Role of P2Y1 purinoceptor in ADP-induced platelet activation.

ADP acts as an agonist of platelet aggregation via specific receptors which are still to be characterised. Amplification by PCR of a human platelet cDNA library confirmed the presence of mRNA of the P2Y1 receptor in platelets. In order to determine if these P2Y1 receptors were involved in ADP-induced platelet activation, we determined the effects of A3P5PS, an antagonist of the P2Y1 receptor, on the binding of [33P]2-MeS-ADP, a potent analogue of ADP. We found that A3P5PS displaced about 27% of [33P]2-MeS-ADP binding, a receptor population which has been shown to be resistant to treatment with clopidogrel, a selective anti-ADP agent. A3P5PS specifically inhibited 2-MeS-ADP-induced shape change and calcium increase but did not affect adenylyl cyclase down-regulation. 2-MeS-ADP-induced platelet aggregation was also inhibited by A3P5PS but was restored when platelets were further activated by serotonin, a non-aggregating compound, therefore suggesting that P2Y1-mediated stimulation is an absolute prerequisite for ADP to induce platelet aggregation and a key event for platelet activation and aggregation to occur. These results therefore show that ADP-induced aggregation cannot be attributed to activation of P2Y1 alone, but must be attributed to the simultaneous activation of the high affinity receptor (P2Y1) and a low affinity receptor of ADP (still to be discovered), each of them essential, but neither able to trigger aggregation alone.

Characterization of P2x1 purinoreceptors on rat platelets: effect of clopidogrel.

This study aimed to determine the binding characteristics of [3H]alpha,beta-Me-ATP, a specific ligand of the P2x1 receptors to rat platelets, and to investigate the effect of clopidogrel, a thienopyridine compound which has been found to selectively inhibit ADP-induced platelet aggregation and adenylyl cyclase ex vivo. Binding of [3H]alpha,beta-Me-ATP to rat platelets was time-dependent and saturable. Scatchard analysis of the saturation binding data indicated that [3H]alpha,beta-Me-ATP bound to one population of specific binding sites with high affinity (KD = 23.6 +/- 1.6 nM; Bmax = 690 +/- 24 fmole/10[8]cells) (n=3). Unlabelled alpha,beta-Me-ATP as well as 2-MeS-ADP and ADP competitively inhibited the specific binding of [3H]alpha,beta-Me-ATP with IC50 values of 19.0 +/- 6.6, 103 +/- 20 and 1120 +/- 80 nM respectively (n=3). Other nucleotide analogues such as ATP, ATP-gammaS, UTP and GTP also antagonized [3H]alpha,beta-Me-ATP binding. When administered orally (10mg/kg, p.o.), clopidogrel inhibited ADP- or 2-MeS-ADP-induced platelet aggregation but did not affect the binding of [3H]alpha,beta-Me-ATP to rat platelets ex vivo. In vitro, alpha,beta-Me-ATP did not induce the aggregation or shape change of rat platelets and did not interfere with ADP-induced platelet aggregation.