FcγRIIa Is Required for Outside-In β3 Integrin Signaling in Human Platelets.

ITAMs (Immunoreceptor Tyrosine-based Activation Motifs) are signaling motifs that are found within the cytoplasmic domains of T and B cell antigen receptors, and in the Fc receptor γ (FcRγ) chain that is associated with Fc receptors for IgG and IgE and with the GPVI collagen receptor on platelets. ITAMs are also an intrinsic component of the cytoplasmic domain of the platelet and leukocyte low affinity receptor for IgG, FcγRIIa. When ITAM-bearing receptors are engaged or cross-linked, ITAM tyrosines undergo Src-dependent phosphorylation, providing a docking site for the protein-tyrosine kinase, Syk. Activation of Syk results in assembly of a multi-protein signaling complex containing the adaptor molecule SLP-76, ultimately resulting in the activation of phospholipase Cγ2 (PLCγ2), which via its lipase activity initiates a multitude of cellular activation responses. Recent studies in platelets and neutrophils have found that ITAM-mediated signaling cascades also play a novel and unexpected role in outside-in integrin signaling, as cells containing mutant forms of Syk, SLP-76, or PLCγ2 fail to become activated normally following integrin engagement. Furthermore, the FcRγ chain and DAP12 have been identified as ITAM-bearing subunits required for integrin signaling in neutrophils and macrophages, and we and others have reported that the GPVI/FcRγ chain complex can function as a weak integrin amplifier in human platelets. Nevertheless, the complement of ITAM-bearing receptors in platelets that are involved in integrin signaling in platelets is not known. To identify candidate receptors that might couple integrins to ITAM signaling, we initiated fibrinogen (Fg) binding to the integrin αIIbβ3 by

1. activating the thrombin receptor, PAR1, on platelets in suspension, or

2. by allowing platelets to spread directly on immobilized Fg.

Interestingly, both forms of platelet activation initiated strong, ligand binding-dependent tyrosine phosphorylation of FcγRIIa, as well as downstream phosphorylation of Syk and PLCγ2. Importantly, prior addition of Fab fragments of the FcγRIIa blocking monoclonal antibody, IV.3, inhibited platelet spreading on immobilized Fg, as well as downstream tyrosine phosphorylation of FcγRIIa, Syk, and PLCγ2. Finally, platelets from a patient with a mild bleeding disorder whose platelets express only 30% of the normal level of FcγRIIa exhibit markedly reduced spreading on immobilized Fg. Taken together, these studies identify FcγRIIa as the major ITAM-bearing receptor in human platelets that supports outside-in integrin signaling.