Integrin alphaIIbeta3 mediates two major functions in platelets: the activated form binds adhesive ligand and promotes platelet aggregation, while the liganded form triggers biochemical reactions required for cytoskeletal reorganization and platelet spreading. The biochemical pathways responsible for alphaIIbeta3 activation and integrin-mediated signaling are poorly characterized. The central hypothesis of this proposal is that specific proteins interact directly with the short cytoplasmic tails of alphaIIbeta3 to regulate the binding and signaling fucntions of the integrin.
One specific aim i s to establish the role of the Rho family of GTPases in alphaIIbeta3 activation. Therefore, constitutively-active or dominant-negative inhibitory formsCdc42, Rac and Rho will be incorporated into permeailized platelets and their effects on fibrinogen receptor function measured using flow cytometric assays that are capable of monitoring alphaIIbeta3 activation and integrin clustering. The second specific aim is to determine the molecular basis for the earliest signaling reactions triggered by integrin ligation. The protein tyrosine kinase, pp72Syk, becomes activated immediately upon binding of fibrinogen to alphaIIbeta3. Therefore, constitutively-active or dominant-negative inhibitory forms of Cdc42, Rac and Rho will be incorporated into permeabilized platelets and their effects on fibrinogen receptor function measured using flow cytometric assays that are capable of monitoring alphaIIbeta3 activation and integrin clustering. The second specific aim is to determine the molecular basis for the earliest signaling reactions triggered by integrin ligation. The protein tyrosine kinase, pp72Syk, becomes activated immediately upon binding of fibriogen to alphaIIbeta3. Therefore, immunoprecipitation, immunoblotting and in vitro assays will be carried out to establish whether Src family kinases are involved in Syk activation in platelets and whether other molecules, such as p52Shc or p140-145SHIP, are members of the nascent integrin signaling unit. Transient transfection experiments in CHO cells will attempt to recapitulate the pattern of integrin-mediated tyrosine phosphorylation in platelets. A third specific aim is to focus directly on the roles of th cytoplasmic tails of aplhaIIbeta and beta3. Synthetic peptides derived from these tails will be introduced into permeabilized platelets to examine their effects on agonist-induced integrin activation. Cytoplasmic tail peptides will also be derivatized, and chemical cross- linking strategies will be employed using permeabilized platelets to identify proteins that interact with the tails in situ. In addition, since recent evidence indicates that the beta3 cytoplasmic tail may become tyrosine-phosphorylated in activated platelets, a yeast two-hybrid system will be modified to screen for human proteins that interact selectively with the tyrosine-phosphorylated beta3 tail. Taken together, these studies should identify and characterize those platelet proteins that regulate the adhesive and signaling functions of alphaIIbeta3.
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