Modified diadenosine tetraphosphates with dual specificity for P2Y1 and P2Y12 are potent antagonists of ADP‐induced platelet activation

Abstract

Diadenosine 5',5'''-P(1),P(4)-tetraphosphate (Ap(4)A), a natural compound stored in platelet dense granules, inhibits ADP-induced platelet aggregation. Ap(4)A inhibits the platelet ADP receptors P2Y(1) and P2Y(12), is a partial agonist of P2Y(12), and is a full agonist of the platelet ATP-gated ion channel P2X1. Modification of the Ap(4)A tetraphosphate backbone enhances inhibition of ADP-induced platelet aggregation. However, the effects of these Ap(4)A analogs on human platelet P2Y(1), P2Y(12) and P2X1 are unclear. To determine the agonist and antagonist activities of diadenosine tetraphosphate analogs towards P2Y(1), P2Y(12), and P2X1. We synthesized the following Ap(4)A analogs: P(1),P(4)-dithiotetraphosphate; P(2),P(3)-chloromethylenetetraphosphate; P(1)-thio-P(2),P(3)-chloromethylenetetraphosphate; and P(1),P(4)-dithio-P(2),P(3)-chloromethylenetetraphosphate. We then measured the effects of these analogs on: (i) ADP-induced platelet aggregation; (ii) P2Y(1)-mediated changes in cytosolic Ca(2+); (iii) P2Y(12)-mediated changes in vasodilator-stimulated phosphoprotein phosphorylation; and (iv) P2X1-mediated entry of extracellular Ca(2+). Ap(4)A analogs with modifications in the phosphate backbone inhibited both P2Y(1) and P2Y(12), and showed no agonist activity towards these receptors. The dithio modification increased inhibition of P2Y(1), P2Y(12), and platelet aggregation, whereas the chloromethylene modification increased inhibition of P2Y(12) and platelet aggregation, but decreased P2Y(1) inhibition. Combining the dithio and chloromethylene modifications increased P2Y(1) and P2Y(12) inhibition. As compared with Ap(4)A, each modification decreased agonist activity towards P2X1, and the dual modification completely eliminated P2X1 agonist activity. As compared with Ap(4)A, tetraphosphate backbone analogs of Ap(4)A have diminished activity towards P2X1 but inhibit both P2Y(1) and P2Y(12) and, with greater potency, inhibit ADP-induced platelet aggregation. Thus, diadenosine tetraphosphate analogs with dual receptor selectivity may have potential as antiplatelet drugs.