An overall goal of this application is understanding the structure/function relationships of coagulation proteins. We propose to make directed mutations of coagulation factor X. These mutations will derive from the molecular models being constructed as part of another subproject of this Program Project. Also, a key question in this grant is the extent to interactions amongst proteins, as opposed to purely phospholipids, regulate the coagulation system. We are specifically interested in the ways in which platelet surface thrombin generation is regulated. For a number of these studies we will use a model system of coagulation that has been developed in our lab. This system uses cellular tissue factor, unactivated platelets, plasma concentrations of coagulation factors and inhibitors. In this system, platelets are activated before coagulation complexes assemble on the activated platelets leading to a burst of thrombin generation. Looking at thrombin generation on platelets from different individuals, we have observed that there are significant, reproducible differences. Also, thrombin generation can be significantly altered by altering the conditions under which platelets are activated. We will use the differences between individuals and the differences seen as a function of activation conditions as tools to allow us to identify components important in promoting the controlled procoagulant activity of platelets. We are also specifically interested in characterizing two specific regulatory proteins: the platelet factor IX binding protein and the endothelial cell factor IX binding protein. In a previous cycle of this PPG, a tight binding site for factor IX was identified on type IV collagen. Since luminal exposure of type IV collagen seems unlikely, we speculate that this type IV collagen domain is a motif that is being reused and propose to determine the nature of the endothelial cell factor IX binding protein.
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