The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts.
P2Y12, a G protein-coupled receptor that plays a central role in platelet activation has been recently identified as the receptor targeted by the antithrombotic drug, clopidogrel. In this study, we further deciphered the mechanism of action of clopidogrel and of its active metabolite (Act-Met) on P2Y12 receptors. Using biochemical approaches, we demonstrated the existence of homooligomeric complexes of P2Y12 receptors at the surface of mammalian cells and in freshly isolated platelets. In vitro treatment with Act-Met or in vivo oral administration to rats with clopidogrel induced the breakdown of these oligomers into dimeric and monomeric entities in P2Y12 expressing HEK293 and platelets respectively. In addition, we showed the predominant association of P2Y12 oligomers to cell membrane lipid rafts and the partitioning of P2Y12 out of rafts in response to clopidogrel and Act-Met. The raft-associated P2Y12 oligomers represented the functional form of the receptor, as demonstrated by binding and signal transduction studies. Finally, using a series of receptors individually mutated at each cysteine residue and a chimeric P2Y12/P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met.
Savi
Pierre
P
Department of Thrombosis and Angiogenesis, Sanofi-Aventis Recherche, 195 Route d'Espagne, 31036 Toulouse, France. pierre.savi@sanofi-aventis.com
Zachayus
Jean-Luc
JL
Delesque-Touchard
Nathalie
N
Labouret
Catherine
C
Hervé
Caroline
C
Uzabiaga
Marie-Françoise
MF
Pereillo
Jean-Marie
JM
Culouscou
Jean-Michel
JM
Bono
Françoise
F
Ferrara
Pascual
P
Herbert
Jean-Marc
JM
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D000077144:Clopidogrel; D006801:Humans; D021962:Membrane Microdomains / Q000187:drug effects* / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058925:Receptors, Purinergic P2Y12; D013988:Ticlopidine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology
C548576:P2RY12 protein, human; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12; D000244:Adenosine Diphosphate; D000077144:Clopidogrel; D013988:Ticlopidine