Aside from its crucial role in the normal hemostatic process, the blood platelet is firmly implicated in the pathogenesis of thrombosis and other vascular diseases; thus it is important to understand the cellular mechanisms which regulate platelet function. Formation of the key platelet activator thromboxane A2 during platelet aggregation is limited by the availability of its chemical precursor, arachidonic acid. This project seeks to clarify the poorly understood mechanism by which arachidonic acid mobilization in human platelets is governed. The roles of three major intracellular signals, Ca2+, the Ca2+/phospholipid-dependent protein kinase (C-kinase), and guanine nucleotide-binding proteins (N-proteins) in the cellular mechanism of arachidonic acid release, will be studied in human platelets in experiments involving intact cells, permeabilized platelets, and platelet-derived subcellular preparations. Previous studies in intact platelets which suggest an involvement of C- kinase in Ca2+-dependent arachidonic acid release will be extended by examining the direct effect of purified C-kinase on the activity of phospholipase A2, the major lipolytic enzyme responsible for arachidonic acid liberation in stimulated platelets. Identification of the C-kinase substrate which mediates activation of phospholipase A2 will be sought. The possible involvement of endogenous phospholipase A2 inhibitors (lipocortins) and their regulation by C-kinase will be studied. And finally, the role of N- proteins will be investigated by using guanine nucleotides and pertussis toxin in permeabilized platelets and studying the effect of purified N-protein subunits on platelet phospholipase A2. Possible interactions among the different signal transducers (Ca2+, C-Kinase, N-proteins) for control of arachidonic acid release will be examined. The goals of this project and future studies are: (1) to identify the cellular factors which control the liberation of arachidonic acid, the precursor for prostaglandins, thromboxanes, and other biologically important substances; and (2) more broadly, to define the intra- and intercellular signalling pathways that regulate platelet aggregation and secretion, as these events are centrally involved in hemostasis and thrombosis.
