SEARCH:   Advanced

Prepublished online as a Blood First Edition Paper on January 30, 2003; DOI 10.1182/blood-2002-10-3027. This Article Full Text (PDF) All Versions of this Article: 2002-10-3027v1 101/10/3908    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Ding, Z. Articles by Kunapuli, S. P Search for Related Content PubMed PubMed Citation Articles by Ding, Z. Articles by Kunapuli, S. P Social Bookmarking                   What's this? Submitted October 18, 2002 Accepted December 18, 2002 Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, C17 and C270 Zhongren Ding, Soochong Kim, Robert T Dorsam, Jianguo Jin, and Satya P Kunapuli* Department of Physiology, Temple University Medical School, Philadelphia, PA, USA Department of Pharmacology, Temple University Medical School, Philadelphia, PA, USA Sol Sherry Thrombosis Research Center, Temple University Medical School, Philadelphia, PA, USA * Corresponding author; email: kunapuli{at}nimbus.temple.edu. Human platelets express two G protein-coupled nucleotide receptors: the P2Y1 receptor, coupled to Gq, and the P2Y12 receptor, coupled to Gi. Although these two receptors have similar pharmacological profiles, they have different reactivities towards thiol reagents. The thiol agent p-chloromercuribenzenesulfonic acid (pCMBS), and the active metabolites from antiplatelet drugs, clopidogrel and CS747, 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. C97S and C175S mutants of the P2Y12 receptor did not express when transfected into CHO-K1 cells, indicating the essential nature of a disulfide bridge between these residues. The C17S, C270S, and C17S/C270S double mutants had similar EC50 values for ADP and 2MeSADP when compared with the wild type P2Y12. Similarly, IC50 values for BzATP, an antagonist of the P2Y12 receptor, also did not differ dramatically among these mutants and the wild type P2Y12 receptor. 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 C17S or C270S receptor mutants, but had no effect on C17S/C270S P2Y12 receptor-mediated inhibition of adenylyl cyclase. These results indicate that, unlike the P2Y1 receptor, which has two essential disulfide bridges linking its extracellular domains, the P2Y12 receptor has two free cysteines in its extracellular domains (C17 and C270), both of which are targets of thiol reagents. We speculate that the active metabolites of clopidogrel and CS747 form disulfide bridges with both C17 and C270 in the P2Y12 receptor, and thereby inactivate the receptor. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: N. F. Ford Clopidogrel Resistance: Pharmacokinetic or Pharmacogenetic? J. Clin. Pharmacol., May 1, 2009; 49(5): 506 - 512. [Abstract] [Full Text] [PDF] Y. Nishiya, K. Hagihara, T. Ito, M. Tajima, S.-i. Miura, A. Kurihara, N. A. Farid, and T. Ikeda Mechanism-Based Inhibition of Human Cytochrome P450 2B6 by Ticlopidine, Clopidogrel, and the Thiolactone Metabolite of Prasugrel Drug Metab. Dispos., March 1, 2009; 37(3): 589 - 593. [Abstract] [Full Text] [PDF] C. Patrono, C. Baigent, J. Hirsh, and G. Roth Antiplatelet Drugs: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition) Chest, June 1, 2008; 133(6_suppl): 199S - 233S. [Abstract] [Full Text] [PDF] P. Savi, J.-L. Zachayus, N. Delesque-Touchard, C. Labouret, C. Herve, M.-F. Uzabiaga, J.-M. Pereillo, J.-M. Culouscou, F. Bono, P. Ferrara, et al. The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts PNAS, July 18, 2006; 103(29): 11069 - 11074. [Abstract] [Full Text] [PDF] J. Turgeon, C. Pharand, and V. Michaud Understanding clopidogrel efficacy in the presence of cytochrome P450 polymorphism Can. Med. Assoc. J., June 6, 2006; 174(12): 1729 - 1729. [Full Text] [PDF] B. T. Ivandic, P. Schlick, P. Staritz, K. Kurz, H. A. Katus, and E. Giannitsis Determination of Clopidogrel Resistance by Whole Blood Platelet Aggregometry and Inhibitors of the P2Y12 Receptor Clin. Chem., March 1, 2006; 52(3): 383 - 388. [Abstract] [Full Text] [PDF] Z. Ding, S. Kim, and S. P. Kunapuli Identification of a Potent Inverse Agonist at a Constitutively Active Mutant of Human P2Y12 Receptor Mol. Pharmacol., January 1, 2006; 69(1): 338 - 345. [Abstract] [Full Text] [PDF] N. von Beckerath, D. Taubert, G. Pogatsa-Murray, E. Schomig, A. Kastrati, and A. Schomig Absorption, Metabolization, and Antiplatelet Effects of 300-, 600-, and 900-mg Loading Doses of Clopidogrel: Results of the ISAR-CHOICE (Intracoronary Stenting and Antithrombotic Regimen: Choose Between 3 High Oral Doses for Immediate Clopidogrel Effect) Trial Circulation, November 8, 2005; 112(19): 2946 - 2950. [Abstract] [Full Text] [PDF] A. R. Giniatullin, S. N. Grishin, E. R. Sharifullina, A. M. Petrov, A. L. Zefirov, and R. A. Giniatullin Reactive oxygen species contribute to the presynaptic action of extracellular ATP at the frog neuromuscular junction J. Physiol., May 15, 2005; 565(1): 229 - 242. [Abstract] [Full Text] [PDF] T. A. Nguyen, J. G. Diodati, and C. Pharand Resistance to clopidogrel: A review of the evidence J. Am. Coll. Cardiol., April 19, 2005; 45(8): 1157 - 1164. [Abstract] [Full Text] [PDF] Z. Ding, F. Tuluc, K. R. Bandivadekar, L. Zhang, J. Jin, and S. P. Kunapuli Arg333 and Arg334 in the COOH terminus of the human P2Y1 receptor are crucial for Gq coupling Am J Physiol Cell Physiol, March 1, 2005; 288(3): C559 - C567. [Abstract] [Full Text] [PDF] F. Krotz, H.-Y. Sohn, and U. Pohl Reactive Oxygen Species: Players in the Platelet Game Arterioscler Thromb Vasc Biol, November 1, 2004; 24(11): 1988 - 1996. [Abstract] [Full Text] [PDF] C. Patrono, B. Coller, G. A. FitzGerald, J. Hirsh, and G. Roth Platelet-Active Drugs: The Relationships Among Dose, Effectiveness, and Side Effects: The Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy Chest, September 1, 2004; 126(3_suppl): 234S - 264S. [Abstract] [Full Text] [PDF] H. Shankar, S. Murugappan, S. Kim, J. Jin, Z. Ding, K. Wickman, and S. P. Kunapuli Role of G protein-gated inwardly rectifying potassium channels in P2Y12 receptor-mediated platelet functional responses Blood, September 1, 2004; 104(5): 1335 - 1343. [Abstract] [Full Text] [PDF] T. Richter, T. E. Murdter, G. Heinkele, J. Pleiss, S. Tatzel, M. Schwab, M. Eichelbaum, and U. M. Zanger Potent Mechanism-Based Inhibition of Human CYP2B6 by Clopidogrel and Ticlopidine J. Pharmacol. Exp. Ther., January 1, 2004; 308(1): 189 - 197. [Abstract] [Full Text] [PDF] J. Lahav, E. M. Wijnen, O. Hess, S. W. Hamaia, D. Griffiths, M. Makris, C. G. Knight, D. W. Essex, and R. W. Farndale Enzymatically catalyzed disulfide exchange is required for platelet adhesion to collagen via integrin {alpha}2{beta}1 Blood, September 15, 2003; 102(6): 2085 - 2092. [Abstract] [Full Text] [PDF]

	Copyright © 2003 by American Society of Hematology         Online ISSN: 1528-0020