidate the structures of the bovine and human coagulation co-factors V and Va. These structures will be used to initially create a homology model of factor VIIIa while the structure of recombinant factor VIIIa (Baxter Healthcare) is completed. These structures will be required for the long term, larger goal of solving the structures of the prothrombinase and intrinsic tenase complexes. An x-ray crystallographic approach will be taken in each case. As detailed in the proposal, diffracting crystals of bovine factor Va have already been obtained and should lead to a complete structure after generating a phase solution. The structure of human factor Va will be determined by similar strategies and should lead to a relatively rapid structure by molecular replacement. Determination of the structure of bovine and human factor Va will allow us to build a homology model of factor VIIIa in order to provide a structural explanation for the numerous functional mutants which have been identified. Crystallization trials will begin immediately using bovine and human factor V as well as recombinant factor VIIIa. The fundamentals of the structure/function relationships of these coagulation cofactors are essential to understanding the molecular basis of defects in the clotting process and should help in designing rational interventions in real life situations.
