Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P(1),P(4)-di(adenosine-5') tetraphosphate (Ap4A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y1, P2Y12, and P2X1 receptors. The resulting structure-activity relationships were used to design Ap4A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y1 and P2Y12 platelet receptors. Unlike Ap4A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap4A to degradation in plasma, thus presenting a new promising class of antiplatelet agents.

BACKGROUND: Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5',5''''-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers. OBJECTIVE: To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma. METHODS: We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma. RESULTS: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers. CONCLUSIONS: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors.

INTRODUCTION: Diadenosine 5',5'''-P(1),P(4)- tetraphosphate (Ap(4)A) is stored in platelet dense granules, but its effects on platelet function are not well understood. METHODS AND RESULTS: We examined the effects of Ap(4)A on platelet purinergic receptors P2Y(1), P2Y(12) and P2X(1). Flow cytometry was used to measure the effects of Ap(4)A in the presence or absence of ADP on: a) P2Y(12)-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y(1)-mediated increase in platelet cytosolic Ca(2+), and c) P2X(1)-mediated intraplatelet entry of extracellular Ca(2+). ADP-stimulated platelet shape change (P2Y(1)-mediated) and aggregation (P2Y(1)- and P2Y(12)-mediated) were measured optically. Ap(4)A inhibited 3 microM ADP-induced: a) platelet aggregation (IC(50) 9.8+/-2.8 microM), b) P2Y(1)-mediated shape change, c) P2Y(1)-mediated increase in platelet cytosolic Ca(2+) (IC(50) 40.8+/-12.3 microM), and d) P2Y(12)-mediated decrease in VASP phosphorylation (IC(50)>250 microM). In the absence of added ADP, Ap(4)A had agonist effects on platelet P2X(1) and P2Y(12), but not P2Y(1), receptors. CONCLUSION: Ap(4)A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y(1) and P2Y(12) receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X(1) and P2Y(12) receptors.