Platelets play an important role in hemostasis, thrombosis, inflammation, and probably in atherosclerosis. In response to aggregating agents such as thrombin or collagen, platelets produce a large number of biologically potent metabolites from arachidonic acid. These metabolites include PGG2, PGH2, thromboxane A2, PGE2, PGD2 and PGF2Alpha, which are associated with hemostasis, thrombosis and the inflammatory process. Arachidonic acid is stored in the cell membrane in esterified form almost exclusively at the 2-position of phospholipids. The cellular level of free arachidonate is low; it is released from the phospholipids primarily via the action of a phospholipase A2 when the cell is stimulated. Thus, phospholipase A2 plays a pivotal role in certain physiological and pathological processes. Yet relatively little is known about the enzyme in platelets or other cell types. In this proposal, we will use human platelets which are easily available in homogeneous and bulk quantities to study the factors regulating the mobilization of arachidonate. Our specific aims are to purify and characterize the multiple forms of phospholipase A2 with emphasis on their regulatory properties, subcellular localization and certain pharmacological and physiological agents. The methods to be used include various purification techniques such as affinity and immunoadsorbant column chromatography, enzymological analysis, and subcellular and immunocytochemical localization. Information gathered from these studies will shed light on the mechanisms controlling the release of free arachidonic acid, which serves as the precursor of all eicosanoids, agents that play important roles in platelet functions.