Characterization of a Novel Function-Blocking Antibody Targeted Against the Platelet P2Y 1 Receptor

Abstract

Objective— Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation and activates platelets through 2 G-protein–coupled receptors, the Gq-coupled P2Y 1 receptor (P2Y 1 R), and the Gi-coupled P2Y 12 receptor. Although the involvement of the P2Y 1 R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use. Approach and Results— Our goal is to determine whether a novel antibody targeting the ligand-binding domain, ie, second extracellular loop (EL2) of the P2Y 1 R (EL2Ab) could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and α granule secretion, and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl 3 -induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose-dependent displacement of the radiolabeled P2Y 1 R antagonist [ 3 H]MRS2500 from its ligand-binding site by EL2Ab. Conclusions— Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y 1 R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y 1 R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies.