Increased platelet reactivity and decreased response to antiplatelet drugs may result in recurrent ischemic events after acute coronary syndrome (ACS). We evaluated laboratory response to aspirin in patients with ACS before and after percutaneous coronary intervention (PCI) and assessed its effect on major adverse clinical events. Sixty-three consecutive patients with ACS were tested for response to aspirin by light transmittance aggregometry (LTA) and the IMPACT-R test (with arachidonic acid) before and 2 to 4 days after PCI and clopidogrel loading. Patients were followed for clinical events up to 15 months from PCI. Response to aspirin improved significantly after PCI and clopidogrel treatment (mean arachidonic acid–induced LTA decreased from 34.9 ± 3.35% before PCI to 15.2 ± 2.2% and surface coverage increased from 2.2 ± 0.27% to 6.2 ± 0.6%, p <0.0001 for the 2 methods). Improved response to aspirin after PCI correlated with response to clopidogrel (LTA and IMPACT-R, p <0.01). Patients with good laboratory response to aspirin before but not after PCI had a significantly lower major cardiovascular event rate during 15-month follow-up in multivariate analysis. In conclusion, laboratory response to aspirin is highly dynamic in patients with ACS. Improved response to aspirin after PCI may result from stabilization of coronary artery disease and/or clopidogrel treatment. Laboratory response to aspirin before PCI and clopidogrel loading is a sensitive marker for platelet reactivity that correlates with clinical outcome in patients with ACS.
Platelets play a central role in the pathogenesis of acute coronary syndrome (ACS). High platelet reactivity in patients presenting with ACS requires dual and sometimes triple antiplatelet treatment. Platelet activation correlates with stability of coronary artery disease, it was shown that patients presenting with ACS have higher platelet reactivity compared to patients with stable disease. Laboratory response to aspirin and clopidogrel can vary between patients and has been shown to carry prognostic implications. Most studies on response to aspirin in coronary artery disease have been performed in patients with elective or nonurgent procedures. Only a few studies have tested response to aspirin in patients presenting with ACS and most of these studies evaluated the response to the combination of aspirin and clopidogrel. Furthermore, laboratory assessment of aspirin responsiveness in most studies was performed by a single measurement during the course of ACS. In addition, those tests were performed at different time points in relation to the coronary intervention. Studies that assessed platelet function on admission for ACS (before percutaneous coronary intervention [PCI]) demonstrated a correlation between higher platelet reactivity and worse clinical outcome by Platelet Function Analyzer 100 (PFA-100). The aim of this study was to follow the trends in response to aspirin in patients with ACS at 2 different time points—during the acute phase before PCI and during a more stable phase after PCI and clopidogrel loading—and assess its relation to clinical outcome.
Methods
Consecutive patients with ACS who underwent PCI were enrolled in this prospective observational study. ACS was defined as typical anginal symptoms with ≥1 of the following: ST-segment deviation on electrocardiogram (≥0.1 mm in 2 consecutive leads) or increased cardiac troponin (troponin T >0.1 ng/ml). Patients with ST-segment elevation myocardial infarction (STEMI) received chewable aspirin before cardiac catheterization and blood draw and underwent primary PCI. Patients with non–ST-segment elevation ACS (NST-ACS) received aspirin 100 mg and low-molecular-weight heparin in the hospital until PCI. Immediately after PCI all patients received a loading dose of clopidogrel 300 mg followed by 75 mg/day and continued with aspirin 100 mg/day. Blood samples for responsiveness to aspirin were drawn immediately before PCI (under treatment with aspirin) and 2 to 4 days after catheterization (under treatment with aspirin and clopidogrel). Patients who received clopidogrel or IIb/IIIa inhibitors before catheterization or patients who did not receive aspirin as indicated in the protocol were excluded from the study. Patients were followed to 15 months after PCI for major adverse clinical events using a computerized hospital database and a telephone questionnaire. Major adverse clinical events were defined as cardiovascular death, nonfatal MI, ischemic stroke, recurrent hospitalization for ACS, or recurrent coronary intervention. The study was approved by the institutional ethics committee.
Whole venous blood anticoagulated with sodium citrate was centrifuged (160 g , 12 minutes) to obtain platelet-rich plasma. Platelet-poor plasma was isolated from the remaining blood sample by centrifugation at 750 g for 5 minutes. Platelet count was not adjusted. Platelet aggregation was evaluated by a turbidimetric aggregometer (PACKS-4, Helena Laboratories, Beaumont, Texas) using adenosine diphosphate (10 μmol/L) and arachidonic acid (1.6 mmol/L) as platelet agonists. The IMPACT-R device (Diamed, Cresier, Switzwerland), i.e., the cone- and plate(let) analyzer, monitors platelet adhesion and aggregation under shear stress. Citrated whole blood (130 μl) was placed in a polystyrene well and subjected to shear (1,800/s for 2 minutes) using a rotating cone. Under these conditions von Willebrand factor and fibrinogen are very rapidly immobilized on the polystyrene surface, serving as a thrombogenic substrate for platelet adhesion. The well was then washed and stained with May–Grünwald stain. Platelet deposition was evaluated by the IMPACT-R image analysis system. Platelet adhesion was measured as surface coverage (percentage) of adherent platelets (single platelets and aggregates). To evaluate response to aspirin or clopidogrel, samples were preincubated with arachidonic acid 0.32 mmol/L or adenosine diphosphate 2.5 μmol/L, respectively, for 1 minute under gentle mixing (10 rpm) ; in patients with poor responses to aspirin, platelet microaggregates formed in the preincubation tube resulted in decreased surface coverage in the IMPACT-R test. Thus, high platelet adhesion reflects a good response to aspirin/clopidogrel using this method.
All statistical analyses were performed using SPSS 17 for Windows (SPSS, Inc., Chicago, Illinois). Differences between responses to aspirin before and after PCI and clopidogrel loading were calculated using paired t test. Differences in patients’ characteristics and response to clopidogrel according to response to aspirin were assessed using chi-square test for categorical variables and 1-way analysis of variance for continuous variables. Differences between STEMI and NST-ACS were assessed by chi-square test for categorical variables and unpaired t test for continuous variables. Univariate event-free survival analysis was performed by Kaplan–Meier analysis with differences in survival curves assessed by log-rank test. Multivariate Cox regression analysis was performed to evaluate the independent effect of prognostic variables on event-free survival. Variables chosen for inclusion in the model were those that tended to be associated with event-free survival on univariate analysis and age. All results are presented as mean ± SEM. A 2-sided p value <0.05 was considered statistically significant for all analyses.
Results
Sixty-three patients with ACS were included in the study. Most patients (72.6%) reported long-term use of low-dose aspirin before hospitalization (75 to 100 mg/day). Forty-seven patients (75%) had NST-ACS. These patients received low-dose aspirin for a mean of 4.1 days before PCI. Sixteen patients (25%) presented with STEMI and were treated with primary PCI; these patients received chewable aspirin 300 mg ≥45 minutes before PCI and blood tests. No difference in response to aspirin was observed between long-term aspirin users and patients who did not receive aspirin before the current ACS (mean arachidonic acid–induced light transmittance aggregometry [LTA] 36.8 ± 4.1 vs 31.6 ± 5.9, p = 0.42).
Clinical characteristics of the study population are presented in Table 1 and were typical for patients with ACS. The study group was divided into tertiles according to arachidonic acid–induced LTA before PCI and clopidogrel loading. The upper tertile represents poor laboratory response to aspirin and the lower tertile represents good laboratory response to aspirin. Good responders had arachidonic acid–induced aggregation <20% and poor responders had >40% ( Table 1 ). Younger patients tended to have a better response to aspirin, whereas women and diabetic patients tended to have a poor response ( Table 1 ). Patients in the lower tertile had also greater surface coverage (preincubation with arachidonic acid) in the IMPACT-R test (3.3 ± 0.65 compared to 1.95 ± 0.35 in upper tertile and 1.24 ± 0.1 in middle tertile, p = 0.005), representing a good response to aspirin in the 2 methods.
| Characteristics | All Patients (n = 63) | Degree of Response ⁎ | p Value | ||
|---|---|---|---|---|---|
| Poor | Intermediate | Good | |||
| (n = 21) | (n = 21) | (n = 21) | |||
| Maximal aggregation of arachidonic acid (%), mean ± SE (range) | 35 ± 3.4 (2.7–100) | 68 ± 4.6 (40–100) | 28 ± 1.3 (20–37.7) | 11 ± 1.2 (2.7–19.5) | <0.001 |
| Mean age (years) | 59.7 | 61.6 | 62.5 | 54.8 | 0.05 |
| Men | 81% | 65% | 91% | 86% | 0.05 |
| Hyperlipidemia † | 81% | 70% | 82% | 91% | 0.22 |
| Diabetes mellitus | 24% | 35% | 27% | 10% | 0.11 |
| Hypertension † | 54% | 65% | 54% | 43% | 0.44 |
| Smokers | 30% | 30% | 32% | 29% | 0.91 |
| Previous ischemic heart disease ‡ | 44% | 40% | 50% | 40% | 0.6 |
| Long-term aspirin use | 73% | 80% | 69% | 70% | 0.61 |
⁎ Patients were divided into tertiles according to arachidonic acid–induced light transmission aggregometry before percutaneous intervention.
Mean amplitude of arachidonic acid–induced LTA before PCI was 34.9 ± 3.35% ( Table 1 and Figure 1 ) and mean surface coverage was 2.2 ± 0.27% (IMPACT-R preincubation with arachidonic acid). Patients in the mid and upper tertiles had an arachidonic acid–induced aggregation of >20% and surface coverage <2%, indicating a low laboratory response to aspirin before PCI.
A striking improvement in response to aspirin was observed 2 to 4 days after PCI and clopidogrel treatment. Mean arachidonic acid–induced LTA of the entire study group decreased to 15.2 ± 2.2% and surface coverage increased to 6.2 ± 0.6% after PCI (p <0.0001 for the 2 methods; Figure 1 ). Mean arachidonic acid–induced platelet aggregations after PCI according to tertiles were 3.3% (range 0 to 6), 10% (range 6.8 to 14.1), and 31.8% (range 14.5 to 91.4) for the lower, middle and upper tertiles, respectively. Forty-eight patients (80%) had arachidonic acid–induced aggregation of <20% after PCI compared to 21 (33%) before PCI and only 4 patients (6.7%) remained in the range of 40% to 100% arachidonic acid–induced LTA (defined as poor responders) .
Adenosine diphosphate–induced aggregation was tested 2 to 4 days after PCI and loading of clopidogrel 300 mg followed by 300 mg 75 mg/day and aspirin 100 mg/day. Mean amplitude of adenosine diphosphate–induced aggregation was 26.69 ± 2.9% (range 0 to 82.3) and mean surface coverage was 5.5 ± 0.5% (range 0.5 to 18, IMPACT-R preincubation with adenosine diphosphate).
To assess the correlation between responses to clopidogrel and aspirin we evaluated adenosine diphosphate–induced aggregation in 3 new tertiles according to laboratory response to aspirin on days 2 to 4 (after PCI and clopidogrel loading). There was a good correlation between responses to clopidogrel and aspirin by LTA and the IMPACT-R test (p = 0.002 and 0.004, respectively) as shown in Figure 2 .
Differences between patients with STEMI and NST-ACS are presented in Table 2 . Patients with STEMI (25%) were younger, had higher rates of a smoking history, and lower rates of dyslipidemia, hypertension, and previous usage of aspirin. However, patients with STEMI had better response to aspirin before PCI compared to patients with NST-ACS by the 2 methods ( Table 2 ). After PCI there was no difference in response to aspirin between the 2 groups. Patients with STEMI had significantly higher baseline platelet adhesion (percent surface coverage with no agonist) compared to NST-ACS before but not after PCI. No difference in clinical outcome between these subgroups was observed.
| Characteristic | NST-ACS | STEMI | p Value |
|---|---|---|---|
| (n = 47) | (n = 16) | ||
| Mean age (years) | 61 ± 1.4 | 53.5 ± 3.2 | 0.03 |
| Men | 79% | 88% | 0.7 |
| Diabetes mellitus | 26% | 19% | 0.74 |
| Hypertension ⁎ | 64% | 25% | 0.009 |
| Smoking | 19% | 63% | 0.003 |
| Hyperlipidemia ⁎ | 87% | 63% | 0.06 |
| History of ischemic heart disease † | 49% | 25% | 0.14 |
| Long-term aspirin use | 83% | 50% | 0.007 |
| Good responders to aspirin before percutaneous coronary intervention | |||
| Arachidonic acid–induced light transmission aggregometry, lower tertile | 24% | 62% | 0.012 |
| Arachidonic acid evaluated by IMPACT-R, upper tertile | 20% | 75% | <0.001 |
| Baseline platelet adhesion (%) before percutaneous coronary intervention (surface coverage, no agonist) | 7.4 ± 0.5 | 17.9 ± 1.8 | <0.001 |
| Baseline platelet adhesion (%) after percutaneous coronary intervention (surface coverage, no agonist) | 8.3 ± 0.5 | 9.2 ± 0.7 | 0.35 |
| Major adverse cardiovascular events | |||
| 6 months | 29% | 20% | 0.5 |
| 15 months | 39% | 33% | 0.7 |
Clinical follow-up was available for 56 of the 63 patients. Event rates were 26.7% at 6 months and 37.5% at 15 months after PCI. One patient had nonfatal MI, 20 patients had recurrent hospitalizations for ACS of whom 15 patients had recurrent revascularization, 10 for in-stent restenosis and 5 for other diseased vessels. No death was recorded. Clinical outcome according to patients’ characteristics is presented in Table 3 . The only significant predictor for major adverse clinical events was a history of diabetes mellitus with a significantly higher event rate in diabetic patients at 15 months ( Table 3 ). There was a trend toward a higher event rate in patients with previous ischemic heart disease and hypertension, which did not reach statistical significance.
| Characteristic | Number of Patients | Event Rate (6 months) | p Value | Event Rate (15 months) | p Value |
|---|---|---|---|---|---|
| All patients | 56 | 26.7 | 37.5 | ||
| Age (<60/>60 years) | 29/27 | 21%/33% | 0.34 | 38%/37% | 0.98 |
| Men/women | 45/11 | 22%/46% | 0.13 | 36%/46% | 0.45 |
| History of diabetes mellitus (yes/no) | 15/41 | 46.7%/20% | 0.06 | 67%/27% | 0.008 |
| History of hypertension (yes/no) | 30/26 | 37%/25% | 0.08 | 47%/27% | 0.11 |
| History of smoking (yes/no) | 15/41 | 20%/29% | 0.55 | 27%/41% | 0.36 |
| History of ischemic heart disease (yes/no) | 24/32 | 37.5%/18.7% | 0.13 | 50%/28% | 0.09 |
| History of hyperlipidemia (yes/no) | 46/10 | 26%/30% | 0.84 | 39%/30% | 0.65 |
| Long-term aspirin use (yes/no) | 40/16 | 25%/28% | 0.95 | 37%/37% | 0.97 |
| Response to aspirin before percutaneous coronary intervention (light transmission aggregometry) | |||||
| Good | 19 | 5% | 0.02 | 11% | 0.01 |
| Intermediate | 19 | 33% | 47% | ||
| Poor | 18 | 45% | 56% | ||
| Response to aspirin before percutaneous coronary intervention (IMPACT-R) | |||||
| Good | 19 | 5% | 0.04 | 26% | 0.2 |
| Intermediate | 19 | 37% | 37% | ||
| Poor | 18 | 39% | 50% | ||
| Response to aspirin after percutaneous coronary intervention (light transmission aggregometry) ⁎ | |||||
| Good | 18 | 17% | 0.47 | 39% | 0.39 |
| Intermediate | 18 | 33% | 44% | ||
| Poor | 18 | 22% | 32% | ||
| Response to aspirin after percutaneous coronary intervention (IMPACT-R) ⁎ | |||||
| Good | 18 | 21% | 0.12 | 26% | 0.13 |
| Intermediate | 18 | 44% | 55% | ||
| Poor | 18 | 17% | 33% |
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