The objective of this proposal is to delineate the mechanisms by which stimuli activate platelets and to determine how agents that increase cAMP (e.g. prostacyclin) inhibit platelet responses to stimulation. Various physiological stimulants (thrombin, collagen, ADP, platelet activating factor) cause platelets to change shape, secrete and aggregate. These responses enable the platelet to fulfill its important role in hemostasis, and may also contribute to thrombosis and atherosclerosis. Ca2+ is vitally involved in enabling or facilitating the biochemical reactions necessary for platelet responses, whereas cAMP opposes all platelet responses. cAMP blocks mobilization of Ca2+ (measured with Quin2) by stimuli, and also inhibits the hydrolysis of phosphatidylinositol 4,5-bisphosphate that generates two important substances; 1,2-diacylglycerol (DG) and inositol 1,4,5-trisphosphate (IP3). DG acts cooperatively with Ca2+ mobilized by ionophores to facilitate protein phosphorylation and secretion. In this project the role of DG in the responses of intact platelets to thrombin, and how its production and actions are affected by physiological stimulants of adenylate cyclase (e.g. prostacylin) will be studied. IP3 is thought to be mediate hormone-induced mobilization of Ca2+ in several cells. In isolated platelet membrane vesicles that have an ATP-dependent pump to accumulate Ca2+ we find that IP3 released Ca2+ providing the first evidence that IP3 may be the second messenger for intracellular Ca2+ mobilization in platelets. This effect of IP3 will be studied in detail to determine if IP3 mobilizes Ca2+ in permeabilized intact platelets, to identify the membrane site(s) and mechanism of action, and whether cAMP affects the action of IP3. The temporal and quantitative relationships between formation of IP3 and Ca2+ mobilization will be studied in control and prostacyclin-treated intact platelets to determine if IP3 can be the second messenger linking stimulation of surface membrane receptors to internal cellular responses. In thrombin-treated platelets the inhibitory action of prostacyclin is antagonized by epinephrine and ADP, thereby affecting Ca2+ mobilization and lipid metabolism. Epinephrine and ADP act through a GTP-binding protein (Ni) that inhibits adenylate cyclase, but which may have other important effects as well. This project shall study the mechanism(s) of action of epinephrine and ADP on Ca2+ mobilization, DG and IP3 formation, and DG and IP3 actions, because these agents may importantly influence the antithrombotic action of prostacyclin.
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