Agonist and antagonist effects of diadenosine tetraphosphate, a platelet dense granule constituent, on platelet P2Y1, P2Y12 and P2X1 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 Ca2+, and c) P2X(1)-mediated intraplatelet entry of extracellular Ca2+. ADP-stimulated platelet shape change (P2Y(1)-mediated) and aggregation (P2Y(1)- and P2Y(12)-mediated) were measured optically. Ap(4)A inhibited 3 mu M ADP-induced: a) platelet aggregation (IC50 9.8 +/- 2.8 mu M), b) P2Y(1)-mediated shape change, c) P2Y(1)-mediated increase in platelet cytosolic Ca2+ (IC50 40.8 +/- 12.3 mu M), and d) P2Y(12)-mediated decrease in VASP phosphorylation (IC50 >250 mu M). 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. (C) 2009 Elsevier Ltd. All rights reserved.