Coactivation of two different G protein-coupled receptors is essential for ADP-induced platelet aggregation

Abstract

ADP is an important platelet agonist causing shape change and aggregation required for physiological hemostasis. We recently demonstrated the existence of two distinct G protein-coupled ADP receptors on platelets, one coupled to phospholipase C, P2Y1, and the other to inhibition of adenylyl cyclase, P2TAC. In this study, using specific antagonists for these two receptors, we demonstrated that concomitant intracellular signaling from both the P2TAC and P2Y1 receptors is essential for ADP-induced platelet aggregation. Inhibition of signaling through either receptor, by specific antagonists, is sufficient to block ADP-induced platelet aggregation. Furthermore, signaling through the P2TAC receptor could be replaced by activation of alpha2A-adrenergic receptors. On the other hand, activation of serotonin receptors supplements signaling through the P2Y1 receptor. Moreover, this mechanism of ADP-induced platelet aggregation could be mimicked by coactivation of two non-ADP receptors coupled to Gi and Gq, neither of which can cause platelet aggregation by itself. We propose that platelet aggregation results from concomitant signaling from both the Gi and Gq, a mechanism by which G protein-coupled receptors elicit a physiological response.

Figure 1

Effect of P2Y1 receptor-selective antagonists on 10 μM ADP-induced platelet aggregation. Human platelets were washed and resuspended in Tyrode’s buffer, and aggregation was measured in the absence of added extracellular calcium, as described in Experimental Procedures. (A) Representative traces of various concentrations of A3P5PS on ADP-induced aggregation of aspirin-treated and washed platelets (8). Additions are shown with an arrow. Concentrations of A3P5PS are shown on the right. (B) Effect of P2Y1 receptor-selective antagonists on the extent of ADP-induced aggregation. The extent of aggregation in the presence of antagonists was normalized to that in the absence of the antagonist (taken as 100%).

Figure 2

Effect of ARL 66096 on ADP-induced inhibition of platelet adenylyl cyclase. Effect of ADP on platelet adenylyl cyclase was determined at various concentrations in the presence and absence of a single dose of 50 nM ARL 66096. The data are normalized to response to the maximal concentration of ADP (taken as 100%).

Figure 3

Mimicking ADP-induced platelet aggregation by activation of two different G protein-coupled receptors on platelets. Aggregation was measured in the absence of added extracellular calcium. Representative aggregation traces are shown with additions as indicated by arrows. The following were used: 5 μM serotonin, 1 μM epinephrine, 10 μM ADP, 300 nM ARL 66096, and 200 μM A3P5PS.

Figure 4

Effect of P2X receptor-selective agonist on platelet aggregation. Representative aggregation traces are shown with additions indicated by arrows. Aggregation was measured as described for Fig. 1.

Figure 5

Model depicting the signal transduction and physiological events mediated by the three ADP receptors on platelets.