The objectives of this proposal are to characterize the mechanisms whereby platelet high molecular weight kininogen (HMWK) can be an extracellular regulator of contact phase zymogen activation, a possible intracellular inhibitor of platelet calpains, and an indirect regulator of endothelial function.
The specific aims of this investigation are as follows: (1) Platelet HMWK present on the external membrane of activated platelets will be quantified and characterized by 125I-monoclonal anti HMWK Fab binding studies; (2) Platelet HMWK will be characterized in unstimulated and activated platelets as to its location and distribution by immunocytochemical staining using colloidal gold; (3) Purified HMWK subunit binding to platelets will be done to determine the portion of the HMWK molecule that binds to platelets and to determine if the affinity of 125I-HMWK-platelet binding is altered when platelets are activated; (4) Coordinate factor XI/XIa-HMWK platelet binding will be performed to determine if HMWK serves as their platelet receptor; (5) The molecular structure of platelet HMWK will be characterized by immunoblot to determine if it is different from plasma HMWK or if it is cleaved by platelet calpain; (6) The effect of purified platelet calpain on purified HMWK will be studied to determine if platelet calpain activates HMWK and produces an altered HMWK with a structure similar to the HMWK seen in platelets, and to determine if HMWk itself can be an inhibitor of platelet calpain; (7) The availability and function of bradykinin derived from platelet HMWK will be ascertained to determine whether it is directly secreted from platelets and to determine whether a sufficient amount can be made available from platelet HMWK to stimulate endothelial cell secretion of prostacyclin. These studies are intended to characterize the platelet surface as a site for contact phase zymogen activation through the expression of HMWK on the platelet external membrane; to demonstrate that HMWK is activated by and is an inhibitor of platelet calpain; and to determine that bradykinin from secreted platelet HMWK is sufficient to stimulate endothelial cell prostacyclin secretion. These objectives are consistent with the hypothesis that platelet HMWK is a mediator and regulator of the surface-activated defense reactions of the proteins of the Hageman factor pathways which participate in the initiation of coagulation, the inflammatory response, and local injury-site blood pressure control.
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