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Original article
High on-treatment platelet reactivity to both aspirin and clopidogrel is associated with the highest risk of adverse events following percutaneous coronary intervention
  1. Nicoline J Breet1,2,
  2. Jochem W van Werkum1,2,
  3. Heleen J Bouman1,2,
  4. Johannes C Kelder1,2,
  5. Ankie M Harmsze3,
  6. Christian M Hackeng2,4,
  7. Jurriën M ten Berg1,2
  1. 1Department of Cardiology, St Antonius Hospital, Nieuwegein, The Netherlands
  2. 2St Antonius Center for Platelet Function research, Nieuwegein, The Netherlands
  3. 3Department of Clinical Pharmacy, St Antonius Hospital, Nieuwegein, The Netherlands
  4. 4Department of Clinical Chemistry, St Antonius Hospital, Nieuwegein, The Netherlands
  1. Correspondence to Jurrien M ten Berg, Department of Cardiology, St Antonius Hospital, P O Box 2500, 3435 CM Nieuwegein, The Netherlands; j.ten.berg{at}antoniusziekenhuis.nl

Abstract

Aim High on-clopidogrel platelet reactivity (HCPR) and high on-aspirin platelet reactivity (HAPR) are associated with atherothrombotic events following coronary stenting. There are, however, few data concerning high on-treatment platelet reactivity to both aspirin and clopidogrel simultaneously. The aim of the present study was to determine the incidence of dual high on-treatment platelet reactivity (DAPR) and its impact on clinical outcome.

Methods On-treatment platelet reactivity was measured in parallel by ADP- and arachidonic acid-induced light transmittance aggregometry (LTA) (n=921) and the point-of-care VerifyNow system (P2Y12 and aspirin) (n=422) in patients on dual antiplatelet therapy undergoing elective stent implantation. HCPR and HAPR were established by receiver-operator characteristic curve analysis. The primary endpoint was a composite of all-cause death, non-fatal acute myocardial infarction, stent thrombosis and ischaemic stroke at 1-year follow-up.

Results The incidence of DAPR varied between 14.7% and 26.9% depending on the platelet function test used. DAPR, assessed by LTA and the VerifyNow system, was highly associated with an adverse clinical outcome. At 1-year follow-up the primary endpoint occurred more frequently in patients with isolated HCPR (11.7%), isolated HAPR (9.6%) or DAPR (10.7%) compared with patients without high on-treatment platelet reactivity (4.2%, all p<0.01) when platelet function was evaluated with LTA. Using the VerifyNow system, patients exhibiting DAPR had the highest risk for the primary endpoint (17.7% vs 4.1% in patients without high on-treatment platelet reactivity, p=0.001).

Conclusions In patients undergoing elective percutaneous coronary intervention, DAPR to aspirin and clopidogrel is present in one in five patients and is associated with a high risk for atherothrombotic events. DAPR measured by the point-of-care VerifyNow system has a higher predictability for atherothrombotic events than LTA.

Clinical Trial Registration Information www.clinicaltrials.gov: NCT00352014.

  • Coronary intervention
  • antiplatelet treatment
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Introduction

Dual antiplatelet therapy with aspirin and clopidogrel is pivotal in preventing atherothrombotic events in patients undergoing percutaneous coronary intervention (PCI) with stent implantation.1 2 However, the individual response to both drugs is heterogeneous and it has been shown that high on-treatment platelet reactivity is associated with an adverse outcome.3–14 Furthermore, several studies have suggested that patients exhibiting high on-treatment platelet reactivity to both aspirin and clopidogrel simultaneously are at an even higher risk of atherothrombotic events.15–18 This is of utmost importance, since several studies have suggested the benefit of tailoring therapy in these patients.9 19 20 The POPular study (The Do Platelet Function Assays Predict Clinical Outcomes in Clopidogrel Pretreated Patients Undergoing Elective PCI) demonstrated that aggregation-based tests were able to predict the occurrence of an adverse cardiovascular event in patients undergoing elective PCI with stent implantation.10 The present subanalysis aimed to explore the incidence of dual high on-treatment platelet reactivity (DAPR) and to assess whether patients exhibiting DAPR have a higher risk of adverse events.

Methods

A detailed description of the clinical characteristics of the patients and the entry and exclusion criteria of the POPular study has been published previously.10 The POPular study was a head-to-head comparison between multiple platelet function assays, gauging the efficacy of clopidogrel, in their capability to predict atherothrombotic events. A recent subanalysis studied the platelet function tests specific for aspirin.21 In brief, the POPular study was a prospective observational study enrolling consecutive patients with established coronary artery disease scheduled for elective PCI with stent implantation. All patients received optimal clopidogrel treatment and all patients were on aspirin 80–100 mg daily for ≥10 days unless they were on long-term anticoagulation with coumarin derivatives. Optimal pretreatment clopidogrel regimens were defined as chronic maintenance therapy of 75 mg for >5 days or a clopidogrel loading dose of 300 mg at least 24 h before PCI or 600 mg at least 4 h before PCI.

The patients on both aspirin and clopidogrel comprised the population in the present analysis. Compliance was verified by a detailed interview upon enrolment (self-reportage) as well as by pharmacy refill data. All interventions were performed according to current guidelines22 and the choice of stent type and periprocedural use of glycoprotein (GP) IIb/IIIa inhibitors was left to the operator's discretion, but the latter were always administered after blood collection. Patients using concomitant medication known to affect platelet function other than aspirin (ie, non-steroidal anti-inflammatory drugs, dipyramidole, upstream GP IIb/IIIa inhibitors), patients with a known platelet function disorder or a whole blood platelet count <150 000/μl were excluded. Clinical follow-up was obtained by contacting all patients at 12 months, which was verified on the basis of source documents from the medical records. The study was conducted according to the principles of the Declaration of Helsinki and the laws and regulations applicable in the Netherlands. The local institutional review board approved the study.

Study design

The present study consisted of two subanalyses. The primary analysis comprised an analysis of the total population on both aspirin and clopidogrel (n=951) using light transmittance aggregometry (LTA) and the point-of-care VerifyNow system (Accumetrics, San Diego, USA) with the aim of establishing whether patients exhibiting DAPR have a higher risk of adverse events than patients without high on-treatment platelet reactivity or high on-treatment platelet reactivity to either aspirin alone or clopidogrel alone. A secondary analysis was performed in those patients in whom both the VerifyNow system and LTA was performed (n=410), with the aim of comparing the predictability of DAPR of both tests.

Follow-up and endpoints

The primary endpoint was defined as a composite of all-cause death, non-fatal myocardial infarction (defined as the occurrence of ischaemic symptoms and a spontaneous (ie, not peri- or post-procedural) troponin T value or creatine kinase MB greater than the upper limit of normal), stent thrombosis (definite stent thrombosis according to the Academic Research Consortium criteria)23 and ischaemic stroke (focal loss of neurological function caused by an ischaemic event).24 An independent committee, blinded to platelet function data, adjudicated all endpoints by reviewoffing the source documents of medical records.

Blood sampling

Before heparinisation, whole blood was drawn from the femoral or radial artery sheath. Blood samples were collected into 3.2% Sarstedt citrate tubes for LTA. The VerifyNow system was performed using Greiner tubes, according to the manufacturer's recommendations. Blood samples for whole blood count were drawn into tubes containing K3-EDTA.

Platelet function measurements

The magnitude of on-treatment platelet reactivity was quantified using the following platelet function tests; LTA using arachidonic acid (AA) and ADP as the agonists and the VerifyNow system using the aspirin and P2Y12 assays. All platelet function measurements were performed within 2 h of blood collection.

Light transmittance aggregometry (LTA)

LTA was performed in non-adjusted platelet-rich plasma on a four-channel APACT 4004 aggregometer (LABiTec, Arensburg, Germany) as previously described.25–29 Platelet-poor plasma was set as 100% aggregation and maximal platelet aggregation (%) was measured using AA in a final concentration of 0.5 mg/ml and ADP in final concentrations of 5 and 20 μmol/l. The cut-off used to identify patients with high on-clopidogrel platelet reactivity (HCPR) was 43% aggregation for 5 μmol/l ADP-induced LTA and 65% for 20 μmol/l ADP-induced aggregation.10 The cut-off used to define high on-aspirin platelet reactivity (HAPR) was calculated by receiver-operator characteristic (ROC) curve analysis based on the 1-year primary endpoint and determined as AA-induced platelet aggregation >20%, which is in line with a previous publication.11 DAPR was defined as exhibiting both HAPR and HCPR.

The VerifyNow system

The VerifyNow system is a whole blood cartridge-based method to determine the magnitude of platelet agglutination induced by either AA in the aspirin assay or ADP and prostaglandin E1 in the P2Y12 assay.30 31 The results are reported in aspirin reaction units and P2Y12 reaction units, respectively. The cut-off used for the VerifyNow P2Y12 assay to identify patients with HCPR was 236 P2Y12 reaction units,10 which is in accordance with previous studies.7 14 The cut-off used for the VerifyNow aspirin assay to define HAPR was calculated by ROC curve analysis based on the 1-year primary endpoint and was determined as 454 aspirin reaction units. DAPR was defined as exhibiting both HAPR and HCPR.

Statistical analysis

Continuous variables are presented as mean (SD) and categorical data are reported as frequencies (percentages). The χ2 test was used to compare categorical data, including the four groups stratified according to platelet reactivity. Normally distributed continuous variables were compared with a two-sided Student t test.

Logistic regression modelling was used to identify independent correlates of the primary endpoint and to adjust for potential confounders (classic cardiovascular risk factors, renal failure, left ventricular ejection fraction <45%, total stent length, number of lesions treated, number of stents implanted, bifurcation lesions, co-medication (including use of clopidogrel loading dose, coumadins, proton pump inhibitors, calcium channel blockers, statins or GP IIb/IIIa inhibitors), laboratory parameters (haemoglobin, platelet count and mean platelet volume) and left anterior descending coronary artery or graft stenting). Platelet reactivity status was entered as a dichotomous variable; patients exhibiting HAPR, HCPR and the interaction term DAPR. All univariate variables with a p value <0.10 were included in the multivariable analysis.

Platelet reactivity status was added to a model including clinical and procedural risk factors. Whether platelet reactivity status according to the various platelet function tests independently contributed to a logistic regression model containing clinical and procedural risk factors was tested with the likelihood ratio test. The Hosmer–Lemeshow goodness-of-fit test was performed to assess the adequacy of the model. Survival analysis for patients with and without DAPR according to the specific definitions was performed using the Kaplan–Meier method and differences between the groups were compared using the log-rank test. All statistical analyses were performed with R Version 2.9 (http://www.r-project.org) and a two-tailed p value of <0.05 was considered significant.

Results

Primary analysis

One thousand and sixty-nine consecutive patients were enrolled, of whom 951 were on aspirin for ≥10 days and clopidogrel. Owing to irregularities in platelet assay supply as well as technical failure in a minority of platelet function tests, not all platelet function assays were performed in every patient. For the LTA, both AA-induced and ADP-induced aggregation data were available in a total of 921 patients using 5 μmol/l ADP (LTA5) and in 923 patients using 20 μmol/l ADP (LTA20). Furthermore, since use of the VerifyNow aspirin cartridge was only started halfway through the POPular study, the VerifyNow aspirin assay was performed in less than half of the population. In a total of 422 patients both the aspirin assay and the P2Y12 assay were performed.

Baseline characteristics of the total cohort are shown in table 1. Clinical outcome at 12 months was available for 949 patients (99.9%). During the 1-year follow-up period, 78 patients (8.2%) achieved the primary endpoint: 16 patients (1.7%) died, 54 (5.7%) had non-fatal acute myocardial infarction, 9 patients (0.9%) presented with definite stent thrombosis and 11 patients (1.2%) had a non-fatal ischaemic stroke.

Table 1

Baseline characteristics

LTA and clinical outcome

The incidence of HCPR was 14.9% using LTA5 and 13.0% using LTA20 (table 2). The prevalence of HAPR was up to twofold higher in patients with HCPR compared with those without HCPR (p<0.0001) (table 3) and approximately one-quarter of the patients had DAPR (table 2).

Table 2

Platelet reactivity status according to the various platelet function tests

Table 3

Incidence of high on-aspirin platelet reactivity in patients with and patients without high on-clopidogrel platelet reactivity

Table 4 summarises the 1-year clinical outcome for patients without high on-treatment platelet reactivity, with HAPR, HCPR or DAPR according to the different platelet function tests. When measured with LTA5 or LTA20, patients with DAPR had numerically (but not statistically significant) more events than patients with HCPR or HAPR. Patients with HCPR, HAPR or DAPR had similarly increased rates of the primary endpoint compared with patients without HPR (table 4). Kaplan–Meier analysis showed that the overall risk for the composite endpoint was significantly higher in patients with isolated HCPR, isolated HAPR or DAPR than in patients without high on-treatment platelet reactivity using LTA5 and LTA20 (figure 1).

Table 4

Clinical outcome

Figure 1

Kaplan–Meier analysis of the event rate of the combined primary endpoint in patients with and without high on-treatment platelet reactivity as measured by multiple platelet function assays. DAPR, high on-clopidogrel platelet reactivity and high on-aspirin platelet reactivity; HAPR, high on-aspirin platelet reactivity; HCPR, high on-clopidogrel platelet reactivity; LTA, light transmittance aggregometry; NPR, neither high on-clopidogrel platelet reactivity nor high on-aspirin platelet reactivity.

VerifyNow and clinical outcome

The incidence of DAPR according to the VerifyNow system was 14.7% (table 2) and the prevalence of HAPR was 2.5-fold higher in patients with HCPR than in those without HCPR (p<0.0001) (table 3). Patients with DAPR according to the VerifyNow system had the highest incidence of the composite primary endpoint, whereas patients with isolated HCPR or HAPR were not at significantly higher risk than patients without high on-treatment platelet reactivity (table 4). DAPR was independently associated with an increased risk of the primary endpoint (figure 2). Kaplan–Meier analysis showed that the overall risk for the composite endpoint was significantly higher in patients with DAPR as assessed by the VerifyNow system compared with those without high on-treatment platelet reactivity (figure 1).

Figure 2

Independent predictors of the primary endpoint. Clinical and procedural factors and platelet reactivity status according to the various platelet function tests. DAPR, high on-clopidogrel platelet reactivity and high on-aspirin platelet reactivity; HAPR, high on-aspirin platelet reactivity; HCPR, high on-clopidogrel platelet reactivity; LTA5, arachidonic acid- and 5 μmol/l ADP-induced light transmittance aggregometry; LTA20, arachidonic acid- and 20 μmol/l ADP-induced light transmittance aggregometry.

Predictive model

Logistic regression modelling was used to identify independent predictors for the primary endpoint (figure 2). Platelet reactivity status according to LTA5 and LTA20 was an independent predictor of the primary endpoint (HCPR, HAPR as well as DAPR). DAPR as assessed by the VerifyNow system was the strongest independent predictor of the primary endpoint. Other variables independently associated with the 1-year primary endpoint were age (calculated for an increase of 10 years), hypertension, left ventricular ejection fraction <45%, graft stenting and a bifurcation lesion. The addition of platelet reactivity status to the predictive model consisting of these clinical and procedural risk factors showed that platelet reactivity measured by LTA (both LTA5 and LTA20) and the VerifyNow system significantly improved the area under the ROC curve. Likewise, the likelihood ratio test showed that platelet reactivity status according to these tests made an additional contribution to the model (table 5). The goodness-of-fit test showed that the predicting model was adequate (all p values >0.20).

Table 5

Area under the curve (AUC) of different backward regression models for the prediction of the primary endpoint at 1-year follow-up

Secondary analysis

LTA5, LTA20 and VerifyNow results were available in 410 patients. During the 1-year follow-up 29 patients (7.1%) achieved the primary endpoint: 8 patients (2.0%) died, 17 (4.1%) had non-fatal acute myocardial infarction, 3 patients (0.7%) presented with definite stent thrombosis and 5 patients (1.2%) had a non-fatal ischaemic stroke.

The addition of platelet reactivity status to the predictive model consisting of clinical and procedural risk factors showed that DAPR as measured with the VerifyNow system significantly improved the area under the ROC curve in this subpopulation (table 6). The goodness-of-fit test confirmed that the predicting model was adequate (p=0.25). In contrast, DAPR as assessed by LTA did not improve the predictive ability of the model and, irrespective of the platelet function assay used, neither HCPR nor HAPR increased the predictability.

Table 6

Area under the curve (AUC) of different backward regression models for the prediction of the primary endpoint at 1-year follow-up in the subpopulation of patients in whom all platelet function tests are available

Discussion

The principal finding of this study is that DAPR occurs with varying prevalence between 14.7% and 26.9% according to the platelet function assay used and is more prevalent than previously assumed.15–18 Of even more importance, DAPR is associated with the occurrence of atherothrombotic events and is a better predictor of an adverse outcome than isolated HCPR or HAPR.

Most studies to date have evaluated the magnitude of platelet reactivity in response to a single antiplatelet drug (either aspirin or clopidogrel) and therefore the observed higher risk for recurrent events may partly have been due to dual resistance.3–14 Measurement of platelet reactivity to multiple agonists comprises the efficacy of dual antiplatelet therapy and may potentially be a more comprehensive method to assess the future risk of an individual patient undergoing PCI.

The mechanisms underlying the high interindividual variability in response to both aspirin and clopidogrel are manifold and include baseline individual variability, genetic polymorphisms and clinical factors.27 32–36 The finding that most patients with HCPR also have HAPR is in accordance with previous studies,37 38 and might be explained by a mechanistic interdependence of the different pathways involved in platelet reactivity since the P2Y12 receptor potentiates the generation of thromboxane-A2.39–41 Another plausible explanation is that patients with DAPR have a generally higher baseline (intrinsic) platelet reactivity status.42–45 A third explanation might be that, in patients with high on-treatment platelet reactivity, increased platelet turnover leads to the release of young platelets that are not inhibited since both aspirin and clopidogrel have a short half-life.46 47

In our study both LTA and the VerifyNow system were used. In line with previous studies,3 4 6 10 LTA was able to discriminate between patients with and without atherothrombotic events at 1-year follow-up. However, LTA, although considered the gold standard for platelet function testing, has a poor reproducibility and is labour-intensive and thus not suitable for daily clinical use.48 We therefore also used the fully automated point-of care VerifyNow system to compare its predictability with LTA. In our study, DAPR according to the VerifyNow system had the largest increase in predictability for the occurrence of adverse events. In addition, a secondary analysis in those patients in whom all platelet function assays were performed (n=410) showed that LTA lost its predictive ability whereas the VerifyNow system remained predictive. The VerifyNow system might therefore be considered a better test to predict clinical events in patients undergoing elective PCI.

Numerous studies have linked a high on-treatment platelet reactivity to atherothrombotic events, and many thresholds to identify patients at higher risk have been established. It has been hypothesised that individual monitoring of platelet reactivity and decreasing the magnitude of platelet reactivity below this threshold might improve the clinical outcome.49 50 Three small studies indeed suggested that individualising therapy based on platelet function might improve outcome.9 20 51 52 The only randomised study thus far is the GRAVITAS (Gauging Responsiveness with a VerifyNow Assay-IMPACT on Thrombosis and Safety) study, a prospective randomised three-arm multicentre trial of 5429 patients undergoing PCI with implantation of a drug eluting stent. Patients exhibiting high on-treatment platelet reactivity 12–24 h after PCI (n=2214) were randomised to either standard maintenance therapy (75 mg) or to an additional loading dose of 600 mg and a double maintenance dose (150 mg). The GRAVITAS study found no benefit of doubling the clopidogrel dose in preventing cardiovascular events in patients with HCPR. However, this study does not rule out the benefit of tailoring therapy based on platelet function testing. On the contrary, since doubling the dose resulted in only a modest reduction in platelet reactivity, these results suggest that the strategy of a double dose of clopidogrel is ineffective in this low-risk population.53 Tailoring therapy based on the use of novel more potent antiplatelet medication (ie, prasugrel or ticagrelor) might be more effective. This is the subject of investigation in the currently ongoing TRIGGER-PCI study (NCT00910299) randomising prasugrel versus clopidogrel. The logical drawback of efficient platelet inhibition is the risk of bleeding complications, and it has been suggested that measuring platelet function might be the solution to define a therapeutic window between bleeding and thrombotic events. Taking the costs and risks associated with bleeding into account, we consider platelet function testing a better option than prescribing all patients more potent antiplatelet therapy. In that case, point-of-care platelet function testing is obviously preferable.54

Some limitations merit mention. First, the present analysis of DAPR has a decreased statistical power owing to a smaller sample size than was used in the POPular study and the stratification into four categories (patients without high on-treatment platelet reactivity vs those with HAPR, HCPR or DAPR) instead of two categories (patients without high on-treatment platelet reactivity vs those exhibiting high on-treatment platelet reactivity). This is most apparent when platelet reactivity status was assessed by the VerifyNow system since the VerifyNow aspirin assay was performed in only half of the patients who were tested with the VerifyNow P2Y12 cartridge. The absence of a higher risk in patients with isolated HAPR or isolated HCPR as measured with LTA might be attributed to the smaller number of patients. This is further elucidated in the secondary analysis in which LTA lost its predictive ability as well. Second, not all of the currently available platelet function tests were included. Third, single time point assessment represents a common limitation to most studies assessing the prognostic value of platelet function testing, including the present one. Last, patients received three different adequate clopidogrel doses. Although previous studies have demonstrated differences in the effect on platelet reactivity of these three dosing regimens, these three regimens are used in daily clinical practice and the present analysis thus reflects the clinical relevance of monitoring platelet function in daily care.

In conclusion, one in five patients undergoing elective PCI exhibits high on-treatment platelet reactivity to both aspirin and clopidogrel. These patients are at higher risk for atherothrombotic events than those with high on-treatment platelet reactivity to either aspirin or clopidogrel. DAPR measured with the point-of-care VerifyNow system might have a higher predictability for atherothrombotic events than when measured by LTA.

References

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Footnotes

  • Competing interests NJB has received a speaker's fee from Siemens; JWvW has received a speaker's fee from Accumetrics and Siemens and a consultancy fee from the Medicines Company; JMtB has received a speaker's fee from Sanofi-Aventis, Eli Lilly, BMS and MSD and consultancy fees from Sanofi-Aventis, Eli Lilly, Schering-Plough and Glaxo Smith Kline. None of the other authors report financial disclosures.

  • Ethics approval This study was conducted with the approval of the Verenigde Commissie Mensgebonden Onderzoek (VCMO), St Antonius Ziekenhuis, Nieuwegein.

  • Provenance and peer review Not commissioned; externally peer reviewed.

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