The active metabolite of Clopidogrel disrupts P2Y12 receptor oligomers and partitions them out of lipid rafts
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.
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.
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 (3–10) or inhibit (11–13) dimer formation, as well as having no effect on preexisting constitutive homo- or heterodimers (14–25).
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).
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 (31–33). 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
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).
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).
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