Ethanol interferes with platelet activation in response to physiological agonists, both in vivo and in vitro. Ethanol-induced platelet dysfunction may account, in part, for bleeding disorders and the increased incidence of strokes in chronic alcoholics. Moreover, moderate ethanol intake may be protective against coronary heart disease, perhaps by the inhibition of platelets. The overall aim of this proposal is to characterize the effect of ethanol on the phospholipase-mediated signal transduction pathways of human platelets, and to determine the relationship of this interaction to defects in platelet function. Ethanol stimulates phosphoinositide-specific phospholipase C in human platelets. The mechanism of this action will be determined in isotopically-labelled preparations of permeabilized platelets and plasma membranes. The studies will emphasize the role of guanine nucleotide binding proteins in this effect. It will also be determined whether the prolonged stimulation of phospholipase C and protein kinase C by ethanol can result in desensitization to agonist stimulation. Platelet responses to be studied include shape change, aggregation, and secretion. Ethanol interferes with arachidonic acid mobilization by an inhibition of phospholipase A2. Studies will determine whether ethanol directly inhibits phospholipase A2 or its regulatory features, which include protein kinase C and G-proteins. Isotopically-labelled and fluorescent-tagged phospholipids will serve as phospholipase A2 substrates. Studies of the effect of ethanol on phospholipase A2 will be extended to human hematopoietic-derived cells. The characteristics of phospholipase D activation will be determined in response to a range of agonists, including phorbol ester. The interaction of ethanol with platelet phospholipase D will be analyzed by measurement of phosphatidylethanol and phosphatidic acid formation. The implications of phosphatidylethanol formation for platelet function will be assessed. Studies will also address the mechanism by which ethanol inhibits platelet secretion that is independent of its effect on phospholipase signalling. Specifically, it will be determined whether ethanol interferes with granule movement and cytoskeletal assembly. Features of the cystoskeleton will be studied by electron microscopy and by biochemical analysis of its individual components.
