The platelet P2Y(12) receptor under normal and pathological conditions. Assessment with the radiolabeled selective antagonist [(3)H]PSB-0413

Abstract

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 [(3)H]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) [(3)H]PSB-0413 bound to 425 ± 50 sites/platelet (K (D) = 3.3 ± 0.6 nM), (2) 0.5 ± 0.2 pmol [(3)H]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 ≫ ADPβS > ADP, while the P2Y(1) ligand MRS2179 and the P2X(1) ligand α,β-Met-ATP did not displace [(3)H]PSB-0413 binding. Patients with severe P2Y(12) deficiency displayed virtually no binding of [(3)H]PSB-0413 to intact platelets, while a patient with a dysfunctional P2Y(12) receptor had normal binding. Studies in mice showed that: (1) [(3)H]PSB-0413 bound to 634 ± 87 sites/platelet (K (D) = 14 ± 4.5 nM) and (2) 0.7 pmol ± 0.3 [(3)H]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, [(3)H]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.

Fig. 1

Pharmacological characterization. a [³H]PSB-0413 was obtained by catalytic hydrogenation using tritium gas with a specific radioactivity of 1.85 to 3.7 TBq/mmol where ³H was fixed on the propyl moieties (black stars). b Washed platelets were activated with 5 μM ADP in the presence of human fibrinogen and in the absence or presence of 100 nM of [³H]PSB-0413. c 1321N1 cell lines transfected with P2Y₁₂, P2Y₁ receptors or control (empty vector) were incubated with 1 nM of [³H]PSB-0413 at 37 °C for 5 min. Binding was performed in the presence (closed square) or absence (open square) of 10 μM of the P2Y₁₂ antagonist AR-C69931MX. Assays were performed in triplicate in three independent experiments

Fig. 2

Binding and pharmacological properties of [³H]PSB-0413 on human intact platelets. a Association kinetic of [³H]PSB-0413 binding on human intact platelets, measured at 37 °C over 30 min. b Dissociation kinetic was measured after [³H]PSB-0413 binding had reached a steady state (15 min) and was initiated by addition of 1 mM ADP. Association and dissociation curves are representative from three independent experiments. c Saturation experiment on human intact platelets from ten healthy subjects was achieved using different concentrations of [³H]PSB-0413 ranging from 0.05 to 50 nM (closed circle). Non-specific binding assessed in the presence of excess ADP (1 mM) and isotherm binding curves were calculated using the software EBDA-LIGAND in the hot-saturation mode. The saturation curve is representative of ten independent experiments. d Competition experiments were performed at 37 °C after a 5-min incubation with 5 nM of [³H]PSB-0413 in the presence of increasing concentration of: AR-C69931MX (closed circle), 2MeSADP (open circle), ADPβS (open square), ADP (closed triangle), α,β-Me-ATP (open diamond), and MRS2179 (closed diamond). Competition curves are the mean of three independent experiments

Fig. 3

Effects of thiol reagents on [³H]PSB-0413 binding to intact human and mouse platelets. a, b Inhibition experiments were performed by incubating 5 nM of [³H]PSB-0413 with intact platelets pretreated for 10 min with increasing concentrations of thiol reagents: apCMBS and b DTT; inhibition curves are the mean of three independent experiments. c Saturation experiment was achieved on intact platelets from control mice (closed circle) or from mice that had been treated with 50 mg/kg clopidogrel (open circle) using concentrations of [³H]PSB-0413 ranging from 0.05 to 50 nM. The saturation curve is representative from three independent experiments

Fig. 4

Binding and pharmacological properties of [³H]PSB-0413 on platelet membranes. Saturation experiments on a human or b mice platelet membrane preparations were achieved by incubating 20 μg of membrane proteins at 25 °C for 60 min with different concentrations of [³H]PSB-0413 ranging from 0.030 to 50 nM. c Binding on platelet membranes from control or clopidogrel (50 mg/kg) treated mice was achieved using 5 nM of [³H]PSB-0413 in the absence (white square) or presence of 1 μM AR-C69931MX (gray square) or 100 μM MRS2179 (black square). Assays were performed in triplicate in three independent experiments