This application is a request for five years funding to determine how aggregation sites are expressed on the membrane surface of activated platelets. Platelet aggregation is a fundamental reaction in hemostasis and thrombosis and appears to be at least a three component system, requiring fibrinogen, Ca++, and a fibrinogen receptor on the membrane surfaces of activated platelets. Several lines of evidence suggest that platelet membrane glycoproteins (GP) IIb and IIIa are at the aggregation sites on platelets and may form part of the fibrinogen receptor. Previous work from our laboratory has led to the hypothesis stating that the increased Ca++ concentrations within activated platelets cause the expression of fibrinogen receptors on the membrane surface by inducing the formation of the GPIIb .IIIa heterodimer complex.
The specific aims of the proposed research are threefold. First, experiments are proposed to test our hypothesis concerning the expression of fibrinogen receptors. Thrombin, purified glycoproteins and monoclonal antibodies will be used as probes to compare the interactions between GPIIb and GPIIIa in control and stimulated platelets. Second, we propose to characterize the Ca++ binding properties of GPIIb and GPIIIa and to localize the Ca++ binding domain of these glycoproteins within membranes. Equilibrium binding of Ca++ to the purified glycoproteins will be determined. Ca++ binding peptides of cleaved glycoproteins will be identified and these domains localized within membranes using peptide-specific antibodies and membrane labeling techniques. Third, experimental protocols are outlined to reconstitute the fibrinogen receptor into phospholipid vesicles. The fibrinogen binding properties of phospholipid vesicles containing either GPIIb.IIIa or additional membrane glycoproteins will be evaluated in an attempt to reconstitute a fibrinogen receptor with similar properties as the receptor on platelet membranes and activated platelets. The relationship of this receptor to platelet aggregation will be evaluated by measuring the binding of phospholipid vesicles containing reconstituted fibrinogen receptors to control and activated platelets and by studying the aggregation properties of thrombasthenic platelets previously fused with fibrinogen receptor-containing vesicles. It is hoped that this study will lead to molecular identification of the fibrinogen receptor and an understanding of how platelets regulate transmembrane receptor function during activation.
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