Adenosine 5’-diphosphate (ADP) activates platelets via the Gq-coupled P2Y1 and Gi-coupled P2Y12 receptors . Thus, any agent antagonizing either of these receptors is expected to inhibit platelet activation and hence can be used as an antithrombotic drug. Clopidogrel [2–4] and prasugrel [5,6] are thienopyridine antithrombotic drugs, metabolized in the liver to active metabolites that irreversibly antagonize the platelet P2Y12 receptor. AR-C69931MX, also known as Cangrelor, is a reversible P2Y12 antagonist .
In this study, we investigated the molecular mechanism by which clopidogrel, prasugrel or AR-C69931MX antagonizes the P2Y12 receptor. The active metabolites derived from clopidogrel and prasugrel contain a reactive thiol group. The formation of a disulfide bond between the thiol group in the active metabolite and the extracellular cysteines of the P2Y12 receptor has been proposed to be the mechanism by which clopidogrel antagonizes the P2Y12 receptor [4,8]. The P2Y12 receptor contains four extracellular cysteines: C17, C97, C175, and C270. The effect of these metabolites on C17 and C270 has not been investigated. Thus, we investigated whether clopidogrel and prasugrel active metabolites also target C17 and C270 of the P2Y12 receptor and antagonize it. In our study, clopidogrel and prasugrel active metabolites antagonized both wild-type (WT), C17S/C270S double mutant P2Y12 receptors expressed in CHO-K1 cells (data not shown). These results suggest that C17 and C270 are not the target sites of thienopyridine-derived antithrombotic agents.
The difference in the results obtained with pCMBS in our previous study  and the active metabolites of clopidogrel and prasugrel (this study) can be explained by the differences in the natures of these compounds. pCMBS is a thiol reagent that can attack any free cysteine residue. However, the active metabolites of thienopyridines are thiol compounds, which can either form a disulfide bridge with free cysteines or reduce an existing disulfide bridge. It appears that the latter is the more effective mechanism by which these active metabolites inactivate the P2Y12 receptor.
Savi et al.  recently reported that clopidogrel active metabolites antagonize the P2Y12 receptor by dissociating P2Y12 receptor oligomers into monomers. However, in their study, oligomer disruption was studied by pretreating CHO-K1 cells expressing P2Y12 receptor and human platelets with clopidogrel active metabolite for 1 h. As the antagonistic effects of clopidogrel and prasugrel active metabolites were profound within 90 s in human platelets (Figs. 1A.1 and B.1) and 10 min in CHO-K1 cells (data not shown), we tested whether P2Y12 oligomers were also disrupted within this time.
In order to evaluate the effect of the clopidogrel active metabolite (CAM) on the oligomeric structures of the P2Y12 receptor, we incubated CHO-K1 cells expressing the WT HA-tagged P2Y12 receptor with 4 and 10 μmol L−1 CAM for 10 and 60 min. The cells were then lysed under non-reducing (−DTT) and reducing (+DTT) conditions. The oligomeric or monomeric forms of the P2Y12 receptor were studied by subjecting lysates to western blot analysis using anti-HA-tag antibodies. Consistent with the results of Savi et al., we also observed the oligomer and the monomer of human P2Y12 receptors in recombinant CHO-K1 cells (Fig. 1A.2). The P2Y12 receptor monomer size is also consistent with results from other investigators [11,12] and was confirmed by immunoprecipitating the P2Y12 receptor with anti-HA-tag antibodies, and then analyzing under reducing conditions (data not shown). As shown in Fig. 1A.2, although CAM disrupted P2Y12 receptor oligomers when incubated for 60 min, it failed to disrupt P2Y12 oligomers in CHO-K1 cells incubated for 10 min, the time within which CAM antagonized the P2Y12 receptor (data not shown). Furthermore, we did observe that P2Y12 receptor oligomers were disrupted after DTT treatment (reducing conditions; Fig. 1A.2, lanes with DTT). These results are consistent with those of Savi et al., and suggest that P2Y12 receptor oligomers are formed by disulphide bonds between the P2Y12 receptor monomers. The above results also indicate that P2Y12 receptor monomers can be detected under our working conditions. Alternatively, we also analyzed whether CAM disrupts P2Y12 receptor oligomers in human platelets. We treated human platelets with 4 and 10 μmol L−1 CAM (Fig. 1A.3) for 90 s (conditions where antagonistic effects are exhibited; Fig. 1A.1). The effect on P2Y12 oligomers was tested under both reducing and non-reducing conditions by western blotting using anti-P2Y12 antibodies. As shown in Fig. 1A.3, both the oligomers (∼150 kDa) and monomers (∼50 kDa) were detected in human platelets. The specificity of the anti-P2Y12 antibodies was evaluated by neutralizing the antibody with a blocking peptide. Treatment of human platelets with 4 or 10 μmol L−1 CAM for 90 s did not affect P2Y12 receptor oligomers (Fig. 1A.3, lanes labeled −DTT). However, these oligomers were disrupted when treated with DTT (Fig. 1A.3, lanes labeled +DTT). Taken together, these results indicate that CAM does not antagonize the P2Y12 receptor by disrupting oligomers to monomers.
We also analyzed whether the prasugrel active metabolite (PAM) antagonizes the P2Y12 receptor by dissociating oligomers. CHO-K1 cells stably expressing WT human P2Y12 receptor were treated with 10 μmol L−1 PAM for 10 min (Fig. 1B.2) and disruption of P2Y12 oligomers was studied as described earlier. Although treatment of CHO-K1 cells with 10 μmol L−1 PAM for 10 min antagonized P2Y12 mediated effects (data not shown), the same treatment failed to dissociate P2Y12 oligomers (Fig. 1B.2, lanes labeled –DTT). However, as shown before under reducing conditions, P2Y12 receptor oligomers were disrupted (Fig. 1B.2, lanes labeled +DTT). Thus, although we were able to detect P2Y12 receptor monomers, PAM by itself failed to disrupt P2Y12 receptor oligomers. We further investigated whether treatment of human platelets with 10 μmol L−1 PAM for 10 min disrupts P2Y12 receptor oligomers, as the same antagonizes P2Y12 receptor (Fig. 1B.1). As shown in Fig. 1B.3 (lanes labeled −DTT), 10 min pretreatment with PAM failed to disrupt P2Y12 oligomers under non-reducing conditions, although the oligomers were disrupted in lysates treated with or without PAM under reducing conditions (Fig. 1B.3, lanes labeled +DTT).
We finally investigated whether a reversible antagonist would affect the oligomeric structures of the P2Y12 receptor and antagonize it. As shown in Fig. 1C (lanes labeled −DTT), treatment of platelets with 100 nM AR-C69931MX for 1 and 10 min did not dissociate the oligomers of the P2Y12 receptor, when lysates were made under non-reducing conditions. However, AR-69931MXC antagonizes the P2Y12 receptor under these conditions (data not shown). Furthermore, DTT disrupted the oligomers (Fig. 1C, lanes labeled +DTT).
Based on these results, we conclude that dissociation of P2Y12 receptor oligomers into monomers is not a required mechanism by which thiol group containing active metabolites antagonize P2Y12 receptor. We speculate that C97 and/or C175 residues in platelet P2Y12 receptor are likely the targeting sites of the active metabolites of clopidogrel and prasugrel.