Physical and functional association of PECAM-1 and Fc RIIa on the platelet plasma membrane

We and others have recently defined that Platelet Endothelial Cell Adhesion Molecule- 1 (PECAM-1/CD31) functions as a negative regulator of platelet-collagen interactions involving the glycoprotein VI/Fc receptor gamma chain (GPVI/FcR-y chain) signaling pathway.1,2 In this study, we hypothesized that PECAM-1 may be physically and functionally associated with FcyRIIa on the platelet membrane. The functional relationship between PECAM-1 and FcyRIIa was assessed by determining the effect of anti-PECAM-1 monoclonal antibody Fab fragments on FcyRIIa-mediated platelet aggregation and heparin-induced thrombocytopenia (HITS)-mediated platelet aggregation. Preincubation of washed platelets with monoclonal antibody fragments of 2BD4 directed against PECAM-1 and IV.3 directed against FcyRIIa completely blocked FcyRIIa-mediated platelet aggregation and HITS-mediated platelet aggregation, whereas anti- CD151 antibody had no blocking effect. Coengagement of FcyRIIa and PECAM-1 resulted in negative regulation of FcyRIIamediated phospholipase Cy2 activation, calcium mobilization, and phosphoinositide 3-kinase-dependent signaling pathways. In addition, the physical proximity of FcyRIIa and PECAM-1 was confirmed by using fluorescence resonance energy transfer and coimmunoprecipitation studies. These results indicate that PECAM-1 and FcyRIIa are colocalized on the platelet membrane and PECAM-1 down-regulates FcyRIIa-mediated platelet responses.