Inhibition of the Platelet P2Y₁₂ Receptor Potentiates the Antiplatelet Effect of Prostacyclin.

Adenosine diphosphate (ADP) plays a key role in platelet function because, although itself a weak platelet agonist, when it is secreted from the platelet dense granules, it amplifies the platelet responses induced by strong platelet agonists. Concomitant activation of two platelet receptors for ADP is necessary for ADP-induced platelet aggregation (PA): the G_q-coupled P2Y₁ receptor and the G_i-coupled P2Y₁₂ receptor. P2Y₁₂ is coupled to inhibition of adenylyl cyclase (AC) through activation of G_αi2. However, inhibition of AC bears no causal relationship to PA: other, partially identified signalling events downstream of P2Y₁₂ are required for ADP-induced PA. P2Y₁₂ plays an important role in thrombogenesis, because its irreversible inhibition by the thienopyridine drug clopidogrel effectively reduces the incidence of cardiovascular events in patients at risk. It is generally held that the antithrombotic effect of anti-P2Y₁₂ drugs is uniquely mediated by inhibition of the P2Y₁₂-dependent PA. However, prevention of inhibition of AC by ADP could play an additional, important antithrombotic role in vivo. In fact, P2Y₁₂ inhibition could amplify the antiplatelet effect of prostacyclin (PGI₂), which increases the platelet levels of cyclic adenosine monophosphate (cAMP), a potent inhibitor of PA, by stimulating AC. To test our hypothesis, we measured the effects of increasing concentrations of PGI₂ (0.01-10uM) on PA of washed, normal human platelets induced by thrombin in the presence or absence of saturating concentrations of ARC69931MX (anti-P2Y₁₂, 1μM) or MRS2500 (anti-P2Y₁, 1μM). We used high concentration of thrombin (0.5 U/ml), which induced PA that was not inhibited by anti-P2Y₁ or anti-P2Y₁₂, both alone and in combination. PGI₂ inhibited PA in the presence of anti-P2Y₁₂, but did not inhibit PA in the presence of anti-P2Y₁ or in the absence of inhibitors. In contrast to PGI₂, dibutiryl-cAMP inhibited PA both in the presence and absence of anti-P2Y₁ or anti-P2Y₁₂. PGI₂ increased platelet cAMP levels in the absence of thrombin or in the presence of thrombin plus anti-P2Y₁₂, but it had no effect on cAMP levels in the presence of thrombin with or without anti-P2Y₁. These results suggest that:

1. PGI₂, at the tested concentrations, does not inhibit PA induced by high concentration of thrombin, because its effect on AC is prevented by the interaction of released ADP with P2Y₁₂;

2. anti-P2Y₁₂, by rescuing AC activity, facilitates the inhibitory effect of PGI₂ on PA: this effect may contribute to the known antithrombotic effect of drugs inhibiting P2Y₁₂.

In the last years, attempts have been made to improve the antithrombotic efficacy of antiplatelet therapy by combined administration of clopidogrel and aspirin (which inhibits the thromboxane A₂ pathway of PA). However, the results of recent clinical trials indicated that this therapeutic strategy is not superior to monotherapy with either clopidogrel or aspirin in some clinical conditions. The unexpected negative results of these clinical trials can be interpreted in light of the proposed, additional antithrombotic mechanism of anti-P2Y₁₂: since aspirin, at the doses that are commonly used to prevent cardiovascular events, inhibits the production of PGI₂ by vascular wall, it could abolish the antithrombotic effect of clopidogrel that is mediated by the potentiation of the inhibitory effect of PGI₂ on PA.