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...

The P2Y 13 receptor has recently been identified as a new P2Y receptor sharing a high sequence homology with the P2Y 12 receptor as well as similar functional properties: coupling to G i and responsiveness to ADP (Communi et al., 2001). In the present study, the pharmacology of the P2Y 13 receptor and its differences with that of the P2Y 12 Similarly, 2MeSADP was more potent than ADP in stimulating IP 3 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 IP 3 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 IP 3 was performed after 30 s, ATP and 2MeSATP seemed to be weak partial agonists of the P2Y 13 receptor expressed in AG32 cells. The stimulatory effect of ADP on the P2Y 13 receptor in AG32 cells was antagonized by reactive blue 2, suramin, pyridoxal-phosphate-6-azophenyl-2Ј,4Јdisulfonic acid, diadenosine tetraphosphate, and 2-(propylthio)-5Ј-adenylic acid, monoanhydride with dichloromethylenebis (phosphonic acid) (AR-C67085MX), but not by N 6

ADP acts as an agonist of platelet aggregation via specific receptors which are still to be characterised. Amplification by PCR of a human platelet cDNA library confirmed the presence of mRNA of the P2Y1 receptor in platelets. In order to determine if these P2Y1 receptors were involved in ADP-induced platelet activation, we determined the effects of A3P5PS, an antagonist of the P2Y1 receptor, on the binding of [ QQ P]2-MeS-ADP, a potent analogue of ADP. We found that A3P5PS displaced about 27% of [ QQ P]2-MeS-ADP binding, a receptor population which has been shown to be resistant to treatment with clopidogrel, a selective anti-ADP agent. A3P5PS specifically inhibited 2-MeS-ADP-induced shape change and calcium increase but did not affect adenylyl cyclase down-regulation. 2-MeS-ADP-induced platelet aggregation was also inhibited by A3P5PS but was restored when platelets were further activated by serotonin, a non-aggregating compound, therefore suggesting that P2Y1-mediated stimulation is an absolute prerequisite for ADP to induce platelet aggregation and a key event for platelet activation and aggregation to occur. These results therefore show that ADP-induced aggregation cannot be attributed to activation of P2Y1 alone, but must be attributed to the simultaneous activation of the high affinity receptor (P2Y1) and a low affinity receptor of ADP (still to be discovered), each of them essential, but neither able to trigger aggregation alone.z 1998 Federation of European Biochemical Societies.

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SummaryThe aim of this study was to describe the pharmacological properties of SR 121787, a new antiaggregating drug which is metabolized in vivo into SR 121566, a potent non-peptide antagonist of Gp IIb/IIIa. In vitro, SR 121566 antagonized the binding of [125I]-fibrinogen (IC50 = 19.8 ± 6.3 nM) and of [125I]-L-692,884, an RGD-containing peptide (IC50 = 291 ± 96 nM) to activated human platelets. SR 121566 inhibited the aggregation of human platelets induced by ADP, collagen, thrombin, arachidonic acid and PAF at concentrations lower than 0.1 μM. Adhesion of human platelets to adhesive proteins was inhibited by SR 121566 (IC50 = 40.3 ± 2.5 nM) only when Gp IIb/IIIa and fibrinogen were involved. No effect was found with regard to other adhesive proteins and/or other integrins. SR 121787 demonstrated a potent and sustained antiaggregating effect when administered intravenously to baboons at a dose 50 μg/kg, and eight hours after the administration of 100 μg/kg, ADP-induced aggregation was still strongly inhibited (more than 80%). A single oral administration of 2 mg/kg of SR 121787 produced a nearly complete inhibition of platelet aggregation for up to 8 h (ED50 at 8 h = 193 ± 20 μg/kg), a significant residual antiaggregating activity being still observed 24h after the administration. When administered orally to rabbits, SR 121787 exhibited a potent antiaggregating (ED50 = 2.3 ± 0.3 mg/kg) and antithrombotic activity in an arterio-venous shunt thrombosis model (ED50 = 10.4 ± 0.8 mg/kg). After oral and IV administration, SR 121787 was well tolerated suggesting that SR 121787, the most potent and long lasting orally active Gp IIb/IIIa antagonist described to date, is a promising antithrombotic compound.