Identification of FcγRIIa as the ITAM-bearing receptor mediating αIIbβ3 outside-in integrin signaling in human platelets

Immunoreceptor tyrosine-based activation motif (ITAM)-containing proteins have recently been demonstrated in macrophages and neutrophils to be required for cell surface integrins to transmit activation signals into the cell. To identify ITAM-bearing proteins that mediate signaling via the platelet-specific integrin alpha IIb beta 3, fibrinogen binding was induced by (1) allowing platelets to spread directly on immobilized fibrinogen, or (2) activating the PAR1 thrombin receptor on platelets in suspension. Both initiated strong, ligand binding-dependent tyrosine phosphorylation of the ITAM-bearing platelet Fc receptor, Fc gamma RIIa, as well as downstream phosphorylation of the protein tyrosine kinase Syk and activation of phospholipase C gamma 2 (PLC gamma 2). Addition of Fab fragments of an Fc gamma RIIa-specific monoclonal antibody strongly inhibited platelet spreading on immobilized fibrinogen, as well as downstream tyrosine phosphorylation of Fc gamma RIIa, Syk, and PLC gamma 2, and platelets from a patient whose platelets express reduced levels of Fc gamma RIIa exhibited markedly reduced spreading on immobilized fibrinogen. Finally, fibrinogen binding-induced Fc gamma RIIa phosphorylation did not occur in human platelets expressing a truncated beta 3 cytoplasmic domain. Taken together, these data suggest that ligand binding to platelet alpha IIb beta 3 induces integrin cytoplasmic domain dependent phosphorylation of Fc gamma RIIa, which then enlists selected components of the immunoreceptor signaling cascade to transmit amplification signals into the cell.