The platelet P2Y12 receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [3H]PSB-0413

Various radioligands have been used to characterize and quantify the platelet P2Y(12) receptor, which share several weaknesses: (a) they are metabolically unstable and substrates for ectoenzymes, (b) they are agonists, and (c) they do not discriminate between P2Y(1) and P2Y(12). We used the [H-3]PSB-0413 selective P2Y(12) receptor antagonist radioligand to reevaluate the number of P2Y(12) receptors in intact platelets and in membrane preparations. Studies in humans showed that: (1) [H-3]PSB-0413 bound to 425 +/- 50 sites/platelet (K (D) = 3.3 +/- 0.6 nM), (2) 0.5 +/- 0.2 pmol [H-3]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 6.5 +/- 3.6 nM), and (3) competition studies confirmed the known features of P2Y(12), with the expected rank order of potency: AR-C69931MX > 2MeSADP a parts per thousand << aEuro parts per thousand ADP beta S > ADP, while the P2Y(1) ligand MRS2179 and the P2X(1) ligand alpha,beta-Met-ATP did not displace [H-3]PSB-0413 binding. Patients with severe P2Y(12) deficiency displayed virtually no binding of [H-3]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y(12) receptor had normal binding. Studies in mice showed that: (1) [H-3]PSB-0413 bound to 634 +/- 87 sites/platelet (K (D) = 14 +/- 4.5 nM) and (2) 0.7 pmol +/- 0.3 [H-3]PSB-0413 bound to 1 mg protein of platelet membranes (K (D) = 9.1 +/- 5.3 nM). Clopidogrel and other thiol reagents like pCMBS or DTT abolished the binding both to intact platelets and membrane preparations. Therefore, [H-3]PSB-0413 is an accurate and selective tool for radioligand binding studies aimed at quantifying P2Y(12) receptors, to identify patients with P2Y(12) deficiencies or quantify the effect of P2Y(12) targeting drugs.