Bidirectional transmembrane modulation of integrin α(IIb)β3 conformations

Abstract

Activation of blood platelets by physiological stimuli (e.g. thrombin, ADP) at sites of vascular injury induces inside-out signaling, resulting in a conformational change of the prototype integrin α(IIb)β3 from an inactive to an active state competent to bind soluble fibrinogen. Furthermore, ligand occupancy of α(IIb)β3 initiates outside-in signaling and additional conformational changes of the receptor, leading to the exposure of extracellular neoepitopes termed ligand-induced binding sites (LIBS), which are recognized by anti-LIBS monoclonal antibodies. To date, the mechanism of bidirectional transmembrane signaling of α(IIb)β3 has not been established. In this study, using our newly developed anti-LIBS(cyt)1 monoclonal antibody, we showed that extracellular ligand binding to α(IIb)β3 on blood platelets induces a transmembrane conformational change in α(IIb)β3, thereby exposing the LIBS(cyt)1 epitope in the α(IIb) cytoplasmic sequence between Lys994 and Asp1003. In addition, a point mutation at this site (P998A/P999A) renders α(IIb)β3 constitutively active to bind extracellular ligands, resulting in fibrinogen-dependent cell-cell aggregation. Taken collectively, these results demonstrated that the extracellular ligand-binding site and a cytoplasmic LIBS epitope in integrin α(IIb)β3 are conformationally and functionally coupled. Such bidirectional modulation of α(IIb)β3 conformation across the cell membrane may play a key role in inside-out and outside-in signaling via this integrin.