ATP Derivatives Are Antagonists of the P2Y1 Receptor: Similarities to the Platelet ADP Receptor

1998 ◽  
Vol 53 (4) ◽  
pp. 727-733 ◽  
Author(s):  
Béatrice Hechler ◽  
Paul Vigne ◽  
Catherine Léon ◽  
Jean-Philippe Breittmayer ◽  
Christian Gachet ◽  
...  
Keyword(s):  
P2y1 Receptor ◽  
Adp Receptor ◽  
Atp Derivatives

Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270

Blood ◽  
2003 ◽  
Vol 101 (10) ◽  
pp. 3908-3914 ◽  
Author(s):  
Zhongren Ding ◽  
Soochong Kim ◽  
Robert T. Dorsam ◽  
Jianguo Jin ◽  
Satya P. Kunapuli
Keyword(s):  
Adenylyl Cyclase ◽  
P2y12 Receptor ◽  
Cysteine Residues ◽  
P2y1 Receptor ◽  
Disulfide Bridges ◽  
Wild Type ◽  
Active Metabolites ◽  
Gtp Binding Protein ◽  
Gtp Binding ◽  
Adp Receptor

Abstract Human platelets express 2 G protein–coupled nucleotide receptors: the platelet adenosine diphosphate (ADP) receptor coupled to stimulation of phospholipase C (P2Y1) via heterotrimeric guanosine 5-triphosphate (GTP)–binding protein Gq, and the platelet ADP receptor coupled to inhibition of adenylyl cyclase (P2Y12) via heterotrimeric GTP-binding protein Gi. Although these 2 receptors are encoded on the same chromosome and have similar pharmacologic profiles, they have different reactivities toward thiol reagents. The thiol agent p-chloromercuribenzene sulfonic acid (pCMBS) and the active metabolites from antiplatelet drugs, clopidogrel and CS-747, inactivate the P2Y12 receptor and are predicted to interact with the extracellular cysteine residues on the P2Y12 receptor. In this study we identified the reactive cysteine residues on the human P2Y12 receptor by site-directed mutagenesis using pCMBS as the thiol reagent. Cys97Ser and Cys175Ser mutants of the P2Y12 receptor did not express when transfected into Chinese hamster ovary (CHO-K1) cells, indicating the essential nature of a disulfide bridge between these residues. The Cys17Ser, Cys270Ser, and Cys17Ser/Cys270Ser double mutants had similar median effective concentration (EC50) values for ADP and 2-methylthio–ADP (2-MeSADP) when compared with the wild-type P2Y12. Similarly, the median inhibitory concentration (IC50) values for BzATP (2′,3′-O-(4- benzoylbenzoyl) adenosine 5′-triphosphate), an antagonist of the P2Y12 receptor, also did not differ dramatically among these mutants and the wild-type P2Y12receptor. pCMBS inactivated the wild-type P2Y12 receptor in a concentration-dependent manner, whereas it had no effect on the P2Y1 receptor. Finally, pCMBS partially affected the Gi coupling of Cys17Ser or Cys270Ser receptor mutants, but had no effect on Cys17Ser/Cys270Ser P2Y12receptor–mediated inhibition of adenylyl cyclase. These results indicate that, unlike the P2Y1 receptor, which has 2 essential disulfide bridges linking its extracellular domains, the P2Y12 receptor has 2 free cysteines in its extracellular domains (Cys17 and Cys270), both of which are targets of thiol reagents. We speculate that the active metabolites of clopidogrel and CS-747 form disulfide bridges with both Cys17 and Cys270 in the P2Y12 receptor, and thereby inactivate the receptor.

The P2Y1 Receptor Is Necessary for Adenosine 5′-Diphosphate–Induced Platelet Aggregation

Blood ◽  
1998 ◽  
Vol 92 (1) ◽  
pp. 152-159 ◽  
Author(s):  
Béatrice Hechler ◽  
Catherine Léon ◽  
Catherine Vial ◽  
Paul Vigne ◽  
Christian Frelin ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Adenylyl Cyclase ◽  
Shape Change ◽  
Jurkat Cells ◽  
Molecular Entity ◽  
Calcium Stores ◽  
P2y1 Receptor ◽  
Adp Receptor ◽  
B10 Cells ◽  
Induced Aggregation

The human P2Y1 receptor heterologously expressed in Jurkat cells behaves as a specific adenosine 5′-diphosphate (ADP) receptor at which purified adenosine triphosphate (ATP) is an ineffective agonist, but competitively antagonizes the action of ADP. This receptor is thus a good candidate to be the elusive platelet P2T receptor for ADP. In the present work, we examined the effects on ADP-induced platelet responses of two selective and competitive P2Y1 antagonists, adenosine-2′-phosphate-5′-phosphate (A2P5P) and adenosine-3′-phosphate-5′-phosphate (A3P5P). Results were compared with those for the native P2Y1 receptor expressed on the B10 clone of rat brain capillary endothelial cells (BCEC) and for the cloned human P2Y1 receptor expressed on Jurkat cells. A2P5P and A3P5P inhibited ADP-induced platelet shape change and aggregation (pA2 = 5) and competitively antagonized calcium movements in response to ADP in fura-2–loaded platelets, B10 cells, and P2Y1-Jurkat cells. In contrast, these compounds had no effect on ADP-induced inhibition of adenylyl cyclase in platelets or B10 cells, whereas known antagonists of platelet activation by ADP such as Sp-ATPαS were effective. These identical signaling responses and pharmacologic properties suggest that platelets and BCEC share a common P2Y1 receptor involved in ADP-induced aggregation and vasodilation, respectively. This P2Y1 receptor coupled to the mobilization of intracellular calcium stores was found to be necessary to trigger ADP-induced platelet aggregation. The present results, together with data from the literature, also point to the existence of another as yet unidentified ADP receptor, coupled to adenylyl cyclase and responsible for completion of the aggregation response. Thus, the term, P2T, should no longer be used to designate a specific molecular entity.

scholarly journals The P2Y1 receptor is an ADP receptor antagonized by ATP and expressed in platelets and megakaryoblastic cells

FEBS Letters ◽  
1997 ◽  
Vol 403 (1) ◽  
pp. 26-30 ◽  
Author(s):  
Catherine Léon ◽  
Béatrice Hechler ◽  
Catherine Vial ◽  
Claude Leray ◽  
Jean-Pierre Cazenave ◽  
...  
Keyword(s):  
P2y1 Receptor ◽  
Adp Receptor

Effect of Dipyridamole on Spontaneous Platelet Aggregation in Whole Blood Decreases with the Time After Venepuncture: Evidence for the Role of ADP

1987 ◽  
Vol 58 (02) ◽  
pp. 744-748 ◽  
Author(s):  
A R Saniabadi ◽  
G D O Lowe ◽  
J C Barbenel ◽  
C D Forbes
Keyword(s):  
Platelet Aggregation ◽  
Correlation Coefficient ◽  
Whole Blood ◽  
Blood Platelet ◽  
Inhibitory Effect ◽  
Time Interval ◽  
Adp Receptor ◽  
Spontaneous Platelet Aggregation

SummarySpontaneous platelet aggregation (SPA) was studied in human whole blood at 3, 5, 10, 20, 30, 40 and 60 minutes after venepuncture. Using a whole blood platelet counter, SPA was quantified by measuring the fall in single platelet count upon rollermixing aliquots of citrated blood at 37° C. The extent of SPA increased with the time after venepuncture, with a correlation coefficient of 0.819. The inhibitory effect of dipyridamole (Dipy) on SPA was studied: (a) 10 μM at each time interval; (b) 0.5-100 μM at 3 and 30 minutes and (c) 15 μM in combination with 100 μM adenosine, 8 μM 2-chloroadenosine (2ClAd, an ADP receptor blocker) and 50 μM aspirin. There was a rapid decrease in the inhibitory effect of Dipy with the time after venepuncture; the correlation coefficient was -0.533. At all the concentrations studied, Dipy was more effective at 3 minutes than at 30 minutes after venepuncture. A combination of Dipy with adenosine, 2ClAd or aspirin was a more effective inhibitor of SPA than either drug alone. However, when 15 μM Dipy and 10 μM Ad were added together, the inhibitory effect of Dipy was not increased significantly, suggesting that Dipy inhibits platelet aggregation independent of Ad. The increase in SPA with the time after venepuncture was abolished when blood was taken directly into the anticoagulant containing 5 μM 2ClAd. It is suggested that ADP released from the red blood cells is responsible for the increased platelet aggregability with the time after venepuncture and makes a serious contribution to the artifacts of in vitro platelet function studies.

The Effect of Agonists and Antagonists of Platelet Aggregation on von Willebrand Factor-Mediated Platelet Agglutination

1991 ◽  
Vol 65 (05) ◽  
pp. 573-577 ◽  
Author(s):  
Jean McPherson ◽  
Marjorie B Zucker ◽  
Evelyn A Mauss ◽  
Sandra Brownlea
Keyword(s):  
Receptor Antagonist ◽  
Intracellular Calcium ◽  
Calcium Ions ◽  
Inhibitory Action ◽  
Platelet Rich Plasma ◽  
Cytoskeletal Proteins ◽  
A 23187 ◽  
Sulphydryl Groups ◽  
Affinity Receptor ◽  
Adp Receptor

SummaryRistocetin-induced platelet agglutination (RIPA) in EDTA-treated citrated platelet-rich plasma was reduced to 49 ± 11% by 1.25 ΜM ADP, 41 ± 14% by 1 ΜM A 23187, and 26 ± 7% by 0.1 Μg/ml platelet activating factor (PAF). The effect of 5-110 ΜM epinephrine was not dose-dependent, but varied between donors, with RIPA from 56-100% of the control. The inhibitory effects of these agonists were not altered by prior treatment of platelets with aspirin. Prior addition of 200 ΜM ATP (an ADP receptor antagonist acting at both high and low affinity ADP receptors) prevented the inhibitory action of ADP but not that of A 23187 or PAF, suggesting that the inhibitory actions of the latter are not mediated by released ADP. As 700 ΜM 8-bromoadenosine 5-diphosphate (an ADP receptor antagonist acting mainly at the high affinity receptor) did not prevent ADP-induced inhibition of RIPA, interaction of ADP with the low affinity receptor is presumably responsible for its inhibitory action. As A 23187, but not phorbol myristate acetate (0.1 ΜM) inhibited RIPA, an increase in intracellular calcium ions rather than direct stimulation of protein kinase C appears to mediate agonist-induced inhibition. Cytochalasin B (10.5-21 ΜM), dibucaine (0.5-1 mM), and prostaglandin E1 (25 nM), added before or after the agonist, prevented or reversed ADP-, A23187-, and PAF-induced inhibition of RIPA, suggesting that the state of the platelet cytoskeleton affects inhibition. N-ethylmaleimide (0.25-0.5 mM), an agent that can penetrate cell membranes and block sulphydryl groups, prevented or reversed ADP, A 23187- and PAF-induced inhibition of RIPA, but 0.5 mM dithionitrobisbenzoic acid, a non-penetrating sulphydryl blocker, had no effect. Diamide (0.1-0.5 mM), an agent that can crosslink cytoskeletal proteins by oxidation of sulphydryl groups, reduced RIPA. Thus an increase in intracellular calcium ions with resultant cytoskeletal changes and reorganisation of intracellular sulphydryl groups may mediate the inhibitory action of agonists on RIPA.

Deficiency of (33P)2MeS-ADP Binding Sites on Platelets with Secretion Defect, Normal Granule Stores and Normal Thromboxane A2 Production

1997 ◽  
Vol 77 (05) ◽  
pp. 0986-0990 ◽  
Author(s):  
Marco Cattaneo ◽  
Rossana Lombardi ◽  
Maddalena L Zighetti ◽  
Christian Gachet ◽  
Philippe Ohlmann ◽  
...  
Keyword(s):  
Binding Sites ◽  
Specific Binding ◽  
Thromboxane A2 ◽  
Normal Subjects ◽  
Congenital Defects ◽  
Normal Production ◽  
Adp Receptor ◽  
Partial Deficiency ◽  
Induced Aggregation ◽  
Platelet Secretion

SummaryBy the term “Primary Secretion Defect” (PSD), we mean a common heterogeneous group of congenital defects of platelet secretion, characterized by a normal primary wave of platelet aggregation induced by ADP and other agonists, a normal concentration of platelet granule contents, and normal production of thromboxane A2. The biochemical abnormalities responsible for PSD are not well known. Since a secretion defect similar to PSD is found in platelets that are severely deficient of binding sites for the ADP analogue 2MeS-ADP and do not aggregate in response to ADP, we tested the hypothesis that PSD platelets have moderately decreased 2MeS-ADP binding sites, which may be sufficient for normal ADP-induced aggregation but not for potentiating platelet secretion. The specific binding of [33P]2MeS-ADP to platelets from 3 PSD patients (347,443 and 490 sites/platelet; KD 2.8-3.9 nM) was lower than to platelets from 24 normal subjects (647 [530-1102]; KD = 3.8 [2.3-7.3]) (median [range]). Normal values were found in a fourth PSD patient (710; KD 3.7). The degree of inhibition of PGE1- induced cAMP increase by 0.1 |μM ADP was lower in patients than in controls. The secretion induced by the endoperoxide analogue U46619 from normal, acetylsalicylic acid-treated platelets under conditions that prevented the formation of large aggregates was potentiated by 1 fimol/1 ADP and inhibited by apyrase. These findings indicate that a partial deficiency of the platelet ADP receptor(s) might be responsible for the defect of platelet secretion in some PSD patients and that ADP potentiates platelet secretion independently of the formation of large aggregates and thromboxane A2 production.

Presence of P2X1 Purinoceptors in Human Platelets and Megakaryoblastic Cell Lines

1997 ◽  
Vol 78 (06) ◽  
pp. 1500-1504 ◽  
Author(s):  
Catherine Vial ◽  
Béatrice Hechier ◽  
Catherine Léon ◽  
Jean-Pierre Cazenave ◽  
Christian Gachet
Keyword(s):  
Intracellular Calcium ◽  
G Proteins ◽  
Cell Lines ◽  
Calcium Influx ◽  
Human Platelets ◽  
P2y1 Receptor ◽  
Calcium Response ◽  
Ionotropic Receptors ◽  
External Calcium

SummaryHuman platelets are thought to possess at least two subtypes of purinoceptor, one of which, coupled to G-proteins, could be the P2Y1 receptor (Léon et al. 1997). However, it has been suggested that the unique rapid calcium influx induced by ADP in platelets could involve P2X1 ionotropic receptors (MacKenzie et al. 1996) and the aim of this study was thus to investigate the presence of P2X purinoceptors in platelets and megakaryoblastic cells. Using PCR experiments, we found P2X1 mRNA to be present in human platelets and megakaryoblastic cell lines. In platelets, the selective P2X1 agonist αβMeATP induced a rise in intracellular calcium only in the presence of external calcium and this effect was antagonized by suramin and PPADS. Repeated addition of a�MeATP desensitized the P2X1 purinoceptor but only slightly affected the ADP response, while no calcium response to αβMeATP was observed in megakaryoblastic cells. These results support the existence of functional P2X1 purinoceptors on human platelets and the presence of P2X1 transcripts in megakaryoblastic cell lines.

scholarly journals Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism

2017 ◽  
Vol 596 (15) ◽  
pp. 3245-3269 ◽  
Author(s):  
Vishaal Rajani ◽  
Yong Zhang ◽  
Venkatesh Jalubula ◽  
Vladimir Rancic ◽  
Shahriar SheikhBahaei ◽  
...  
Keyword(s):  
Ventilatory Response ◽  
P2y1 Receptor ◽  
Hypoxic Ventilatory Response ◽  
Bötzinger Complex

Personalized ADP-receptor inhibition strategy and outcomes following primary PCI for STEMI (PASTOR study)

2016 ◽  
Vol 202 ◽  
pp. 463-466 ◽  
Author(s):  
Jussi Mikkelsson ◽  
Tuomas Paana ◽  
Aino Lepantalo ◽  
Pasi P. Karjalainen
Keyword(s):  
Primary Pci ◽  
Receptor Inhibition ◽  
Adp Receptor

P2Y13: Identification and Characterization of a Novel Gαi-Coupled ADP Receptor from Human and Mouse

2002 ◽  
Vol 301 (2) ◽  
pp. 705-713 ◽  
Author(s):  
Fang L. Zhang ◽  
Lin Luo ◽  
Eric Gustafson ◽  
Kyle Palmer ◽  
Xudong Qiao ◽  
...  
Keyword(s):  
Adp Receptor ◽  
Identification And Characterization ◽  
Human And Mouse
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