Platelet stimulation results in the activation of phospholipases which initiate the subsequent intracellular synthesis of bioactive metabolites such as eicosanoids and platelet activating factor. During the current grant interval we have purified a novel phospholipase A2 which is completely activated by physiologic increments in calcium ion concentration (800nM) and has a substrate specificity which is anticipated to result in the highly selective release of arachidonic acid and initiate the synthesis of PAF. During the course of these experiments we have also demonstrated the presence of latent calcium independent phospholipase A2 and transacylase activities. The overall goal of the proposed research is the identification of the biochemical mechanisms which regulate the activities of these enzymes and elucidation of the chemical interactions which underlie their remarkable kinetic properties. The first objective of the proposed research is the purification and characterization of calcium independent phospholipase A2 and transacylase activities. Attention will subsequently focus on the biochemical mechanisms responsible for expression of latent enzymic activity. Next, the possibility that calcium dependent phospholipase A2 is activated by intracellular translocation, isoform interconversion or specific phospholipase A2-protein interactions will be explored. The primary sequence of this calcium dependent phospholipase A2 will be delineated by molecular cloning of this enzyme and the specific chemical interactions responsible for its catalytic activity, calcium sensitivity and substrate specificity will be delineated by site directed mutagenesis. The complete sequence of the human gene(s) encoding calcium dependent phospholipase A2 will be determined. The contribution of platelet plasmenylethanolamine to arachidonic acid release during platelet stimulation will be determined utilizing novel radiolabels we have synthesized. Taken together, the proposed studies will provide new information on the regulation of key enzymes which mediate the release of arachidonic acid during platelet stimulation and are important modulators of such diverse processes as thrombosis, atherosclerosis and inflammation.
