The objective of this research proposal is to understand the role of vinculin in platelet activation and microfilament organization. Our working hypothesis is that vinculin exist in several forms and following platelet activation one or more of these forms is modified to allow its interaction with actin, other actin associated proteins, or the plasma membrane. To test this hypothesis we plan to isolate cytoskeletal and membrane fractions from unactivated and activated platelets and probe for vinculin's distribution and localization by immunoblot analysis. We also will study the in vitro interaction of the soluble platelet vinculin we have purified with actin and actin binding proteins using viscometric, fluorometric, sedimentation, affinity matrix and gel overlay analyses. Further, we will study the effects of calcium and phosphorylation on the distribution and activity of vinculin in unactivated, activated, adherent, and aggregated platelets. These studies should provide fundamental information on the role of vinculin in cytoskeletal organization and also in platelet activation.
