Association between ligand-induced conformational changes of integrin αIIbβ3 and αIIbβ3-mediated intracellular Ca2+ signaling

Platelet alpha(IIb)beta(3) is a prototypic integrin and plays a critical role in platelet aggregation. Occupancy of alpha(IIb)beta(3) with multivalent RGD ligands, such as fibrinogen, induces both expression of ligand-induced binding sites (LIBS) and alpha(IIb)beta(3) clustering, which are thought to be necessary for outside-in signaling. However, the association between LIES expression and outside-in signaling remains elusive. In this study, we used various alpha(IIb)beta(3)-specific peptidomimetic compounds as a monovalent ligand instead of fibrinogen and examined the association between LIES expression and outside-in signaling such as alpha(IIb)beta(3)-mediated intracellular Ca2+ signaling. Using a set of monoclonal antibodies (MoAbs) against LIES, we showed that antagonists can be divided into two groups. In group I, antagonists can induce LIES on both alpha(IIb) and beta(3) subunits. In group II, antagonists can induce LIES on the alpha(IIb) subunit, but not on the beta(3) subunit. Inhibition studies suggested that group I and group II antagonists interact with distinct but mutually exclusive sites on alpha(IIb)beta(3) 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 alpha(IIb)beta(3) 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 A(2) production. This increase in [Ca2+](i) was not observed in thrombasthenic platelets, which express no detectable alpha(IIb)beta(3) Thus, only the group I antagonists, albeit a monovalent ligand, can initiate alpha(IIb)beta(3)-mediated intracellular Ca2+ signaling in the presence of thrombin stimulation. Our findings strongly suggest the association between beta(3) LIES expression and alpha(IIb)beta(3)-mediated intracellular Ca2+ signaling in platelets. (C) 1998 by The American Society of Hematology.