Directly Activating the Integrin αIIbβ3 Initiates Outside-In Signaling by Causing αIIbβ3 Clustering

alpha IIb beta 3 activation in platelets is followed by activation of the tyrosine kinase c-Src associated with the carboxyl terminus of the beta 3 cytosolic tail. Exogenous peptides designed to interact with the alpha IIb transmembrane (TM) domain activate single alpha IIb beta 3 molecules in platelets by binding to the alpha IIb TM domain and causing separation of the alpha IIb beta 3 TM domain heterodimer. Here we asked whether directly activating single alpha IIb beta 3 molecules in platelets using the designed peptide anti-alpha IIb TM also initiates alpha IIb beta 3-mediated outside-in signaling by causing activation of beta 3-associated c-Src. Anti-alpha IIb TM caused activation of beta 3-associated c-Src and the kinase Syk, but not the kinase FAK, under conditions that precluded extracellular ligand binding to alpha IIb beta 3. c-Src and Syk are activated by trans-autophosphorylation, suggesting that activation of individual alpha IIb beta 3 molecules can initiate alpha IIb beta 3 clustering in the absence of ligand binding. Consistent with this possibility, incubating platelets with anti-alpha IIb TM resulted in the redistribution of alpha IIb beta 3 from a homogenous ring located at the periphery of discoid platelets into nodular densities consistent with clustered alpha IIb beta 3. Thus, these studies indicate that not only is resting alpha IIb beta 3 poised to undergo a conformational change that exposes its ligand-binding site, but it is poised to rapidly assemble into intracellular signal-generating complexes as well.