- The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts

The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts


Pierre Savi,* Jean-Luc Zachayus, Nathalie Delesque-Touchard,* Catherine Labouret,* Caroline Hervé,* Marie-Françoise Uzabiaga,§ Jean-Marie Pereillo,§ Françoise Bono,* Pascual Ferrara, and Jean-Marc Herbert* Jean-Michel Culouscou,
Departments of *Thrombosis and Angiogenesis,
Genomic Sciences, and
§Analytical Research, Sanofi-Aventis Recherche, 195 Route d’Espagne, 31036 Toulouse, France
Edited by Barry S. Coller, The Rockefeller University, New York, NY, and approved May 4, 2006
To whom correspondence should be addressed. E-mail: [email protected]
Deceased August 8, 2004.
Author contributions: P.S. and J.-L.Z. designed research; J.-L.Z., N.D.-T., C.L., and C.H. performed research; N.D.-T., M.-F.U., and J.-M.P. contributed new reagents/analytic tools; P.S., J.-L.Z., N.D.-T., C.L., J.-M.C., and P.F. analyzed data; and P.S., J.-M.C., F.B., and J.-M.H. wrote the paper.
Received December 5, 2005.

Freely available online through the PNAS open access option.



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.

Keywords: mechanism of action, platelet, antiaggregant




Many G protein-coupled receptors (GPCRs) have been shown to assemble as homodimers, heterodimers, as well as larger oligomers (1, 2). The existence of such oligomeric entities raises questions as to their functional consequences as well as their physiological relevance. Heterologous expression systems have provided a variety of answers concerning ligand-dependent regulation of GPCR oligomeric states. Ligand binding, depending on the GPCR studied, can promote (310) or inhibit (1113) dimer formation, as well as having no effect on preexisting constitutive homo- or heterodimers (1425). The fact that heterodimerization may alter the pharmacological properties of a GPCR along with its internalization and signal transduction behavior is of critical importance (26, 27).

Clustering, even for nonheptahelical receptors, now appears as a common feature of cell signaling. Specialized structures such as clathrin-coated pits, caveolae, and lipid rafts contain high concentrations of signaling molecules. Rafts represent dynamic assemblies of proteins and lipids, mostly sphingolipids and cholesterol (28, 29). Proteins such as glycophosphatidylinositol-anchored proteins, nonreceptor tyrosine kinases, Gα subunits of heterotrimeric G proteins, and palmitoylated proteins appear to localize to these microdomains (30). In addition, recent studies have shown that partitioning of proteins in and out of rafts can depend on their state of activation or dimerization (3133). A variety of GPCR have also been identified in caveolae or rafts. These include α and β-adrenergic receptors (34, 35), adenosine A1 receptor (36), angiotensin II type 1 receptor (37), muscarinic receptor (38), EDG1 receptor (39), bradykinin B1 and B2 receptors (33, 40, 41), endothelin receptor (43), rhodopsin (44), and N-formyl peptide receptor (45). In the majority of cases, this location was found to be sensitive to ligand stimulation, clustering in raft being either increased or decreased.

Platelets are key elements in hemostasis and thrombosis. Diverse agonists are known to activate platelet aggregation and fibrinogen binding to the subsequently activated integrin GPIIb-IIIa complex. In this process, ADP is of particular importance, because it is released by damaged cells and activated platelets, thus enhancing the action of many platelet activators. ADP mediates platelet aggregation through its binding to two GPCRs P2Y1 and P2Y12, acting together to achieve complete aggregation (46). P2Y12 is expressed in platelets, megakaryocytes, and neuronal cells (47). Upon activation, P2Y12 triggers a cascade of signaling events including adenylyl cyclase inhibition and PI3K activation (48). P2Y12 knockout mice are particularly protected against thrombosis (49). In humans, two genetic P2Y12 deficiencies have been described, associated with a hemorrhagic phenotype and a pronounced impairment of ADP-induced platelet aggregation (50, 51).

P2Y12 is the target of clopidogrel, a well known antithrombotic compound that has demonstrated its efficacy and favorable safety profile in an extensive clinical program, by preventing ischemic events such as cardiovascular death, myocardial infarction, or stroke in atherothrombotic patients, on top of standard treatment (52). Clopidogrel does not, by itself, exhibit direct antiaggregant activity in vitro. Indeed, in vivo studies have demonstrated that clopidogrel has to undergo hepatic metabolization to obtain an active metabolite (53). This active metabolite (Act-Met) has been isolated, and its structure has been elucidated (54). It contains a free thiol function, and its activity is lost when the thiol is derivatized (55), suggesting its possible interaction with cysteine-containing sequences. In vitro, Act-Met inhibits the binding of 2MeS-ADP to platelets and ADP-induced aggregation of platelets. In a recent study, Act-Met was found to inhibit the binding of 2MeS-ADP to P2Y12 (56). This inhibition was shown to be irreversible and selective for P2Y12 (57, 58).

Here, we have further determined the mechanism of action of clopidogrel and of its active metabolite on the P2Y12 receptor. We have found that these compounds act on this receptor by an original mechanism, by interfering with P2Y12 assembly and its localization in lipid rafts. This allowed us to demonstrate the importance of oligomerization and membrane localization on the function of this receptor. Finally, we provide evidence for the molecular interaction between Act-Met and P2Y12.


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