Activating Individual αIIbβ3 Molecules Using An Anti-αIIb-Transmembrane Domain Peptide Causes αIIbβ3 Oligomerization and αIIbβ3-Mediated Outside-In Signaling

Activating αIIbβ3 in platelets is followed by αIIbβ3 clustering and αIIbβ3-mediated outside-in signaling. However, the molecular bases for the latter two processes are not well understood. It has been found that activating αIIbβ3 in platelets is followed by trans-autoactivation of the tyrosine kinase c-Src constitutively associated with the β3 cytosolic tail. In turn, activated c-Src initiates a signaling cascade that culminates in the re-organization of the platelet cytoskeleton, platelet spreading, and fibrin clot retraction. Previously, we observed that replacing Gly708 in the β3 transmembrane helix with Asn causes constitutive αIIbβ3 activation, as well as ligand binding-independent αIIbβ3 clustering when the β3 mutant is expressed in CHO cells. Here, we have asked whether activating αIIbβ3 in platelets in the absence of ligand binding is sufficient to cause c-Src activation. Exogenous peptides that interact with the membrane-embedded segments of αIIbβ3, such as the CHAMP (Computed Helical Anti-Membrane Protein) peptide anti-αIIb TM, cause agonist-independent activation of single αIIbβ3 molecules. To test whether anti-αIIb TM causes c-Src activation in the absence of ligand binding to αIIbβ3, we suspended gel-filtered human platelets in buffer containing an inhibitory cocktail of EDTA, apyrase, and eptifibatide that completely prevents anti-αIIb TM-stimulated platelet aggregation as well as FITC-labeled fibrinogen binding to αIIbβ3 and extracted aliquots of the stimulated platelets with 1% NP-40 in a buffer containing a cocktail of protease and phosphatase inhibitors. c-Src was immunoprecipitated from the extracts and immunoblotted for phosphorylation of c-Src tyrosine residue 416, an indicator of c-Src activation. We found that stimulating platelets with anti-αIIb TM caused Tyr416 phosphorylation. This effect required the presence of αIIbβ3 because it did not occur when β3-knockout mouse platelets were incubated with the peptide. Further, Tyr416 phosphorylation was prevented by the Src-family kinase inhibitor PP2, but not by the inactive analogue PP3, implying that it resulted from the trans-autophosphorylation of β3-associated c-Src. Tyr416 phosphorylation was not dependent on an anti-αIIb TM-induced rearrangement of the platelet cytoskeleton because it was not inhibited by the actin polymerization inhibitors cytochalasin D and latrunculin A. Similar results were seen when immunoprecipitated β3 was immunoblotted for phosphorylated Tyr416. The tyrosine kinase Syk is also associated with the β3 cytosolic tail. Like c-Src, we found that incubating platelets with anti-αIIb TM caused Syk phosphorylation that was independent of ligand binding to αIIbβ3, was catalyzed by activated c-Src because it was inhibited by PP2 but not PP3, and like c-Src trans-autophosphorylation, was unaffected by cytochalasin D and latrunculin A. By contrast, we found that anti-αIIb TM-induced αIIbβ3 activation was not sufficient to cause phosphorylation of FAK when ligand binding to αIIbβ3 was prevented by EDTA, apyrase, and eptifibatide. Anti-αIIb TM-induced c-Src activation in the absence of ligand binding to αIIbβ3 implies that the peptide causes ligand-binding-independent αIIbβ3 clustering. To demonstrate that this is the case, we incubated suspended CHO cells expressing wild-type αIIbβ3 with anti-αIIb TM, fixed the cells with formalin, and stained the cells with the FITC-labeled αIIbβ3-specific monoclonal antibody A2A9. Like CHO cells expressing β3 Gly708Asn, the αIIbβ3 of CHO cells incubated with anti-αIIb TM was present in patches distributed over the cell surface, consistent with the formation of αIIbβ3 clusters. By contrast, in the absence of anti-αIIb TM, αIIbβ3 was present in a homogenous ring at the CHO cell periphery. These studies demonstrate that the CHAMP peptide anti-αIIb TM can activate β3-associated c-Src and Syk under conditions to preclude ligand binding to αIIbβ3. This indicates that activation of individual αIIbβ3 molecules can initiate αIIbβ3 oligomerization in the absence of ligand binding and cytoskeletal reorganization. Thus, these studies suggest that not only is resting αIIbβ3 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.