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Prepublished online as a Blood First Edition Paper on January 30, 2003; DOI 10.1182/blood-2002-10-3027.
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Blood, 15 May 2003, Vol. 101, No. 10, pp. 3908-3914
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Inactivation of the human P2Y12 receptor by thiol
reagents requires interaction with both extracellular cysteine
residues, Cys17 and Cys270
Zhongren Ding,
Soochong Kim,
Robert T. Dorsam,
Jianguo Jin, and
Satya P. Kunapuli
From the Department of Physiology, the
Department of Pharmacology, and The Sol Sherry Thrombosis Research
Center, Temple University School of Medicine, Philadelphia,
PA.
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 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 Cys17Ser or Cys270Ser receptor mutants, but
had no effect on Cys17Ser/Cys270Ser P2Y12
receptor-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.
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