Increasing evidence strongly suggests that genetic variation is an important determinant of the wide interindividual variability in the response to clopidogrel therapy. Previous investigation in our laboratory indicated that ~70% of the variability in on-clopidogrel ADP-induced platelet aggregation, a widely accepted surrogate of clopidogrel response, can be attributed to heritable factors [3
]. Indeed, a genome-wide association study and several candidate gene studies have been carried out in both healthy individuals and patients with cardiovascular disease who have reported common polymorphisms significantly associated with active metabolite levels, platelet aggregation, and/or rates of adverse cardiovascular events [3
]. From these investigations and others, the most consistently replicated and perhaps clinically actionable genetic variant identified is the common loss-of-function cytochrome P450 2C19 (CYP2C19
)*2 variant (rs4244285), which accounts for ~12% of the interindividual variability in clopidogrel response [3
]. Other CYP2C19
loss-of-function variants (*3–*8) are much less common in the population, but would be expected to have a similar effect on clopidogrel response as CYP2C19
]. The effect of the common gain-of-function CYP2C19
*17 variant and variants in other candidate genes (e.g. ABCB1
, and P2RY12
) on-clopidogrel response have been evaluated, with mixed results [11
]. Despite these efforts, heritability estimates suggest that a large proportion of the variability in clopidogrel response explained by genetic factors remains unknown.
The oxidation of clopidogrel prodrug into 2-oxo-clopidogrel, followed by subsequent hydrolysis into its active thiol metabolite, is a two-step reaction catalyzed by cytochrome P450 enzymes. CES1 hydrolyzes a large number of endogenous and therapeutic compounds that contain functional thioester, carboxylic acid ester, and amide functional groups [26
]. Recent investigations have shown that CES1 is the primary enzyme responsible for hydrolyzing the methyl ester of clopidogrel, 2-oxo-clopidogrel, and the active metabolite, yielding carboxylic acid derivatives that are biologically inactive [11
]. Furthermore, the production of these inactive carboxylic acid derivatives occurs at a considerably higher rate compared with the formation of the clopidogrel active metabolite [11
]. Therefore, genetic variation affecting the activity or expression of CES1 may have important clinical relevance.
variant at position 143 results in a glycine-to-glutamic acid amino acid substitution that, on the basis of its location within the enzyme, would be predicted to considerably affect the catalytic function of CES1. X-ray crystallography data show that glycine 143 of CES1 constitutes, in part, an oxyanion hole near the active site catalytic triad (serine 221, histidine 468, and glutamic acid 354) [28
]. Oxyanion holes are used to stabilize substrate–enzyme intermediates and several investigations of other esterases show that abrogation of these moieties alters the catalytic function significantly [29
]. Consistent with these findings, previous in-vitro expression studies have shown that the 143E substitution resulted in a complete loss of catalytic function when methylphenidate was used as a substrate [6
]. Furthermore, the same study showed that when p
-nitrophenol acetate was used, the catalytic function (Vmax
) of CES1
143E was reduced to 21.4% compared with wild-type CES1 (143G). Clopidogrel or its active metabolites were not tested as substrates in this study.
To our knowledge, this is the first investigation to examine the effect of the CES1 G143E variant on-clopidogrel metabolism, ADP-induced platelet aggregation, and cardiovascular outcomes. In this study, we observed that PAPI participants carrying the decreased function CES1 143E-allele had significantly higher plasma levels of the clopidogrel active metabolite compared with 143G-allele homozygotes. All individuals received the same dose of parent clopidogrel and no difference in the prodrug level was observed between genotype groups. Consistent with metabolite data, we observed in two independent studies that CES1 143E-allele carriers had lower on-treatment ADP-induced platelet aggregation in response to clopidogrel treatment but not at baseline. Interestingly, we observed a moderate difference in the cardiovascular event rates between genotype groups (13.7% in CES1 143G-allele homozygotes vs. 0% in 143E-allele carriers) that did not achieve statistical significance in our sample size. Given that the frequency of the 143E carriers in the population is relatively rare (~1–2%), larger studies will be required to further elucidate whether they are more responsive to the antiplatelet actions of clopidogrel.
Although the CES1 G143E variant is relatively uncommon, it appears to exert a large effect on both clopidogrel metabolism and ADP-induced platelet aggregation even in its heterozygous state. In fact, its magnitude of effect on these traits is comparable, if not larger than that of the CYP2C19*2 variant. As reported previously in the same PAPI study, the absolute β value for the effect of CYP2C19*2 on ADP-induced platelet aggregation was 7.4, compared with 11.6 for CES1 143E ().
Effect of CES1 143E, CYP2C19*2, and CYP2C19*17 variants on postclopidogrel ADP-stimulated platelet aggregation in participants of the Amish Pharmacogenomics of Anti-Platelet Intervention study
Importantly, a recent meta-analysis has shown that carriers of the gain-of-function CYP2C19
*17 variant had an increased risk of experiencing an adverse bleeding event compared with noncarriers (odds ratio=1.25, 95% CI 1.07–1.47; P
]. In the PAPI study, the CES1
143E-allele has a much larger effect on on-clopidogrel ADP-induced aggregation than CYP2C19
*17, thus leading us to hypothesize that CES1
143E carriers may also be at a greater risk for bleeding events (). However, it is important to keep in mind that given the small number of CES1
143E-allele carriers in this study, it is also possible that the effect size of this variant has been overestimated. Given that prasugrel is inactivated pre-dominantly by CES2
and thus its activity would not be expected to be significantly affected by the 143E CES1
variant, additional studies will be required to investigate these hypotheses further [32