FcγRIIa and the Integrin β3 Cytoplasmic Domain: Two Prerequisites for Thrombocytopenia and Thrombosis Following Administration of the Fibrinogen Receptor Antagonist, Eptifibatide.

Thrombocytopenia and thrombosis following treatment with the glycoprotein αIibβ3 antagonist, eptifibatide, are rare complications that are thought to be caused by patient antibodies specific for ligand-occupied αIibβ3. Whether such antibodies induce platelet clearance by simple opsonization, by inducing mild platelet activation, or both, is not well understood. To gain insight into the mechanism by which eptifibatide-dependent antibodies cause platelet clearance, we incubated normal platelets with serum derived from a 73 year old patient who developed profound thrombocytopenia following eptifibatide administration. This patient’s serum was found to contain an αIibβ3-specific eptifibatide-dependent antibody that induced significant platelet secretion and aggregation in the presence, but not in the absence, of eptifibatide. Interestingly, a number of signaling molecules recently shown to function downstream of integrin engagement, including the integrin β3 cytoplasmic domain, the protein-tyrosine kinase Syk, and phospholipase Cγ2 (PLCγ2) each became tyrosine phosphorylated in platelets subjected to patient serum plus eptifibatide. Because the Immunoreceptor Tyrosine-based Activation Motif (ITAM)-bearing platelet Fc receptor, FcγRIIa, plays a prominent role in antibody-induced platelet activation, we examined its role in eptifibatide-induced platelet activation and clearance. We found that (1) FcγRIIa became rapidly and strongly tyrosine phosphorylated following incubation of normal platelets with patient serum+eptifibatide and (2) pre-incubation of platelets with Fab fragments of the FcγRIIa-specific monoclonal antibody, IV.3, completely blocked both phosphorylation of FcγRIIa, Syk, and PLCγ2, as well as platelet secretion and aggregation induced by patient serum+eptifibatide. Because amounts of patient sera are often limiting, a model system comprised of the αIibβ3 complex-specific mAb, AP2, and the ligand mimetic peptide, RGDW was employed, and found to faithfully mimic these reactions. Thus, intact, but not Fab fragments of, AP2 caused tyrosine phosphorylation of FcγRIIa, Syk, PLCγ2, as well as platelet secretion and aggregation, and these reactions could be completely blocked with Fab fragments of mAb IV.3, further implicating FcγRIIa in eptifibatide-induced thrombocytopenia/thrombosis. Finally, to gain further insight into how integrins functionally link to FcγRIIa to activate platelets, we subjected platelets from a patient with a variant form of Glanzmann thrombasthenia whose platelets express normal levels of αIibβ3 and FcγRIIa, but lack most of the β3 cytoplasmic domain, to similar treatment. Neither patient serum+eptifibatide nor AP2+RGDW were able to activate this patient’s platelets, implicating the β3 cytoplasmic domain and its associated tyrosine kinases in eptifibatide-induced platelet activation. Taken together, these data suggest a novel mechanism involving both FcγRIIa and the cytoplasmic domain of integrin β3 in thrombocytopenia and thrombosis following administration of fibrinogen receptor antagonists.