Stimulation of platelets results in the activation of phospholipases which initiate the subsequent intracellular synthesis of bioactive metabolites such as eicosanoids and platelet activating factor. Vinyl ether and alkyl ether phospholipids are the major storage depots for archidonic acid in platelets. Furthermore, alkyl ether choline glycerophospholipid is the chemical precursor of platelet activating factor. Several lines of evidence suggest that vinyl ether and alkyl ether phospholipid classes are specific substrates for phospholipases which are activated after cell stimulation. However, the existence of such enzymes or the mechanism of their activation after cell stimulation has not been directly demonstrated. Recently we have identified a platelet phospholipase A2 whose activity is markedly stimulated by thrombin utilizing alkyl ether and vinyl ether but not diacyl phospholipid substrates. Furthermore, we have demonstrated that physiologic increments of Ca2+ ion (2 MuM) activate platelet phospholipase A2 activity utilizing plasmalogen but not diacyl phospholipid substrates. Thus enzymes which catalyze these activities are likely responsible, at least in part, for the selective release of arachidonic acid and the synthesis of platelet activating factor after platelet stimulation. The first objective of the proposed research is the identification of the molecular mechanism through which thrombin activates platelet phospholipase A2 activity resulting in the selective hydrolysis of phospholipid classes which are highly enriched in arachidonic acid. The second objective of the proposed research is the determination of the molecular mechanism which underlies the 100 fold difference in the calcium sensitivity of platelet phospholipase A2 toward plasmalogen and diacyl phospholipid substrates and delineation of its physiologic significance in the selective hydrolysis of arachidonic acid after cell stimulation. The third objective of the proposed research is the purification and characterization of platelet alkyl ether specific transacylase activity and identification of endogenous regulatory factors which modulate its activity. Taken together, these studies will identify the biochemical mechanisms responsible for the activation of platelet class specific phospholipase A2 and transacylase activities which likely regulate the production of biologically active eicosanoic and platelet activating factor. The identification and characterization of the enzyme systems which regulate their production should facilitate the pharmacologic manipulation of several pathologic processes such as thrombosis, atherosclerosis and inflammation.
