Introduction

Aspirin therapy significantly reduces the incidence of ischemic stroke.1) Despite the demonstrated benefits of aspirin in primary and secondary prevention, some individuals do not obtain the anticipated anti-platelet effect of aspirin and recur ischemic stroke. Various laboratory tests for platelet function have shown variability among individuals in the potency of anti-platelet effect with conventional doses of aspirin.10) The significant proportion of patients with low response to aspirin are prone to fail to prevent recurrent ischemic stroke.10) However, there is no formal definition of low response to aspirin. The clinical failure of aspirin to protect individuals from ischemic stroke is known as clinical aspirin low-responsiveness, while laboratory results indicating the failure of aspirin to inhibit platelet activity are known as laboratory aspirin low-responsiveness.3) There are conflicting reports on the incidence of laboratory aspirin low-responsiveness, and the reported rates vary with the test methods used. Furthermore, there is uncertainty regarding the clinical relevance of low response to aspirin.4) Moreover, fewer studies have examined the response to aspirin in patients with cerebrovascular disorders compared to cardiovascular ones. Therefore, we evaluated the incidence of laboratory aspirin low-responsiveness in patients treated with aspirin for secondary prevention of cerebrovascular events using the VerifyNow£— Aspirin assay and correlated these results with a relapse of ischemic stroke in a cross-sectional study.

Subjects and Methods

Participants

We recruited 74 patients with recurrent infarction despite of continuous aspirin medication who were admitted to the Gil Medical Center between August 2006 and May 2008. We also enrolled 88 non-recurred subjects with aspirin medication as a control group. All controlled subjects signed written informed consents and underwent the blood sampling for aspirin response test, standard battery of laboratory and physical examinations. Inclusion criteria for this study were continuous treatment with aspirin 100~300 mg a day for at least one month before blood testing. The patients with recurrent infarction were named as the recurrent stroke group (RG) and the control group without recurrence was named as non-recurrent stroke group (NRG). The medication used for the secondary prevention of stroke was enteric-coated aspirin. Concomitant cerebrovascular risk factors of these patients included hypertension (defined as systolic blood pressure¡Ã140 mmHg and diastolic blood pressure¡Ã90 mmHg or antihypertensive treatment), diabetes mellitus (defined as fasting blood glucose¡Ã126 mg per dL, postprandial plasma glucose¡Ã200 mg per dL, or treatment), hypercholesterolemia (blood cholesterol levels¡Ã200 mg on diet or treatment with statins), smoking (currently smoking), and body mass index (BMI). Aspirin compliance was determined in patient interviews. We excluded patients who had stroke of cardio-embolism, undetermined, or other determined etiology according to TOAST classification. We also excluded patients with any kind of history of gastrointestinal bleeding, hemorrhagic stroke, coagulopathy, platelet count ¤100,000/mm3, hematocrit ¤25%, creatinine¤‚4 mg/dL, current use of non-steroidal anti-inflammatory drugs, anticoagulants, or antiplatelet drugs other than aspirin or discontinuation of aspirin before testing. Fifty-two subjects (n= 32 in RG, n=20 in NRG) were excluded from the analysis. Therefore, RG consisted of 42 patients and NRG contained 68 patients. The local ethics committee on human research approved the protocol, and all patients provided written informed consent.

Platelet function analysis

Aspirin-induced platelet inhibition was measured using a commercial point-of-care assay, the VerifyNow£— Rapid Platelet Function Analyzer. The VerifyNow£— system is a simple, rapid (running time within 5 minutes), point-of-care system that requires a low sample volume and no sample preparation.10) To run the assay, citrate-anticoagulated blood (2 ml) was added to cartridges that contain fibrinogen-coated beads and platelet agonists. If aspirin has produced the expected antiplatelet effect, the fibrinogen-coated beads will not agglutinate, and light transmission will not increase. The results are expressed as aspirin reaction units (ARU).15) An ARU¤‚550 indicates the absence of aspirin-induced platelet dysfunction based on the correlation with epinephrine-induced light transmission aggregometry. The sensitivity and specificity of this assay are 92% and 85%, respectively.3)

Statistics

The two-sided t-test and chi-square test were used to analyze differences in demographic features and aspirin low-responsiveness between RG and NRG. Multivariate logistic regression analysis was used to assess the clinical factors predicting a low response to aspirin. All statistical analyses were performed using SPSS for Windows (ver. 12.0 SPSS) and ¥ñ¤0.05 was regarded as statistically significant.

Results

The clinical and demographic characteristics of the patients are listed in Table 1. There were 110 patients on aspirin for secondary prevention with a mean treatment duration of 17 months. The mean age of RG was older than that of NRG (69¡¾7 vs. 65¡¾11 years; ¥ñ=0.03). In addition, the prevalence of a laboratory low response to aspirin was significantly higher in RG than in NRG (26.2 vs. 5.8%; ¥ñ=0.01). A laboratory low response to aspirin was significantly associated with thrombotic events with an odds ratio (OR) of 5.68 and a 95% confidence interval of 1.67 to 19.27(data not shown). Although the mean duration of aspirin treatment was longer in RG than in NRG, the difference was not significant (¥ñ=0.13). The presence of hypertension, diabetes, hyperlipidemia, current smoking, and obesity did not differ significantly between RG and NRG.

To test whether laboratory aspirin low-responsiveness is an independent predictor of cerebral ischemic events in RG, we performed a multivariate logistic regression analysis including age, systemic hypertension, diabetes mellitus, hyperlipidemia, and smoking. Laboratory aspirin low-responsiveness was associated independently with recurrent ischemic stroke (¥â coefficient 1.629; ¥ñ=0.016) (Table 2).

The variability in the laboratory response to aspirin is shown in Fig. 1. The mean ARU was higher in RG than in NRG (495¡¾70 vs. 455¡¾59; ¥ñ=0.003; Fig. 2). There were significantly more patients who currently smoke in the aspirin-resistant group than in the non-resistant group (73.3 vs. 35.7%; ¥ñ=0.006 two-sided t-test). Smoking was associated with a significantly higher incidence of laboratory aspirin low-responsiveness. The odds ratio for a laboratory low response to aspirin for current smoking was 4.93 with a 95% confidence interval of 1.46 to 16.69. A multivariate logistic regression analysis including other possible factors like age, systemic hypertension, diabetes mellitus, hyperlipidemia, and aspirin dose revealed that only current smoking was independently associated with laboratory aspirin low-responsiveness (¥â coefficient 1.954; p=0.003; Table 3).

Discussion

We found that up to 26.2% of patients with recurrent stroke are laboratory aspirin low-responsive (as measured with the VerifyNow£— Aspirin assay) despite chronic aspirin therapy. The patients with recurrent stroke had an increased prevalence of laboratory aspirin low-responsiveness compared with patients without stroke recurrence (¥ñ=0.01). The fact that laboratory aspirin low-responsiveness was independently related to relapse of ischemic stroke after controlling of other stroke risk factors suggests that this low-responsiveness is an independent predictor of recurrent cerebrovascular events. Therefore, particular attention is needed with this high-risk subgroup. Previous studies reported that the prevalence of a laboratory low response to aspirin ranged from 5~60% of the population.9) The absence of standardized diagnostic criteria and a single validated method for identifying patient response to aspirin have led to a wide range of population estimates.

The VerifyNow£— Aspirin assay is a quantitative method for determining the effect of aspirin on platelet reactivity that has been approved by the US Food and Drug Administration (FDA). This assay uses arachidonic acid as the agonist and is aspirin specific, because arachidonic acid-induced platelet aggregation requires the activity of cyclooxygenase-1, which is specifically blocked by aspirin.12) Although there is increasing interest in the use of platelet function tests to monitor the effects of aspirin, there are markedly fewer studies of patients with cerebrovascular disease than of patients with cardiovascular disorders. In addition, only a few studies have examined the low response to aspirin in patients with cerebrovascular disease using the VerifyNow£— test.8)14) In our study, 13.6% of the patients showed laboratory aspirin low-responsiveness. This proportion is comparable to the 12% low-responsiveness reported in another study of Koreans with a history of ischemic stroke that also used the VerifyNow£— assay.14) However, no studies have used this test to examine the association between laboratory aspirin low-responsiveness and failure of prevention in cerebrovascular events. Two cerebrovascular studies using other methods to evaluate the platelet response to aspirin therapy attempted to correlate laboratory aspirin low-responsiveness with clinical outcomes. Grotemeyer et al. evaluated a post-stroke cohort for 2 years and found that among 174 patients with complete follow-up, aspirin non-responders had a 10-fold increase in the risk for recurrent ischemic vascular events compared with aspirin responders.5) More recently, using a retrospective PFA-100 analysis, a cross-sectional study reported that in 53 patients treated with aspirin for the secondary prevention of transient ischemic attack or stroke, the rate of laboratory aspirin low responsiveness was significantly higher (34%) in those with recurrent cerebrovascular events compared with those without recurrence (0%).6) The outcomes of these studies and our results suggest that laboratory aspirin low-responsiveness increases the risk for recurrent vascular events.

Furthermore, our results demonstrate that laboratory aspirin low-responsiveness measured with the VerifyNow£— test is associated with the clinical failure of aspirin treatment. Although some studies have suggested associations of aspirin low-responsiveness with non-compliance,2) smoking,13) hypertension,14) hypercholesterolemia,13)or age7), no study has identified definite clinical predictors of aspirin responsiveness. In this study, we found that current smoking was the only parameter correlated with laboratory aspirin low-responsiveness. Smoking promotes isoprostane synthesis and reduces the effect of aspirin on platelet aggregation.16) The strong association of smoking with a laboratory low response to aspirin suggests that aspirin low- responsiveness is one mechanism by which smoking generates a risk of thrombotic events.11) Although some previous studies showed that the dose of aspirin influenced the prevalence of aspirin low responsiveness,4)2) in our study, the aspirin dose did not significantly affect the laboratory low response to aspirin (¥ñ=0.47). However, no patient treated with 300 mg of aspirin had laboratory aspirin low-responsiveness (Table 4). It is possible that the failure of aspirin to prevent stroke arises from an inadequate dose of aspirin. Additional studies to determine whether laboratory aspirin low-responsiveness is dose dependent are needed.

Our study has several potential limitations. First, the study population was small and confined to single Korean hospital. Second, our study did not have a prospective randomized design and unrecognized confounders might have influenced the occurrence of stroke in addition to aspirin responsiveness. Third, the antiplatelet effect of aspirin may fluctuate in patients on a consistent dose, and a single baseline measurement may not reflect the extent of platelet inhibition over long periods of time. Finally, the acute stroke state in RG may have led to changes in hemostasis that in turn affected the platelet response.6) Prospective investigations are required to define aspirin low-responsiveness and its relationship to clinical outcome.

In summary, our study has significant clinical implications. Laboratory aspirin low-responsiveness may be associated with clinical failure to prevent recurrent ischemic cerebrovascular diseases, known as clinical aspirin low-responsiveness. Identifying laboratory aspirin low-responsiveness might be useful for preventing recurrent ischemic stroke.

References

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