Thrombin generation at the human platelet surface is effected through the assembly and function of the enzymatic complex, Prothrombinase, consisting of a 1:1 stoichiometric, Ca/2+ complex of the cofactor factor Va and the serine protease factor Xa. Subsequent to platelet activation, released platelet factor V(a) and/or plasma-derived factor Va bind to the platelet membrane surface and in so doing form at least part of the receptor for factor Xa. Thus, several protein/protein and protein/membrane interactions dictate functional complex assembly. A goal of this research project is to define how platelets actively participate in and regulate complex assembly. Because platelets have been shown to express the factor Xa membrane receptor Effector Protease Receptor-1 (EPR-1) following agonist-induced activation, substantial effort will be placed on defining its role in, and regulation of, a functional Prothrombinase complex. The platelet's ability to regulate events critical for enzyme complex assembly subsequent to agonist-induced activation will be assessed as well, specifically as related to 1) functional EPR-1 expression and 2) expression of a platelet factor Va molecule resistant to activated protein C inactivation, and perhaps other proteases as well. Because thrombin once formed positively regulates Prothrombinase complex assembly and function through platelet activation, a second goal is to understand how thrombin interactions with platelet membrane proteins affect its function. Effort will be placed on defining and isolating the thrombin high affinity binding site which appears to render the thrombin molecule transiently inactive and delineating the relationship of glycoprotein Ib/IX to this site. Finally, the observation that both a thrombomodulin-like molecule constitutively expressed on the platelet membrane and platelet-derived factor Va inhibit fibrinolysis through a thrombin-dependent mechanism will be explored, especially as related to the recently identified Thrombin Activatable Fibrinolysis Inhibitor (TAFI). The ultimate goal of this research project is to define the mechanisms by which platelets actively regulate both the generation and function of thrombin at their membrane surface. Defining the cellular and molecular events which regulate thrombin production is fundamental to our overall understanding of the hemostatic and thrombotic processes.
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