Concurrent signaling from Gαq- and Gαi-coupled pathways is essential for agonist-induced αvβ3 activation on human platelets

Summary.  The integrin αvβ3 mediates platelet adhesion to the matrix protein osteopontin and likely is the predominant integrin mediating platelet adhesion to the matrix protein vitronectin. To address the mechanism that regulates αvβ3 activity in platelets, we measured the effect of the P2Y₁ antagonist adenosine 3'-phosphate-5'-phosphate (A3P5P) and the P2Y₁₂ antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y₁ and P2Y₁₂ was required to activate αvβ3. The reducing agent dithiothreitol and Mn²⁺ also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates αvβ3 activity by perturbing the conformation of its extracellular domain. The actin polymerization inhibitors cytochalasin D and latrunculin A also induced platelet adhesion to osteopontin and vitronectin. Thus, αvβ3 activity in resting platelets appears to be constrained by the platelet cytoskeleton. Moreover, the effect of these agents was inhibited by A3P5P and AR-C66096 at micromolar and subnanomolar concentrations, respectively, suggesting that subthreshold platelet stimulation by ADP was required. Our data suggest that signals from both Gα_q- and Gα_i-coupled receptors converge to release cytoskeletal constraints on αvβ3. We propose that the release of cytoskeletal constraints and a concurrent increase in affinity for ligands is responsible for αvβ3-mediated platelet adhesion.