HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Adenosine diphosphate (ADP)–induced thromboxane A2 generation in human platelets requires coordinated signaling through integrin IIb 3 and ADP receptors

Adenosine diphosphate (ADP) is a platelet agonist that causes platelet shape change and aggregation as well as generation of thromboxane A2, another platelet agonist, through its effects on P2Y1, P2Y12, and P2X1 receptors. It is now reported that both 2-propylthio-D-dichloromethylene adenosine 5 -triphosphate (AR-C67085), a P2Y12 receptor–selective antagonist, and adenosine-2 -phosphate-5 -phosphate (A2P5P), a P2Y1 receptor–selective antagonist, inhibited ADP-induced thromboxane A2 generation in a concentration-dependent manner, indicating that coactivation of the P2Y12 and P2Y1 receptors is essential for this event. SC49992, a fibrinogen receptor antagonist, blocked ADP-induced platelet aggregation and thromboxane A2 production in a concentration-dependent manner. Similarly, P2 receptor antagonists or SC49992 blocked ADP-induced arachidonic acid liberation. Whereas SC49992 blocked arachidonic acid–induced platelet aggregation, it failed to inhibit thromboxane A2 generation induced by arachidonic acid. Thus, ADPinduced arachidonic acid liberation, but not subsequent conversion to thromboxane A2, requires outside-in signaling through the fibrinogen receptor. The Fab fragment of ligand-induced binding site–6 (LIBS6) antibody, which induces a fibrinogen-binding site on the integrin IIb 3, caused both platelet aggregation and thromboxane A2 generation. Inhibitors of phosphoinositide 3-kinase, Syk, Src kinases, or protein tyrosine phosphatases inhibited platelet aggregation but not thromboxane A2 generation, indicating that these signaling molecules have no significant role in phospholipaseA2 activation. In the presence of P2 receptor antagonists A2P5P or AR-C67085, LIBS6 failed to generate thromboxane A2, suggesting that inside-out signaling through ADP receptors is necessary for this event. It was concluded that both outside-in signaling from the fibrinogen receptor and inside-out signaling from the P2Y1 and P2Y12 receptors are necessary for phospholipase A2 activation, resulting in arachidonic acid liberation and thromboxane A2 generation. (Blood. 2002; 99:193-198)