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, P2T AC . In this study, using specific antagonists for these two receptors, we demonstrated that concomitant intracellular signaling from both the P2T AC 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 P2T AC receptor could be replaced by activation of ␣ 2A -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 G i and G q , neither of which can cause platelet aggregation by itself. We propose that platelet aggregation results from concomitant signaling from both the G i and G q , a mechanism by which G protein-coupled receptors elicit a physiological response.ADP is the first small molecular weight platelet agonist to be identified (1). When stimulated with ADP, platelets undergo shape change, release granule contents, and produce thromboxane A 2 (2, 3). In addition, ADP activates the fibrinogen receptor, causing platelets to bind fibrinogen and aggregate (2, 3). The receptors through which extracellular nucleotides elicit physiological responses have been classified as P2 receptors and are divided into P2X ligand-gated ion channels and P2Y G protein-coupled receptors (4). These receptor subtypes are numbered in the order of cloning, and to date 7 subtypes of P2X receptors and 10 subtypes of P2Y receptors have been cloned (5, 6). Sage and coworkers (7) recently demonstrated a P2X receptor in platelets and proposed that this receptor is the P2X1 receptor subtype mediating rapid calcium influx. We have provided evidence for two distinct G protein-coupled ADP receptors, one coupled to the inhibition of adenylyl cyclase, the P2T AC receptor, and the other coupled to the activation of phospholipase C, the P2Y1 receptor, in human platelets (8,9). Several compounds, including ARL 66096, ticlopidine, and clopidogrel, have been utilized to block ADPinduced inhibition of adenylyl cyclase and subsequent platelet aggregation both in vitro and in vivo (3,8), suggesting that the P2T AC receptor mediates ADP-induced platelet aggregation.Here, we delineate the role of the three ADP receptors, the P2T AC , P2Y1, and P2X1 receptors, in ADP-induced human platelet aggregation and demonstrate that some agonistinduced physiological responses may require simultaneous activation of multiple receptor subtypes by the same agonist, resulting in converging signal transduction pathways leading to a physiological response.

Adrenaline enhances in vitro platelet activation and aggregation in blood samples from ticagrelor‐treated patients

“…ADP binds to the G‐protein‐coupled receptors P2Y 1 22 and P2Y 12 23, 24 located on the platelet surface membrane. For a normal ADP‐induced activation and aggregation, responses from both pathways are required 25, 26, 27. When a P2Y 12 ‐receptor antagonist such as ticagrelor has bound to the platelet receptor, the ADP‐induced activation of P2Y 12 is inhibited.…”

“…When a P2Y 12 ‐receptor antagonist such as ticagrelor has bound to the platelet receptor, the ADP‐induced activation of P2Y 12 is inhibited. Adrenaline binds to the α 2A ‐receptor, activating a pathway which mimics the P2Y 12 ‐receptor mediated pathway 26, 28, 29. This may explain that the combination of addition of ADP and adrenaline to blood samples from patients on ongoing treatment with ticagrelor results in a better aggregation response compared to either substance alone, as responses from both signaling pathways are achieved.…”

P2 receptors and platelet function

“…Co-activation of the P2Y 1 and P2Y 12 receptors is necessary for normal ADP-induced platelet aggregation since separate inhibition of either of them with selective antagonists results in a dramatic decrease in aggregation [22,46,80]. The P2Y 1 and P2Y 12 receptors are differentially involved in platelet aggregation induced by other agonists, with the P2Y 1 playing only a minor role, except in the case of collagen-induced activation, while P2Y 12 supports amplification of these responses.…”

Platelets at the vascular interface

“…Similarly, the ADP receptor P2Y1, which is not very abundant on the platelet surface[19], is rapidly desensitized[20,21]. Thus, it can only cause a rapid but short-lived rise of the cytosolic calcium concentration and needs co-signaling through the P2Y12 receptor to support platelet aggregation[22,23]. So both PAR1 and P2Y1 are very sensitive and provide a fast but reversible activation response, while PAR4 and P2Y12 are required to sustain the signal.…”