This proposal aims at elucidating the biochemical mechanism responsible for the release of arachidonate in human platelets. It is proposed that thrombin or collagen turns on phosphatidylinositol metabolism through the phospholipae C pathway and activates a calcium activated, diglyceride and phospholipid dependent protein kinase which catalyzes the phosphorylation of a 40K protein. This protein appears to be a phospholipase A2 inhibitory protein which is inactivated and dissociated from phospholipase A2 following phosphorylation thereby relieving phospholipase A2 from inhibition. The consequence is to allow phospholipase A2 to catalyze the release of arachidonic acid from various phospholipids. The role of phospholipase C and lipases pathway in releasing arachidonate is to provide the earliest molecules of arachidonate and metabolites to augment the diglyceride formation and stimulatory process. Therefore, we plan to isolate the key enzyme, phospholipase C, in a homogeneous state and to study its regulatory properties. We will also produce monoclonal antibodies to localize this enzyme during platelet activation using immunocytochemical techniques. Furthermore, we will examine how 40 K protein phosphorylation can be related to arachidonate release and provide some evidences that this protein is an endogenous phospholipase A2 inhibitory protein and its inhibitory activity is regulated by phosphorylation. The result of this research program will provide a basic understanding of the platelet activation process and may lead to devising new ways to control platelet functions and disorders.
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