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

Abstract

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 P2Y12͞P2Y13 receptor, we pointed out the involvement of cysteine 97 within the first extracellular loop of P2Y12 in the mechanism of action of Act-Met. mechanism of action ͉ platelet ͉ antiaggregant M any 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).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, G␣ 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 ␣ and ␤-adrenergic receptors (34, 35), adenosine A1 receptor (36), angiotensin II type 1 receptor (37), muscarinic receptor (38), EDG1 receptor (39), bradykinin B1 and B2 receptor...

P2X1 receptor-mediated inhibition of the proliferation of human coronary smooth muscle cells involving the transcription factor NR4A1

“…Our present results demonstrate for the first time that the P2X 1 receptor agonist α,β-methylene-ATP inhibits both proliferation and migration of human coronary smooth muscle cells. Blockade of P2Y 12 receptors by antagonists such as ticagrelor [61] or by active metabolites of clopidogrel or prasugrel [62,63] may direct effects of endogenous adenine nucleotides to inhibition of the progression of arteriosclerosis by an action on smooth muscle P2X 1 receptors.…”

P2 receptors and platelet function

“…The active metabolite of clopidogrel [107] covalently binds cysteine residues of the P2Y 12 receptor, thus precluding the binding of ADP [108][109][110]. Moreover, it has been recently reported that clopidogrel's active metabolite disrupts homopolymers of the P2Y 12 receptor expressed in lipid rafts and partitions them out of lipid rafts [111], pointing to the importance of oligomerization and membrane localization on the function of this receptor. Further studies are however required to confirm these findings.…”

Ticagrelor binds to human P2Y12 independently from ADP but antagonizes ADP‐induced receptor signaling and platelet aggregation

“…Thus, contamination with trace amounts of ATP seems to be more likely. However, as the presence of lipid rafts may be crucial for the activity of P2Y 12 [23], it would be interesting to study whether high ADP concentrations affect the formation or composition of lipid rafts.…”