Association Between Ligand-Induced Conformational Changes of Integrin αIIbβ3 and αIIbβ3-Mediated Intracellular Ca2+ Signaling

Abstract

AbstractPlatelet αIIbβ3 is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of αIIbβ3 with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and αIIbβ3 clustering, which are thought to be necessary for outside-in signaling. However, the association between LIBS expression and outside-in signaling remains elusive. In this study, we used various αIIbβ3-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIBS expression and outside-in signaling such as αIIbβ3-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIBS, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIBS on both αIIb and β3 subunits. In group II, antagonists can induce LIBS on the αIIb subunit, but not on the β3 subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on αIIbβ3. Neither group I nor group II antagonist increased intracellular Ca2+concentrations ([Ca2+]i) in nonactivated platelets. All antagonists at nanomolar concentrations abolished the increase in [Ca2+]i in 0.03 U/mL thrombin-stimulated platelets, which is dependent on both fibrinogen-binding to αIIbβ3 and platelet-aggregation. However, only group I antagonists at higher concentrations dose-dependently augmented the [Ca2+]i increase, which is due to aggregation-independent thromboxane A2 production. This increase in [Ca2+]i was not observed in thrombasthenic platelets, which express no detectable αIIbβ3. Thus, only the group I antagonists, albeit a monovalent ligand, can initiate αIIbβ3-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between β3LIBS expression and αIIbβ3-mediated intracellular Ca2+ signaling in platelets.