The hypothesis that this proposal addresses is that high molecular weight kininogen (HMWK) on or about the platelet surface is an important regulator of plasma serine and cellular cysteine proteases. The objectives of this proposal are to identify and characterize the molecular interactions of HMWK with platelets. Studies will be performed to determine the mechanisms of interaction between HMWK and platelets or platelet constituents.
The specific aims of this proposal are (1) to characterize the availability of platelet HMWK expressed on the external membrane of activated platelets by monoclonal anti-HMWK125 I- Fab binding, immunocytochemical localization using colloidal gold, and molecular structure studies on activated platelet membranes as well as determine whether platelet HMWK can be a source of bradykinin for endothelial cell activation; (2) to investigate the interaction of exogenous HMWK with platelets to determine the molecular portion(s) of HMWK that binds to platelets, to identify the platelet receptor for HMWK, and to determine whether platelet-bound HMWK serves as a receptor for factors XI/XIa; (3) to study the interaction of HMWK with platelet calpain to determine the kinetics of HMWK's inhibition of platelet calpain, to characterize the domain(s) on HMWK that inhibit platelet calpain, to ascertain the mechanism of activation of HMWK by platelet calpain, and to determine whether platelet calpain liberates bradykinin from HMWK; and (4) to characterize the mechanism by which kallikrein inhibits platelet function and to ascertain the influence of HMWK on kallikrein's inhibition of platelet function. The proposed studies specifically address the molecular interactions of platelet and plasma HMWK on a physiologic surface. Since HMWK is a cofactor for activation of some serine zymogens (factor XII, prekallikrein, and factor XI) and an inhibitor of cysteine proteases (calpain and cathepsins) these studies will determine how HMWK associated with platelets can regulate these proteolytic systems. These studies on the interaction of HMWK with platelets should indicate new specific pathways by which the serine proteases, kallikrein and factor XIa, and cysteine proteases, calpains, could influence inflammation, intrinsic coagulation, fibrinolysis and blood pressure regulation.
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