This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Healthy volunteers will be used for the studies. Blood will be drawn and platelets isolated. Experiments to assess the role of calpain, spectrin, and skelemin will involve inhibiting specific pathways and interactions within platelets and assessing the functional conseqences. Pathways will be inhibited with specific pharmacological inhibitors and by the introduction of palmitylated peptides. The latter peptides will be designed to ablate interactions of interest, such as those between spectrin SH3 domain and identified binding partners, that between skelemin and integrin, or that between calpain and identified binding partners. Consequence of ablating specific signaling molecules or interactions will be assessed by meausuring the ability of the integrin aIIbb3 to be activated, the ability of platelets to adhere or the ability of platelets to spread. Detailed effects of inhibition will be assessed by immnofluorescent studies identifying the redistribution of integrin, actin filaments, spectrin, calpain and skelemin in spreading cells. In other studies, platelets will be used for assays of specific signaling molecules such as calpain, Rac, cdc42. Isolation of purified proteins from platelets will be performed for in vitro binding assays, raising antibodies, and for in vitro assays identifying calpain cleavage products.
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