The surface membrane of the platelet is the site at which agonists stimulate aggregation and secretion, and it is the surface upon which thrombin generation is facilitated. Platelets are activated upon the binding of agonists to specific surface receptors. The long term goal of this proposal is to better characterize the structural changes within the plasma membrane that occur subsequent to the binding of an agonist and that culminate in the platelet's hemostatic responses. Three general approaches will be taken. First, because the platelet receptor for epinephrine has already been well characterized pharmacologically, the structural features of this Alpha2-adrenergic receptor will be explored. Specifically, the receptor will be solubilized and an attempt will be made to identify the native receptor using polyacrylamide gel electrophoresis under non-denaturing conditions. In addition, the receptor will be purified by affinity chromatography using both and Alpha2-adrenergic antagonist and anti-Alpha2-receptor monoclonal antibodies as the affinity reagents. The receptor will also be reconstituted into artificial membranes in order to examine the influence of specific ions, membrane lipids and proteins on receptor function. Second, because platelets contain saturable and high affinity binding sites for Ca++ on their surface, the effect of receptor stimulation on the binding of Ca++ to specific proteins on the surface membrane will be examined. These membrane Ca++ binding proteins will be identified using 45Ca++ as well as the lanthanides, gadolinium and terbium, which are known for their affinity for Ca++ binding sites. Third, the relationship between the binding of Ca++ to the surface membrane and specific Ca++ dependent membrane changes, such as phospholipid hydrolysis and protein phosphorylation will be examined. These changes may occur subsequent to epinephrine receptor stimulation and membrane Ca++ association and appear to be involved in platelet activation. The above studies will be carried out using normal platelets as well as platelets with known membrane lipid and protein structural defects. As a result, we hope to clarify not only the role of membrane-bound Ca++ in receptor-mediated platelet activation, but also the pathophysiology of some congenital and acquired disorders of platelet function.
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