Platelet activation at sites of vascular injury involves a signaling cascade that includes G proteins, phospholipases, and a variety of secondary messengers. Upon stimulation by thrombin, the subsequent activation of protein kinase C causes the immediate phosphorylation of a 40-47 kDa protein, called pleckstrin. The physiologic role for pleckstrin, which is the principal substrate for protein kinase C in activated platelets, has been an elusive goal for platelet biologists. The cloned sequence for pleckstrin contains two so-called """"""""pleckstrin homology domain"""""""" that may direct protein:protein interactions, analogous to other protein targeting motifs such as SH2 or SH3. The pleckstrin homology domain is found in a variety of proteins that have roles in signal transduction or cytoskeletal function, including proteins that associate with G proteins and ras. Our preliminary data, that form the baseline of this project, demonstrate a direct association between the pleckstrin homology motif and the G-beta-gamma subunit of heterotrimeric G proteins. The major focus will be to identify the role of pleckstrin, and pleckstrin homology domains, in platelet activation and vascular biology. Particular emphasis will be placed on the regulation of GTP binding proteins. Our specific scientific goals are to: (I) To further establish and more fully characterize the interaction between pleckstrin and G proteins, 2) To determine whether pleckstrin associates with low molecular weight GTP binding proteins, (3) To analyze the regulation of pleckstrin in platelets.
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