Clopidogrel is a key antiplatelet agent that inhibits the second step of platelet aggregation through blockade of the P2Y12-adenosine diphosphate (ADP) receptor.1 Its use has enabled percutaneous coronary intervention (PCI) to expand by dramatically reducing thrombotic complications in acute coronary syndromes (ACSs).2,3 Since the first report by Jaremo et al. of a large inter-individual variability in the inhibitory effect of clopidogrel on platelet reactivity (PR), a number of studies have highlighted the critical role of PR inhibition in the prevention of thrombotic and bleeding complications in patients with an ACS undergoing PCI.4–12
As clopidogrel is a prodrug that only exerts a mild inhibitory effect after biotransformation in the liver, it has been observed that a significant number of patients are unable to reach an optimal level of PR inhibition after drug intake, even when high loading dose is used. Several mechanisms are involved in what was called ‘clopidogrel resistance’, but better fits the term ‘high on-treatment platelet reactivity’ (HTPR). Intrinsic and extrinsic factors are responsible for HTPR and can act alone or in combination, causing insufficient inhibition of PR by clopidogrel.13 In particular, genetic polymorphisms have been shown to have a critical role in shaping the individual’s response to the drug. Importantly, both PR and genetic testing have been shown to be predictive of thrombotic and bleeding complications following PCI.
These recent findings prompted the cardiology community to replace the ‘one fits it all’ dogma with the potential benefits of individualised antiplatelet regimens. Despite this, whether genetic or platelet function testing should be used routinely, if it all, remains undecided. Further, the potential usefulness of such individualised therapy remains to be put into perspective in view of the availability of new antiplatelet agents.
Clopidogrel is a prodrug that requires bio-transformation in the liver into an active metabolite to exert its antiplatelet properties. Most of the clopidogrel absorbed (~85–90%) is hydrolysed by carboxylase into an inactive carboxylic acid metabolite; whereas the remaining ~10–15% is metabolised by hepatic cytochrome (CYP) P450 isoenzymes in a two-step process. In the first step, the thiophene ring of clopidogrel is oxidized to 2-oxo-clopidogrel, which is then hydrolysed to a labile active metabolite, which has both carboxylic acid and thiol groups.14–16 CYP2C19, CYP1A2 and CYP2B6 participate in the first metabolic step. CYP2C19, CYP2C9, CYP2B6 and CYP3A are responsible for the second step.14,15
The highly unstable active metabolites generated specifically bind to the platelet P2Y12 receptor, resulting in an irreversible blockade of the receptor and thus of platelet activation-aggregation.17 This complex biotransformation is responsible for the slow onset of clopidogrel action and its large inter-individual variability.
Inter-individual Variability in Clopidogrel Responsiveness
Variable and insufficient active metabolite generation are the primary explanations for the variability of response to clopidogrel. The mechanisms responsible for HTPR are numerous and include clinical factors, baseline individual variability and genetic polymorphism (see Table 1). The main clinical factors are poor compliance, inadequate doses and high body mass index. In addition, patients suffering from an ACS have an increased baseline PR and therefore more often exhibit HTPR.
Individual differences or drug–drug interactions in intestinal absorption or in cytochrome P450 (CYP450) isoenzyme activity may also account for variability in response to the drug. Indeed, co-administration of CYP450 isoenzyme-inhibiting drugs (such as a proton-pump inhibitor or calcium-channel blocker) may reduce the efficacy of clopidogrel.13
Internal factors of inter-individual variability include genetic polymorphisms of platelet receptors, GP IIb/IIIa receptors, liver cytochrome P450 isoenzymes and up-regulation of platelet activation pathways. The key role of 2C19*2 polymorphism has recently been demonstrated and is consistent with its involvement in both steps of clopidogrel biotransformation.18–20 Studies have observed that the 2* allele is a loss-of-function polymorphism that is responsible for HTPR and the 17* allele is a gain-of-function polymorphism associated with a high level of PR inhibition. Thus, this single gene determination could be useful in predicting response to clopidogrel.18
HTPR is mainly related to a high baseline PR or reduced clopidogrel effect. As baseline platelet reactivity and the impact of the drug cannot be clinically predicted, there is the need for platelet reactivity or genetic testing to assess whether PR inhibition is optimal.
The Predictive Value of Platelet Reactivity for Thrombosis and Bleeding
Jaremo et al. first described the lack of uniform response to clopidogrel between patients using ADP-evoked platelet fibrinogen binding using a flow cytometry technique with two ADP solutions.4 Following this report, Barragan et al. demonstrated a link between the level of PR inhibition achieved and thrombotic events after PCI using the vasodilator-stimulated phosphoprotein (VASP) index.5 These findings led the way to several other studies using different platelet assays that confirmed the predictive value of a single measurement of PR before PCI for short- and long-term outcomes.6–12
For years, there was controversy over which test and which cut-off value to use. Recently, authors have been able to identify PR thresholds to predict thrombotic events with a very high negative value using receiver operating characteristic (ROC) curve analysis. For example, with the VerifyNow P2Y12 device, the cut-off value is 235–240 PRU. This device exhibited a predictive value of 100% in a study by Price et al.8
Following these findings, a consensus on the definition of HTPR was recently proposed by summarising the results of all these studies21 (see Table 2). Thus, the question of which test and which cut-off value to use was answered. The consensus statement enables clinicians to use PR thresholds that have a clinical value and to differentiate patients with a high and low risk of events following PCI.
Achieving a high level of PR inhibition is critical to prevent thrombotic events; however, excessive inhibition could in theory lead to increased bleeding. In fact, more potent agents such as prasugrel or ticagrelor are associated with decreased thrombotic events compared with clopidogrel, but at the price of increased bleeding.22,23 In addition to this, Sibbing et al. have recently observed a link between the level of PR inhibition and major bleeding due to non-coronary artery bypass graft-related thrombosis in myocardial infarction (TIMI).24 These consistent data are in line with a relationship between excessive PR and bleeding with P2Y12 ADP receptor-blockers.
Therefore, accumulating data supports a therapeutic window of PR inhibition to prevent both thrombotic and bleeding events. Finally, the use of PR to tailor therapy could be greatly beneficial in patients undergoing PCI to prevent under- or overdosage of the drug and its clinical consequences.
Genetics to Predict Thrombosis and Bleeding
Among the intrinsic factors associated with HTPR, the recent focus has been on CYP 2C19 polymorphisms. In fact, in healthy volunteers a 32.4% relative reduction (p<0.001) of active clopidogrel metabolites in the plasma and a relative reduction of approximately 25% in mean platelet aggregation (p<0.001) was observed in carriers of the reduced-function CYP2C19 allele.25–27 These observations suggest that CYP2C19*2 heterozygotes have one normal CYP2C19 allele and maintain partial enzymatic activity, while CYP2C19*2 homozygotes have little or no enzymatic activity.28
Accordingly, among patients with ACS undergoing stenting and treated with clopidogrel in the TRITON–TIMI 38 study, the reduced-function CYP2C19 allele carriers had a higher rate of major adverse cardiac events (hazard ratio [HR] 1.53; p=0.01), including stent thrombosis (HR 3.09; p=0.02), compared with non-carriers.25 Of importance, the frequency of this allele in the community is high: 30% of Caucasians and 40% of Asians carry it.
Similarly, Sibbing et al. demonstrated that CYP2C19*2 carriers had a significantly higher cumulative 30-day incidence of stent thrombosis compared with CYP2C19 wild-type homozygotes (HR 3.81; p<0.007).29 These findings have been confirmed by a recent meta-analysis showing a HR of 1.61 (p<0.001) for major adverse cardiac events in carriers of CYP2C19*2 alleles.20 The risk was greater in CYP2C19*2 heterozygotes and homozygotes compared with carriers wild-type alleles (HR 1.50; p=0.016 and HR 1.81; p=0.004). On the other hand, the 17* allele of the same gene is a gain-of-function allele associated with significantly lower ADP-induced platelet aggregation following clopidogrel intake (p<0.039).30 Of importance, 17* carriers have an approximately two-fold increase in risk of TIMI major and minor bleeding.26,27,30
The fact that these single-nucleotide polymorphisms are not related to baseline platelet reactivity is consistent with their key role as genetic mediators of clopidogrel response.31
Despite these important data, however, no single study has demonstrated a conclusive link between:
- the presence of a genetic polymorphism;
- the generation clopidogrel’s active metabolite (pharmacokinetic measurement);
- clopidogrel responsiveness (pharmacodynamic measurement); and
- clinical outcomes.
Similarly, very little is known about the less common loss-of-function variants including *3, *4, *5, *6, *7 and *8.
The heritability of clopidogrel response is approximately 70% and the CYP2C19*2 genotype only accounts for about 12% of variability in clopidogrel response. For this reason, the majority of factors, both genetic and non-genetic, influencing variability of response to clopidogrel are not taken into account by the assessment of this single allele. To date, it has been demonstrated that the sensitivity of the *2 carrier state for detecting HTPR is only 56%.31
Since genetic polymorphism of CYP2C19 only accounts for a small part of HTPR (12%),31 it is probable that genetic testing for 2C19 polymorphisms would be inferior to PR assessment, which takes into account the external and internal factors responsible for HTPR. In addition, a genetic test that would require several genes to be tested for would be very complex to perform compared with a single measurement of PR at the time of PCI.
It must be noted there are a lack studies comparing the predictive value of PR and genetic testing for thrombotic and bleeding events.
There is a large body of evidence demonstrating that HTPR is associated with an increased risk of thrombotic events. Therefore, various therapeutic strategies have been proposed to decrease ischaemic risk in patients with HTPR, including:
- increasing clopidogrel dose or tailoring therapy according to PR monitoring;32–34
- switching to ticlopidine;35
- the addition of cilostazol;36
- periprocedural platelet glycoprotein (GP) IIb/IIIa inhibition;37 and
- the use of novel P2Y12 receptor inhibitors, such as prasugrel, ticagrelor or elinogrel.38–42
Increasing the dose of clopidogrel may accelerate the time course and enhance the magnitude of subsequent platelet inhibition. However, despite doses of up to 2,400mg approximately 10% of patients still had HTPR according to the cut-off value of ≤50% using the VASP index.32,33 A small number of patients with HTPR will become good responders when using a clopidogrel maintenance dose of 150g/day using same cut-off value.34
Although periprocedural GP IIb/IIIa receptor blockade is effective in reducing peirprocedural ischaemic events among these patients,37 30- day and long-term outcomes are not available. Thus, it is likely that because GP IIb/IIIa inhibitors have a short half life, the rate of events after infusion is stopped will be high.
New agents are now becoming available and could be of great interest in preventing HTPR. Prasugrel, a third-generation thienopyridine, provides more effective P2Y12 receptor inhibition than clopidogrel. This has been related to its faster and more uniform PR inhibition.38 Importantly, the biological response to prasugrel and its clinical outcome are not influenced by CYP2C19*2-carrier status.39 Ticagrelor, a novel reversible cyclopentyl triazolopyrimidine nonthienopyridine, is a direct-acting P2Y12 receptor inhibitor. It has been shown to be highly effective in patients with HTPR and to provide optimal PR inhibition.41,42
Prasugrel and ticagrelor are also very effective in reducing thrombotic events in ACS patients, although at the price of a higher bleeding rate. This fact mirrors the statement made by the US Food and Drug Administration (FDA) urging clinicians to ‘consider use of other antiplatelet medications or alternative dosing strategies for clopidogrel. ’Finally, elinogrel is a reversible, direct-acting P2Y12 receptor inhibitor. It significantly reduced aggregation and the portion of subjects with HPR among clopidogrel non-responders.43 However, outcome data are not available.
The clinical impact of tailoring antiplatelet therapy according to PR or genetic testing is on the horizon. To date only small-scale studies have determined whether PR testing to individualise therapy could improve outcome. In these two studies, loading dose adjustment was performed according to PR monitoring. The goal was to obtain a VASP index below the thrombotic threshold of 50% in order to reach optimal PR inhibition in patients before PCI. This strategy was efficient in 90% of patients with HTPR, irrespective of their CYP2C19 status.44 This therapeutic strategy was clinically beneficial, causing a dramatic reduction in sub-acute stent thrombosis rates without any increase in bleeding. Larger studies are being performed to confirm these preliminary results.
Several large-scale studies, including the Gauging Responsiveness With A VerifyNow Assay – Impact On Thrombosis And Safety (GRAVITAS) and Thrombocyte Activity Reassessment and Genotyping for PCI (TARGET PCI), will help to answer the question of whether individualised therapy is clinically benefical. The results of the GRAVITAS studies will soon be available and are likely to confirm preliminary studies.
There is currently a lack of direction in the use of genetic testing to tailor clopidogrel therapy in poor metabolisers on the basis of genotyping. In a recent multicentre prospective study it was demonstrated that genetic testing did not add to PR monitoring in tailoring clopidogrel therapy to obtain optimal PR inhibition in ACS patients undergoing PCI. In this trial, homo- and heterozygote carriers of CYP 2C19 responded similarly to wild-type carriers with regards to tailored therapy (see Figure 1). Thus, it seems that when a very high dose of clopidogrel is used, genetic status no longer matters. From this study it can be hypothesised that PR testing is enough to tailor therapy. This is supported by the fact that no appropriate therapeutic response to the presence of CYP2C19*17 has yet been defined. No study to date has compared the use of PR and genetic testing in tailored therapy. This will be the goal of the TARGET PCI trial.
It must be noted that in a recent study, the influence of both HTPR and genotyping on clinical outcomes were evaluated separately. Although CYP2C19*2 and HTPR had comparatively high specificity (72% and 79%, respectively), each factor independently only identified 46% of patients with events. Interestingly, 75% of patients with events were identified when both risk factors were combined.31 The latter observation suggests that both genotyping and ex vivo platelet function testing may be more predictive than either alone.
Finally, the potential clinical interest of such functional or genotype testing will be dependent on the antiplatelet agents that are prescribed in ACS in the near future. Prasugrel and ticagrelor are faster and more potent P2Y12 ADP receptor blockers. With these new drugs, the rate of patients with HTPR and thrombotic events is greatly decreased; thus reducing potential interest in these tests. In addition to this, CYP 2C19 would be of no interest since it does not affect the metabolism of ticagrelor and prasugrel.
PR is a promising tool in the search to overcome inter-individual variability in clopidogrel responsiveness and improve patient outcomes in the near future. Studies have allowed the determination of the optimal level of PR to prevent bleeding and thrombotic complications. A recent consensus has defined the optimal level of PR inhibition, which could allow widespread use of these techniques. In addition to this, small scale studies have suggested clinical benefit with dose adjustment according to PR monitoring. PR monitoring could also be useful with the use of new antiplatelet agents. Genetic testing may also help to predict the laboratory and clinical response to clopidogrel, but is unable to encompass all of the factors influencing the effect of clopidogrel on platelets. In addition to this, genetic testing is of no use with new antiplatelet agents and does not predict the response to dose adjustment. Therefore, it is likely that for individualised therapy PR testing will be superior to genetic testing.