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 two G-Protein Coupled Receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor (P2Y12R). While the involvement of the P2Y1R 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, i.e., second extracellular loop (EL2) of the P2Y1R [abbreviated as 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 alpha granule secretion and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3 induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose dependent displacement of the radiolabelled P2Y1R antagonist [3H]MRS25000 from its ligand binding site by EL2Ab. Conclusions Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies.

Investigation of a Thromboxane A 2 Receptor–Based Vaccine for Managing Thrombogenesis

“…Mouse platelets were prepared, as discussed previously 42, 43. Briefly, mouse blood was collected from a ventricle, and the citrated (0.38%) blood was mixed with PBS, pH 7.4, and was incubated with prostaglandin I 2 (10 ng/mL; 5 minutes), followed by centrifugation at 237 g for 10 minutes at room temperature.…”

“…Platelets (with counts adjusted, as previously described42, 43, 44) were stimulated with 1 μmol/L U46619, 5 μmol/L ADP, or 80 μmol/L TRAP4, and aggregation was measured. Each experiment was repeated at least 3 times, with blood pooled from at least 3 separate groups of 6 to 8 immunized mice.…”

“…These experiments were performed, as we previously described 42, 43, 44. Platelets from vaccinated mice (C‐EL2, C‐EL2r, and KLH) were prepared, as previously described (250 μL; 2.5×10 8 /mL), before being placed into siliconized cuvettes and stirred for 5 minutes at 37°C at 237 × g .…”

“…Flow cytometric analysis was performed on platelets from vaccinated mice (C‐EL2, C‐EL2r, and KLH), as previously discussed 42, 43, 44. Briefly, platelets (2×10 8 ) were fixed with 2% formaldehyde for 30 minutes at room temperature, before incubation with anti‐mouse glycoprotein IIb‐IIIa, anti–glycoprotein Ib, or anti–glycoprotein VI antibodies at room temperature for 30 minutes in the dark.…”

“…Flow cytometric analysis was performed on platelets from vaccinated mice (C‐EL2, C‐EL2r, and KLH), as previously discussed 42, 43, 44. Briefly, platelets (2×10 8 ) were stimulated with 1 μmol/L U46619, 5 μmol/L ADP, or 80 μmol/L TRAP4 for 3 minutes.…”