Competitive Inhibition by Adenosine 5′‐triphosphate of the Actions on Human Platelets of 2‐chloroadenosine 5′‐diphosphate, 2‐azidoadenosine 5′‐diphosphate and 2‐methylthioadenosine 5′‐diphosphate

Cusack, N. J.; Hourani, S.M.O.

doi:10.1111/j.1476-5381.1982.tb09302.x

Cited by 46 publications

(16 citation statements)

References 14 publications

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“…Synthetic agonists of the P2T AC receptor, generally 2-substituted derivatives of ADP, are also inducers of platelet aggregation (13,14). Antagonists of this receptor, such as ATP and ARL 66096, have been shown to block both ADP-induced adenylyl cyclase inhibition (8,15) and platelet aggregation (15,16).…”

Section: Resultsmentioning

confidence: 99%

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

Jin

Kunapuli

1998

Proc. Natl. Acad. Sci. U.S.A.

494

449

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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.

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Section: Resultsmentioning

confidence: 99%

“…Platelets also contain several other G proteins, including, G z , G s , G 12 , G 13 , and G 16 (32,33). Although increase in cAMP levels through activation of G s inhibits agonist-induced platelet aggregation (23), the functional role of the other G proteins in this response needs to be determined.…”

Section: Cell Biology: Jin and Kunapulimentioning

confidence: 99%

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

Jin

Kunapuli

1998

Proc. Natl. Acad. Sci. U.S.A.

494

449

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“…The possibility that direct antagonism of GPCR activation occurs by extracellular signaling molecules has been suggested by observations that ATP is a competitive antagonist of the hP2Y 12 -R (Cusack and Hourani, 1982;Bodor et al, 2003) and of the hP2Y 4 -R (Bogdanov et al, 1998;Kennedy et al, 2000). The physiological relevance of UDP antagonism at the hP2Y 14 -R will be important to investigate, as will the idea that UDP simultaneously activates the hP2Y 6 -R while inhibiting the hP2Y 14 -R.…”

Section: Discussionmentioning

confidence: 99%

UDP Is a Competitive Antagonist at the Human P2Y₁₄Receptor

Fricks¹,

Maddileti²,

Carter³

et al. 2008

J Pharmacol Exp Ther

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G protein-coupled P2Y receptors (P2Y-R) are activated by adenine and uracil nucleotides. The P2Y 14 receptor (P2Y 14 -R) is activated by at least four naturally occurring UDP sugars, with UDPglucose (UDP-Glc) being the most potent agonist. With the goal of identifying a competitive antagonist for the P2Y 14 -R, UDP was examined for antagonist activity in COS-7 cells transiently expressing the human P2Y 14 -R and a chimeric G␣ protein that couples Gi-coupled receptors to stimulation of phosphoinositide hydrolysis. UDP antagonized the agonist action of UDP-Glc, and Schild analysis confirmed that the antagonism was competitive (pK B ϭ 7.28). Uridine 5Ј-O-thiodiphosphate also antagonized the human P2Y 14 -R (hP2Y 14 -R) with an apparent affinity similar to that of UDP. In contrast, no antagonist activity was observed with ADP, CDP, or GDP, and other uracil analogs also failed to exhibit antagonist activity. The antagonist activity of UDP was not observed at other hP2Y-R. In contrast to its antagonist action at the hP2Y 14 -R, UDP was a potent agonist (EC 50 ϭ 0.35 M) at the rat P2Y 14 -R. These results identify the first competitive antagonist of the P2Y 14 -R and demonstrate pharmacological differences between receptor orthologs.

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“…AR-C66096 was the sample synthesized previously (3). Antisera against G-protein subunits were purchased from the following suppliers: antiserum G o /1 (for G␣ o ), EC/2 (for G␣ i3 ), AS/7 (for G␣ i1 and G␣ i2 ), and RM/1 (for G␣ s and G␣ olf ) from PerkinElmer Life Sciences; antisera S-20 (for G␣ 12 ), A-20 (for G␣ 13 ), and C-16 (for G␤ 1 ) from Santa Cruz Biotechnology; antisera 371727 (for G␣ i2 ), 371754 (for G␣ q and G␣ 11 ), 371783 (for G␣ 16 ) from Calbiochem-Novabiochem; antisera PC64 (for G␣ z ), G51829 (for G␣ t1 ), 3A-195 (25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40) Ci/mmol), the secondary antibodies used, and the ECL detection kit were from Amersham Biosciences. All other chemicals were of analytical grades from established commercial sources.…”

Section: Methodsmentioning

confidence: 99%

“…In B10 cells (3) ATP and 2-ClATP are weak partial agonists, and 2-MeSATP is a very potent full agonist (EC 50 , 3.5 nM), whereas in the platelet these and other adenosine triphosphates are antagonists (13,14). It may be, therefore, that these endothelial cells contain a different subtype related to the P2Y 12 receptor, or even a quite different, previously unknown receptor of the P2Y family, whereas other explanations are also possible (3).…”

mentioning

confidence: 99%

Characterization and Channel Coupling of the P2Y12Nucleotide Receptor of Brain Capillary Endothelial Cells

Simon¹,

Filippov²,

Göransson³

et al. 2002

Journal of Biological Chemistry

103

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B10 cells are an endothelial clonal line derived spontaneously by culture and selection of rat brain microvascular endothelial cells (1), a source of considerable pharmacological potential. The B10 cells respond to extracellular adenine (but not uracil) nucleotides with intracellular Ca 2ϩ mobilization, mediated by a G-protein-coupled P2Y 1 receptor (2). Whereas the known rat P2Y 1 receptor cDNA could be isolated from the B10 cells (2), no transcript for any other then-known P2Y receptor subtype was detectable in them. However, the B10 cells were found to exhibit another second messenger response, namely the inhibition of stimulated adenylyl cyclase, but with a nucleotide agonist specificity very similar to that of the P2Y 1 receptor (2). It was further demonstrated (3) that selective antagonists of the P2Y 1 receptor, such as adenosine 3Ј-phosphate 5Ј-phosphate, were unable to affect that response, although it showed high sensitivity to 2-propylthio-D-␤,␥-difluoromethylene-ATP (AR-C66096), 1 a specific antagonist (4, 5) of the adenylyl cyclase-inhibitory P2Y T or "P2T" receptor for ADP, which was known as an important functional component of blood platelets. That study (3) demonstrated that a second P2Y receptor activity is present in the B10 cell, with some of its functional features in common with the platelet P2Y T receptor.Recently, a cDNA encoding a nucleotide receptor with a novel seven-transmembrane sequence was identified independently by two groups, starting from a human platelet cDNA library or an orphan human DNA sequence, and shown to be distantly but significantly related to the known P2Y receptors (6, 7). This was designated as the P2Y 12 receptor. Its amino acid sequence lies on a previously unrecognized separate branch of the P2Y family (8). It was characterized in both studies to show its adenine nucleotide specificity and its inhibition of forskolin-stimulated adenylyl cyclase, in which it corresponds to the platelet P2Y T receptor (6, 7). That human P2Y 12 receptor cDNA was expressed again in cell line hosts by other groups, confirming the reported series of agonists but with higher potencies in the cAMP decrease (9) and showing their affinities by competition with the binding of radiolabeled 2-MeSADP (9, 10). In the original identification of the human P2Y 12 receptor, a similar cDNA was also derived from a rat platelet library (6), and its RNA was shown to express in Xenopus oocytes, but this receptor was not further characterized.The question, therefore, obviously arises as to whether the cyclase inhibitory receptor for adenine nucleotides of the B10 capillary endothelial cell is in fact the P2Y 12 receptor. However, this cannot necessarily be assumed, because the P2T receptor has commonly been described in the literature as specific to the platelet (11,12). Furthermore, the adenylyl cyclase-inhibitory P2Y receptor activity in the B10 cell differs in an important

show abstract

Competitive Inhibition by Adenosine 5′‐triphosphate of the Actions on Human Platelets of 2‐chloroadenosine 5′‐diphosphate, 2‐azidoadenosine 5′‐diphosphate and 2‐methylthioadenosine 5′‐diphosphate

Cited by 46 publications

References 14 publications

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

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

UDP Is a Competitive Antagonist at the Human P2Y₁₄Receptor

Characterization and Channel Coupling of the P2Y12Nucleotide Receptor of Brain Capillary Endothelial Cells

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Competitive Inhibition by Adenosine 5′‐triphosphate of the Actions on Human Platelets of 2‐chloroadenosine 5′‐diphosphate, 2‐azidoadenosine 5′‐diphosphate and 2‐methylthioadenosine 5′‐diphosphate

Cited by 46 publications

References 14 publications

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

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

UDP Is a Competitive Antagonist at the Human P2Y14Receptor

Characterization and Channel Coupling of the P2Y12Nucleotide Receptor of Brain Capillary Endothelial Cells

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UDP Is a Competitive Antagonist at the Human P2Y₁₄Receptor