Platelet activity plays a key role in arterial thrombosis, leading to acute thrombotic events after coronary intervention.1 Dual antiplatelet therapy with acetylsalicylic acid (ASA) and clopidogrel is currently the standard treatment for prevention of thromboischaemic events in patients undergoing coronary angioplasty2 and provides additional inhibition of platelet activity.3

However, a substantial number of patients do not show adequate platelet inhibition and develop postinterventional thromboischaemic events despite standard treatment.4 5 Recently, the term “antiplatelet drug resistance” has emerged and increasing data show that dual antiplatelet therapy does not produce an adequate response in a relevant number of patients. The prevalence of antiplatelet drug resistance has been described with a broad variety in 5–45% of the patients.4 6 Dual drug resistance has been investigated in only small collectives and was found in up to 6% of study patients.7 The purpose of this study was to evaluate residual platelet activity by measurement of sensitivity to platelet agonists in an unselected, consecutively investigated cohort of patients with coronary stenting for acute and stable coronary artery disease. Furthermore, we aimed to characterise factors that influence an increased residual platelet activity (IRPA) despite dual antiplatelet therapy.

PATIENTS AND METHODS

Study group

We consecutively enrolled patients undergoing percutaneous coronary intervention (PCI) with coronary stent implantation for symptomatic coronary artery disease. High-risk acute coronary syndrome (ACS) was diagnosed if one of the following criteria were fulfilled: unstable angina (clinical symptoms and new ECG changes, but no markers of myocardial necrosis), acute myocardial infarction with markers of myocardial necrosis (troponins or CK-MB mass) including ST elevation myocardial infarction (STEMI) and non-ST elevation myocardial infarction (NSTEMI). The diagnosis of high-risk ACS was determined by blinded investigators and before the degree of response to antiplatelet therapy was noted. Stable angina was defined as a history of cardiac chest pain and a positive exercise test. The study protocol was approved by the local ethics committee and signed informed consent was obtained from all patients. Patients with known platelet function disorders, thrombocytopenia (<10⁵ cells/mm³) or any contraindications against ASA or clopidogrel were excluded from the study. A loading dose of 600 mg clopidogrel was given to all patients before PCI, followed by a daily dose of 75 mg for at least 3 months. Patients who had been treated with glycoprotein IIb/IIIa (GP IIb/IIIa) inhibitors within 1 week before possible measurement were excluded from the study. All patients received 500 mg ASA intravenously (IV) before PCI followed by 100 mg once a day. The majority of patients (around 90%) were already being given chronic aspirin therapy (100 mg once a day).

Blood sampling and platelet aggregation

Patient blood was collected ⩾6 hours (median 26.5 hours) after the first administration of 600 mg clopidogrel loading dose and ASA 500 mg IV, when maximum platelet inhibition was expected.8^–10 There was no significant time difference between PCI and aggregometry between patients with ACS and stable angina pectoris. Blood samples were collected in 3.8% citrate plasma as described.8 After centrifugation at 1000 rpm for 10 minutes to obtain platelet-rich plasma (PRP) and additionally 10 minutes at 3500 rpm to recover platelet-poor plasma (PPP), the platelet concentration of PRP was adjusted to 2×10⁵/µl by adding homologous PPP. The percentage platelet aggregation was assessed by the turbidometric method using a Chronolog Lumi aggregometer with Aggro-Link Software (Chronolog Corp., Havertown, PA, USA) 5 minutes after addition of adenosine diphosphate (ADP) 20 µmol/l or collagen 5 µg/ml.11 12

We categorised residual platelet aggregation by building tertiles according to the distribution of total aggregation values. IRPA was defined for patients in whom platelet aggregation induced by ADP (20 µmol/l) or collagen (5 µg/ml) respectively was measured in the highest tertile (fig 1).

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Figure 1 Scatter plot showing adenosine diphosphate (ADP)- and collagen-induced post-treatment aggregation of 480 patients enrolled in the study (A) 53 patients (11.0%) were measured with increased residual aggregation (IRPA) by ADP, 42 patients (8.8%) with IRPA by collagen, 14 patients (2.9%) showed IRPA by both agonists (B).

Statistical methods

Categorical variables are presented as frequencies and percentages. Continuous data with a normal distribution are expressed as mean (SD). Platelet aggregation is expressed as the median. The χ² test was used for dichotomous analysis of categorical data. Means between two normally distributed variables were compared by a two-tailed unpaired t-test. A Wilcoxon rank-sum (Mann–Whitney) test was performed to analyse differences between platelet aggregation.

Multivariate logistic regression analysis was performed to evaluate the association of relevant factors with IRPA-collagen, IRPA-ADP and IRPA-collagen/ADP (ASA/clopidogrel). Variables entered into the model included gender, age (>65 years), cardiovascular risk factors (diabetes, hyperlipidaemia, hypertension, smoking status), left ventricular dysfunction (ejection fraction ⩽30%) and the diagnosis of ACS at the time of enrolment. All probability values reported are two sided, and a value of p<0.05 was considered to indicate significance. Statistical analysis was performed with SPSS software, version 13 for windows (SPSS, Inc, Chicago, IL, USA).

RESULTS

Four hundred and eighty patients were consecutively enrolled in this study. Two hundred and fifty-nine patients (54.0%) underwent elective procedure for stable angina, and subsequent stent implantation was performed. Two hundred and twenty-one patients (46.0%) were treated by PCI for ACS (unstable angina, NSTEMI, STEMI). Table 1 shows the demographic characteristics of the patients.

Post-treatment response to ADP and collagen showed a high interindividual variability (fig 1A). We divided the values into tertiles of residual platelet aggregation. The lower border of the upper tertile was 63.7% for ADP and 66.3% for collagen. Thus, 53 patients (11.0%) were identified with IRPA in response to ADP and 42 patients (8.8%) identified with IRPA in response to collagen. In 14 patients (2.9%) both IRPA-collagen and ADP was determined (fig 1B).

Patients with ACS had a significantly higher prevalence of IRPA-ADP and collagen and both IRPA-ADP/collagen than patients with stable angina pectoris (IRPA-ADP: 14.9% vs 7.7%; IRPA-collagen: 11.8% vs 6.2%; IRPA-ADP/collagen 6.0% vs 1.3%; p<0.05). Myocardial infarction (STEMI + NSTEMI) had the highest impact on residual platelet activity. Of patients measured in the upper tertile of ADP-induced aggregation 35.4% were STEMI patients (vs 21.1% of total patients), 33.3% were NSTEMI patients (vs 15.7%) and 2.1% had unstable angina (vs 7.4); p<0.001. The distribution of STEMI patients in the upper tertile of collagen-induced aggregation was 28.6% (vs 21.0), for NSTEMI patients 19.0% (vs 15.9%) and for patients with unstable angina 9.5% (vs 7.4), p = 0.36. Among the patients with very high residual platelet aggregation (IRPA-ADP/collagen) there were 46.2% (vs 21.0%) with STEMI, 30.8% (vs 15.9) with NSTEMI, and none of the patients had unstable angina (vs 7.4% in total), p = 0.02.

In univariate analysis ACS showed a significant higher prevalence in groups with IRPA-collagen, IRPA-ADP and IRPA-collagen/ADP than in patients with normal platelet activity (IRPA-collagen 61.9%; IRPA-ADP 62.3%; IRPA-collagen/ADP 78.6% vs 43.4%; p<0.05). Distribution of cardiovascular risk factors and co-medication was not significantly different between groups except a more frequent administration of β-blockers in patients with normal response to antiplatelet therapy (table 1).

In multivariate regression analysis ACS was independently associated with IRPA-ADP, IRPA-collagen and IRPA-collagen/ADP after adjustment for cardiovascular risk factors (diabetes, hypertension, hypercholesterolaemia, tobacco use), severe left ventricular dysfunction (<30%), age and gender (IRPA-ADP, odds ratio (OR) = 2.4; 95% confidence interval (CI) 1.3 to 4.4; p<0.01; IRPA-collagen, OR = 2.3, 95% CI 1.2 to 4.5, p = 0.02, IRPA-collagen/ADP, OR = 4.5; 95% CI 1.2 to 16.9; p = 0.02; figs 2A–C).

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Figure 2 Odds ratios for the influence of cardiovascular risk factors, acute coronary syndrome at enrolment, age (>65 years) and gender on residual platelet aggregation by adenosine diphosphate (ADP) (A), collagen (B) and both ADP and collagen (C). Acute coronary syndrome is an independent factor associated with increased agonist-induced residual platelet aggregation and very high residual aggregation (IRPA-ADP/collagen). IRPA, increased residual platelet activity; LV, left ventricular.

Among the factors that were not significant at the level p<0.05, diabetes showed the strongest influence on IRPA-ADP (OR = 1.7, 95% CI 0.9 to 3.3, p = 0.08), IRPA-collagen (OR = 1.9, 95% CI 0.9 to 3.7, p = 0.08) and on IRPA-ADP/collagen (OR = 2.25, 95% CI 0.70 to 7.20, p = 0.17; figs 2A–C).

DISCUSSION

The major finding of this study is that a relevant number of patients with coronary stenting for ACS have a high residual platelet aggregation despite conventional dual antiplatelet therapy. We consecutively investigated post-treatment platelet activity in an unselected cohort of patients with symptomatic coronary artery disease treated with coronary stent implantation. Thus, we demonstrate by a single measurement that about 8–11% of the patients show increased residual platelet aggregation measured either by collagen- or ADP-induced aggregometry. Furthermore, we observed a very high residual platelet activity defined by increased sensitivity to both platelet agonists in 3% of the patients.

Different methods have been used to characterise platelet response to antiplatelet therapy. Thus, a broad range of prevalences for antiplatelet drug resistance have been reported.4 6 13^–15 In this study we aimed to evaluate post-treatment aggregation to evaluate the degree of platelet activity in patients treated with antiplatelet therapy. We performed optical aggregometry which has been established as reliable method for monitoring platelet function.16 By a single measurement of post-treatment platelet aggregation we characterised the effects of platelet inhibition in patients treated with a 600 mg loading dose of clopidogrel and a standard dose of aspirin. ADP-induced platelet aggregation has been frequently employed to define low response to the ADP-receptor blocker clopidogrel. In this study we used high concentrations (20 µmol/l) of ADP in order to assess the final irreversible rather than a transient platelet aggregation. The rate of low response to clopidogrel has been shown dependent on concentrations of platelet agonists. Accordingly, if defined by high residual platelet aggregation (eg, upper tertile of residual platelet activity) our data fit well with previously reported rates of low responders to clopidogrel characterised by aggregometry studies using equivalent ADP concentrations.17 Recently, we and others demonstrated that monitoring of ADP-induced residual platelet aggregation after clopidogrel loading can help to identify patients with increased risk for recurrent thromboischaemic events, including stent thrombosis.13 18 19

The majority of the patients (90%) in our cohort were chronic aspirin users. Influences of chronic aspirin treatment on aspirin sensitivity have been reported.20 As a limitation we did not assess the individual time of aspirin treatment which might have affected the measurement of platelet function.

We demonstrate that high-risk ACS is independently associated with the presence of a high post-treatment platelet aggregation. Therefore, conventional dual antiplatelet therapy might not produce an adequate response in a subgroup of patients with symptomatic coronary artery disease. This is of utmost clinical importance since antiplatelet drug resistance has been found to be associated with recurrent thromboischaemic events.5 17 18 21

One reason for our observation of increased platelet post-treatment reactivity in patients with ACS might be a higher pretreatment platelet activity in coronary ischaemic events.22 23 Gurbel and coworkers demonstrated a correlation between pretreatment activity and post-treatment platelet response to clopidogrel.24 Thus, patients with ACSs might show inadequate response to antiplatelet therapy, partly, because of a higher degree of pretreatment platelet activity. We did not evaluate pretreatment platelet aggregation nor did we estimate individual platelet response over the course of time, which has been described as variable up to 30 days after PCI. However, our data imply that in the acute phase, around 24 hours after stent implantation when in most of our patients with ACS platelet aggregation profiles were determined, dual antiplatelet therapy is not effective in around 8–11% of the patients. Patients with ACS might therefore benefit from an intensified antiplatelet regimen. The improved prognosis of an additional antiplatelet therapy with GP IIb/IIIa inhibitors (abciximab) for risk reduction for cardiovascular events in patients with ACS has been recently demonstrated by the ISAR-REACT 2 trial.25 In the CLEAR PLATELETS trial Gurbel and coworkers measured additional platelet inhibition by GP IIb/IIIa inhibitors (eptifibatide) in coronary stent patients already treated with ASA and 600 mg loading dose.26

Another approach would be a dose increase of oral antiplatelet therapy. The ALBION study showed an additional effect of a 900 mg loading dose27 and patients already chronically treated with clopidogrel showed a significant additional inhibition of ADP-induced platelet aggregation by a repeated 600 mg loading dose.28 Accordingly, a dose-dependent effect has been demonstrated on platelet inhibition measured by platelet function tests.29 30 Additional studies are necessary to weigh the clinical benefit of a dose increase against the haemorrhagic risk in patients with increased residual platelet activity despite conventional antiplatelet therapy.

CONCLUSIONS

The present study suggests that ACS is independently associated with a higher post-treatment platelet aggregation measured by reactivity to platelet agonists early after stent implantation. Furthermore, we observed high residual activity to ADP and collagen in a considerable number of patients, indicating the ineffectiveness of dual antiplatelet therapy. Since there is growing evidence for an association of inadequate response to antiplatelet therapy with the incidence of recurrent thromboischaemic events after PCI, patients with acute coronary events might therefore benefit and have an improved clinical prognosis from an alternative or intensified antiplatelet regimen. Further systematic studies are needed to evaluate the effects of an adopted antiplatelet therapy in this jeopardised group of patients.