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.

Thromboxane A 2 is a positive feedback lipid mediator produced following platelet activation. The G q -coupled thromboxane A 2 receptor subtype, TP␣, and G i -coupled TP␤ subtype have been shown in human platelets. ADPinduced platelet aggregation requires concomitant signaling from two P2 receptor subtypes, P2Y1 and P2T AC , coupled to G q and G i , respectively. We investigated whether the stable thromboxane A 2 mimetic, (15S)-hydroxy-9,11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619), also causes platelet aggregation by concomitant signaling through G q and G i , through co-activation of TP␣ and TP␤ receptor subtypes. Here we report that secretion blockade with Ro 31-8220, a protein kinase C inhibitor, completely inhibited U46619-induced, but not ADP-or thrombin-induced, platelet aggregation. Ro 31-8220 had no effect on U46619-induced intracellular calcium mobilization or platelet shape change. Furthermore, U46619-induced intracellular calcium mobilization and shape change were unaffected by A3P5P, a P2Y1 receptor-selective antagonist, and/or cyproheptadine, a 5-hydroxytryptamine subtype 2A receptor antagonist. Either Ro 31-8220 or AR-C66096, a P2T AC receptor selective antagonist, abolished U46619-induced inhibition of adenylyl cyclase. In addition, AR-C66096 drastically inhibited U46619-mediated platelet aggregation, which was further inhibited by yohimbine, an ␣ 2A -adrenergic receptor antagonist. Furthermore, inhibition of U46619-induced platelet aggregation by Ro 31-8220 was relieved by activation of the G i pathway by selective activation of either the P2T AC receptor or the ␣ 2A -adrenergic receptor. We conclude that whereas thromboxane A 2 causes intracellular calcium mobilization and shape change independently, thromboxane A 2 -induced inhibition of adenylyl cyclase and platelet aggregation depends exclusively upon secretion of other agonists that stimulate G i -coupled receptors.Upon exposure to activating agonists (e.g. thrombin, ADP, and collagen), platelets liberate arachidonic acid stored as phospholipid in the platelet plasma membrane that is converted into thromboxane A 2 by sequential oxygenation of arachidonic acid by cycloxygenase and thromboxane A 2 synthase (1). The released thromboxane A 2 acts as a positive feedback mediator in the activation and recruitment of more platelets to the primary hemostatic plug (2). Thromboxane A 2 exerts its actions via specific G protein-coupled receptors and has been described as either a potent platelet agonist (2, 3) or as a weak agonist with an important role in amplifying the response of platelets to more potent agonists (4).Pharmacological studies indicate the presence of two potential thromboxane A 2 receptor (TP receptor) 1 subtypes on human platelets (5, 6). The TP receptor gene has been cloned and encodes two subtypes of the TP receptor that result from alternative splicing of the primary transcript (7). The subtypes share the identical first 293 amino acids but possess different carboxyl-terminal domains. A complete cDNA of the 343 amino acid T...

The serine-threonine kinase Akt has been established as an important signaling intermediate in regulating cell survival, cell cycle progression, as well as agonistinduced platelet activation. Stimulation of platelets with various agonists including thrombin results in Akt activation. As thrombin can stimulate multiple G protein signaling pathways, we investigated the mechanism of thrombin-induced activation of Akt. Stimulation of platelets with a PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin resulted in Thr 308 and Ser 473 phosphorylation of Akt, which results in its activation. This phosphorylation and activation of Akt were dramatically inhibited in the presence of AR-C69931MX, a P2Y 12 receptor-selective antagonist, or GF 109203X, a protein kinase C inhibitor, but Akt phosphorylation was restored by supplemental G i or G z signaling. Unlike wild-type mouse platelets, platelets from G␣ q -deficient mice failed to trigger Akt phosphorylation by thrombin and AYPGKF, whereas Akt phosphorylation was not affected by these agonists in platelets from mice that lack P2Y 1 receptor. However, ADP caused Akt phosphorylation in G␣ q -and P2Y 1 -deficient platelets, which was completely blocked by AR-C69931MX. In contrast, ADP failed to cause Akt phosphorylation in platelets from mice treated with clopidogrel, and thrombin and AYPGKF induced minimal phosphorylation of Akt, which was not affected by AR-C69931MX in these platelets. These data demonstrate that G i , but not G q or G 12/13 , signaling pathways are required for activation of Akt in platelets, and G i signaling pathways, stimulated by secreted ADP, play an essential role in the activation of Akt in platelets. Akt (also known as protein kinase B (PKB))1 is a 57-kDa serine/threonine kinase, which is the cellular homologue of the viral oncogene v-akt of the acutely transforming retrovirus Akt8 (1). Akt plays an important role in mediating the antiapoptotic effect of many growth factors, and it is also overexpressed in several cancer forms (2, 3). Akt contains a pleckstrin homology domain adjacent to a centrally located catalytic domain that is connected to a short C-terminal tail (4). The catalytic domain of Akt contains a Thr 308 phosphorylation site and displays high homology to the catalytic domains of cAMPdependent protein kinase A and protein kinase C. A second phosphorylation site, Ser 473 , is located in the C-terminal tail (4). There are at least three different Akt isoforms identified in humans, which display more than 80% sequence homology and are named Akt1 (PKB␣), Akt2 (PKB␤), and Akt3 (PKB␥) (5). It has been shown that Akt1 (PKB␣) and Akt2 (PKB␤) are expressed in human platelets, whereas Akt3 (PKB␥) is not present in human platelets (6). Both translocation of Akt to cell membranes and phosphorylation of both Thr 308 and Ser 473 are required for full enzyme activity. Akt is activated by various agonists including platelet-derived growth factor, epidermal growth factor, insulin, nerve growth factor, U46619 (a thromboxan...

Thrombin is an important agonist for platelet activation and plays a major role in hemostasis and thrombosis. Thrombin activates platelets mainly through protease-activated receptor 1 (PAR1), PAR4, and glycoprotein Ib. Because adenosine diphosphate and thromboxane A 2 have been shown to cause platelet aggregation by concomitant signaling through G q and G i pathways, we investigated whether coactivation of G q and G i signaling pathways is the general mechanism by which PAR1 and PAR4 agonists also activate platelet fibrinogen receptor (␣IIb␤3). APAR1-activating peptide, SFLLRN, and PAR4-activating peptides GYPGKF and AYPGKF, caused inhibition of stimulated adenylyl cyclase in human platelets but not in the presence of either Ro 31-8220, a protein kinase C selective inhibitor that abolishes secretion, or AR-C66096, a P2Y12 receptor-selective antagonist; ␣-thrombin-induced inhibition of adenylyl cyclase was also blocked by Ro 31-8220 or AR-C66096. In platelets from a P2Y12 receptor-defective patient, ␣-thrombin, SFLLRN, and GYPGKF also failed to inhibit adenylyl cyclase. In platelets from mice lacking the P2Y12 receptor, neither ␣-thrombin nor AYPGKF caused inhibition of adenylyl cyclase. Furthermore, AR-C66096 caused a rightward shift of human platelet aggregation induced by the lower concentrations of ␣-thrombin and AYPGKF but had no effect at higher concentrations. Similar results were obtained with platelets from mice deficient in the P2Y12. We conclude that (1) thrombin-and thrombin receptoractivating peptide-induced inhibition of adenylyl cyclase in platelets depends exclusively on secreted adenosine diphosphate that stimulates G i signaling pathways and ( IntroductionPlatelet activation plays a major role in hemostasis and thrombosis. Several agonists, including adenosine diphosphate (ADP), thrombin, and thromboxane A 2 , can activate platelets. 1 These agonists cause platelets to change their shape, to aggregate, and to release the contents of granules. Thrombin, generated at the site of vascular damage by extrinsic and intrinsic coagulation cascades, is an important agonist for platelet activation. Thrombin mediates its cellular effects primarily through a family of G protein-coupled protease-activated receptors (PARs). These receptors are activated by a unique mechanism in which the protease creates a new extracellular amino-terminus that functions as a tethered ligand, resulting in intramolecular activation. 2,3 Three of the 4 known PARs, PAR1, PAR3, and PAR4, are activated by thrombin. PAR1 is detected in human platelets and has a major role in activation of human platelets by thrombin, but it plays no role in mouse platelets. 4 PAR2 functions as a receptor for trypsin but not for thrombin. 5 PAR3 is necessary for mouse platelets to be activated by lower concentrations of thrombin. 6 PAR3 functions as a cofactor for the activation of PAR4 by thrombin in mouse platelets. 7 There is another receptor, PAR4, which appears to function in both mouse and human platelets. 4,8 PAR4 also mediates platelet respo...

Rationale In the failing heart, persistent β-adrenergic receptor (βAR) activation is thought to induce myocyte death by protein kinase A (PKA)-dependent and PKA-independent activation of calcium/calmodulin-dependent kinase II (CaMKII). β-Adrenergic signaling pathways are also capable of activating cardioprotective mechanisms. Objective This study used a novel PKA inhibitor peptide (PKI) to inhibit PKA activity to test the hypothesis that βAR signaling causes cell death through PKA-dependent pathways and cardioprotection through PKA-independent pathways. Methods and Results In PKI transgenic mice, chronic isoproterenol (ISO) failed to induce cardiac hypertrophy, fibrosis, myocyte apoptosis and depressed cardiac function. In cultured adult feline ventricular myocytes (AFVMs), PKA inhibition protected myocytes from death induced by β1-AR agonists by preventing cytosolic and SR Ca2+ overload and CaMKII activation. PKA inhibition revealed a cardioprotective role of β-adrenergic signaling via cAMP/EPAC /Rap1/Rac/ERK pathway. Selective PKA inhibition causes protection in the heart after myocardial infarction (MI) that was superior to β-blocker therapy. Conclusion These results suggest that selective block of PKA could be a novel heart failure therapy.