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.

Pharmacological characterization of the human P2Y13 receptor.

The P2Y13 receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y12 receptor as well as similar functional properties: coupling to Gi and responsiveness to ADP (Communi et al., 2001). In the present study, the pharmacology of the P2Y13 receptor and its differences with that of the P2Y12 receptor have been further characterized in 1321N1 cells (binding of [33P]2-methylthio-ADP (2MeSADP) and of GTPgamma[35S]), 1321N1 cells coexpressing Galpha16 [AG32 cells: inositol trisphosphate (IP3) measurement, binding of GTPgamma[35S]) and Chinese hamster ovary (CHO)-K1 cells (cAMP assay)]. 2MeSADP was more potent than ADP in displacing [33P]2MeSADP bound to 1321N1 cells and increasing GTPgamma[35S] binding to membranes prepared from the same cells. Similarly, 2MeSADP was more potent than ADP in stimulating IP3 accumulation after 10 min in AG32 cells and increasing cAMP in pertussis toxin-treated CHO-K1 cells stimulated by forskolin. On the other hand, ADP and 2MeSADP were equipotent at stimulating IP3 formation in AG32 cells after 30 s and inhibiting forskolininduced cAMP accumulation in CHO-K1 cells. These differences in potency cannot be explained by differences in degradation rate, which in AG32 cells was similar for the two nucleotides. When contaminating diphosphates were enzymatically removed and assay of IP3 was performed after 30 s, ATP and 2MeSATP seemed to be weak partial agonists of the P2Y13 receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y13 receptor in AG32 cells was antagonized by reactive blue 2, suramin, pyridoxal-phosphate-6-azophenyl-2',4'disulfonic acid, diadenosine tetraphosphate, and 2-(propylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (AR-C67085MX), but not by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate (MRS-2179) (up to 100 microM). The most potent antagonist was N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-5'-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (ARC69931MX) (IC50 = 4 nM), which behaved in a noncompetitive way. The active metabolite of clopidogrel was unable to displace bound 2MeSADP at concentrations up to 2 microM.