Original Article

Variability in Platelet Response to A Single Daily Dose of 150 mg Enteric Coated Aspirin in A High Risk Population

H Mardikar*, D Deo*, N Deshpande*, Manjusha Mardikar*, A Ghosh*, Khushboo Munot*, S Steinhubl**, D Mukherjee**

*Spandan Heart Institute and Research Center, Nagpur, India. **Gill Heart Institute, University of Kentucky, Lexington, United States of America.
Received : 23.10.2007; Revised : 25.1.2008; Accepted : 3.3.2008

Abstract

Purpose : Previous studies have reported inadequate anti-platelet effect in 0.4-35% of patients taking aspirin. Such studies have arbitrarily defined the terms “semi-responders”, “non-responders” or “resistant” to variable doses of aspirin on the basis of absolute values derived from different ex-vivo platelet aggregation (PA) methods. Our objective was to define response to 150-mg dose of aspirin in terms of normally distributed values using an ex-vivo measure of PA in a population at high risk for vascular events.
Methods : We prospectively studied high risk patients with either established coronary artery disease (CAD) or stroke or transient ischemic attack (TIA) or peripheral vascular disease or with multiple atherothrombotic risk factors like diabetes plus one of the following - hypertension, increased total cholesterol, cigarette smoking, micro-albuminuria, low-high density lipoprotein (HDL), family history of CAD and receiving single 150 mg dose of aspirin daily. PA was assessed by chronolog lumi-aggregometer (490-2D) using arachidonic acid (AA) reagent.
Results : 130 patients were studied. The response of subjects to aspirin followed a normal, bell shaped distribution curve with a mean and standard deviation (S.D.) of 13.1 ± 4.4%. 3.1% patients had PA values more than 2 S.D. of the mean, hence termed as hypo-responders to aspirin while another 3.1% patients had PA values less than 2 S.D. of the mean, hence termed as hyper-responders to aspirin.
Conclusion : There is minimal inter-individual variability in the response to aspirin when tested with AA as the reagent. The response to aspirin follows a normal Gaussian distribution. The prevalence of hypo-responders to aspirin in high risk population is only 3.1%. This is the first study to document “hypo” and “hyper-responders” to single daily dose of 150mg aspirin. The clinical relevance of these findings remains to be determined. ©

INTRODUCTION

It is a well known fact that many patients taking aspirin continue to have adverse cardiovascular events. One potential explanation for aspirin failure is variable response of individual patients to aspirin and inadequate platelet inhibition.1,2 Previous studies have estimated that adequate anti-platelet effect is not achieved in 0.4 - 35% patients taking aspirin.3 Aspirin non-responsiveness or resistance is linked with increased platelet reactivity4,5 and these patients are at high risk for adverse cardiovascular events.2,6,7 The CURE trial8 showed that adding another anti-platelet agent - clopidogrel to aspirin improved outcomes in patients with Non ST Elevation Acute coronary Syndromes (NSTE ACS). However CHARISMA9 trial showed that clopidogrel plus aspirin is not significantly more effective than aspirin alone in patients at high risk for atherothrombotic events in terms of reducing rate of myocardial infarction, stroke or death. The trial also showed that the combination may be harmful for primary prevention. In view of these facts, treatment of patients at high risk for atherothrombotic events with optimal anti-platelet therapy remains enigmatic. There is a great need to appropriately identify aspirin “hypo-responders” so that therapy can be individualized. Prior studies regarding aspirin resistance have arbitrarily defined the terms “hypo-responders” or “non-responders” to aspirin on the basis of absolute values derived from different ex-vivo platelet assessment methods. This ex-vivo platelet responsiveness is influenced by a number of genetic and environmental influences. A recent study by Gurbel et al10 concluded that the assessment of aspirin resistance is highly assay dependent. Hence it is expected that there would be a large inter-individual variation in response to aspirin therapy depending on use of particular assays and definitions of aspirin resistance. To define aspirin responsiveness, a better approach would be - defining normally distributed values indicative of aspirin response as done in the standard laboratory practice. This implies classifying all the values outside two standard deviations of the mean or median as “hypo-responders” or “hyper-responders”.

METHODS

This was a single centre, prospective, observational study. After taking informed consent, we enrolled patients with established coronary artery disease (CAD) or Stroke or Transient Ischemic Attack (TIA) or Peripheral vascular disease or with multiple atherothrombotic risk factors like diabetes plus one of the following - hypertension, increased total cholesterol, cigarette smoking, micro-albuminuria, low-High Density Lipoprotein (HDL), family history of CAD. All patients were receiving ≥ 5 days of aspirin and were compliant with the therapy. Patients who were on other anti-platelet drugs (clopidogrel, ticlopidine, dipyridamole), those on long term non-steroidal anti-inflammatory drugs therapy, those who had received antithrombotic medications within 24 hours before enrollment, patients with recent acute coronary syndrome, patients having surgical procedure within 1 week before enrollment, patients with family or personal history of bleeding disorders, patients with platelet count <150 × 103/µl or >450 × 103/µl, patients with hemoglobin <8g/dl, patients with history of myeloproliferative disorders, malignant paraproteinemias or history of heparin-induced thrombocytopenia were excluded. A total of 130 patients were enrolled over a period of 4 months.
Samples of whole blood were collected in 3.2% sodium citrate vacutainer tubes at the time of enrolment. One tube of blood anticoagulated with ethylene-diaminetetraacetic acid was collected for baseline hemoglobin and platelet count. Whole-blood specimens were centrifuged for 10 min at 200 g to obtain platelet-rich plasma (PRP). Platelet-poor plasma (PPP) was obtained on the remaining specimen by re-centrifugation at 2,000 g for 15 min. 0.5 mM Arachidonic Acid (AA) reagent (5 µL AA in 500 µL of PRP) was used to stimulate platelet aggregation in the PRP. Platelet aggregation was assessed by Chronolog Lumi-Aggregometer model 490-2D. All blood samples were processed within 3 hours of blood collection. Aspirin response was defined for 150mg aspirin in terms of AA induced platelet aggregation.
Statistical analysis
Histogram was derived from the data of 130 patients and all patients having values within two standard deviations from the mean were classified as “Normal responders”. Response was termed “hypo” if a value was less than two standard deviations from the mean and “hyper” if the value was more than two standard deviations from the mean. Baseline characteristics of each group of responders were expressed as mean ± 1 standard deviation or as percentages when appropriate. Differences in the mean values between respective groups were assessed by Chi square test (for categorical variables) and ANOVA for continuous variables. All calculated p values are considered as significant when < 0.05. Statistical analysis was performed with SPSS 15.0 for Windows (SPSS, Chicago, IL).

RESULTS

The response of subjects to aspirin followed a normal, bell shaped distribution curve with a mean and standard deviation of 13.1 ± 4.4% (Fig. 1). Out of the 130 patients studied, 4 (3.1%) patients had platelet aggregation values more than two standard deviations of the median and hence termed as hypo-responders to aspirin while another (3.1%) patients had platelet aggregation values less than two standard deviations of the median and hence termed as hyper-responders to aspirin.
Baseline characteristics of patients within each group are mentioned in Table 1. The small proportion of patients in the “hypo-response” and “hyper-response” category limits our ability to draw significant conclusions from this table. The only statistically significant difference between the groups was in the sex distribution, with half of the hypo-responders as females.

DISCUSSION

This study shows that response to aspirin when tested by optical platelet aggregometry using AA as the agonist followed a normal, bell shaped distribution curve. The variability in the response to 150 mg single daily dose of aspirin was minimal with only 3.1% as hypo-responders and 3.1% as hyper-responders.

AA - arachidonic acid, PA - platelet aggregation, N - number of subjects.
Fig. 1 : Response of subjects to 150mg enteric coated aspirin.

Aspirin resistance in Indian population has not been a subject of extensive research. The standard dose of aspirin prescribed in India is 150 mg QD, which is different from the doses used in western trials on aspirin resistance. Apart from our study, Sadiq et al11 is the only other study which has evaluated the prevalence of aspirin resistance in Indian patients on 150 mg daily dose of aspirin. This study recruited 50 patients with stable coronary artery disease and defined aspirin resistance as a mean aggregation of ≥ 70% with 10 mM of adenosine di-phosphate and a mean aggregation of ≥ 20% with 0.5 mg/ml of AA on optical aggregometry. Aspirin semi responders were defined as those meeting only one of the criteria. Based on these criteria, 2.08% patients were found to be aspirin-resistant, 39.58% were aspirin semi responders and 58.33% were aspirin responders. The mean platelet aggregation with AA was 13.58 ± 21.40%. In contrast to this study, our study demonstrated a much less variability 13.1 ± 4.4%, although the mean platelet aggregation is similar.
In our study, we assessed the response to aspirin using a fixed concentration of a single platelet agonist, AA. Prior studies have demonstrated the presence of residual platelet reactivity to agonists other than AA (like collagen or ADP, or rapid shear-induced aggregation as measured by PFA-100) in patients treated with aspirin.12-15 Aspirin is an irreversible cyclo-oxygeanse 1 (COX-1) inhibitor and prevents activation of platelets primarily through the AA-thromoboxane-COX1 pathway. Hence the presence of residual platelet reactivity to agonists other than AA may not directly indicate residual COX-1 activity and should not be interpreted as evidence for aspirin resistance. The results of our study and other recently conducted studies10 show that aspirin resistance measured with AA as agonist is very low and that platelet COX-1 activity is inhibited in the majority of patients. However, aspirin may have a role in reducing platelet activation through mechanisms other than COX-1 inhibition. Whether this can be assessed by using multiple agonists in addition to AA is not clear.
Limitations of the Study
In this study we have not used other agonists like ADP, Collagen and Epinephrine which may help to identify aspirin response independent of COX-1 pathway. These were not included in the study as our intention was to study COX-1 inhibition by aspirin. Another limitation is that, we did not evaluate aspirin response before and after aspirin therapy. This was done because most of the patients at high risk for vascular disease are already on aspirin.

CONCLUSION

This study shows that there is minimal inter-individual variability in the response to aspirin when platelet aggregation is tested using AA as the agonist. The variability in the response to aspirin followed a normal, bell shaped distribution curve. The prevalence of hypo-responders to aspirin in high risk Indian population with this method is only 3.1%. This is the first study to document “hypo” and “hyper-responders” to aspirin in high risk Asian Indians on a standard dose of 150 mg enteric coated aspirin, although its clinical relevance remains to be determined. Further studies are needed to determine if the small fraction of hypo-responders have worse clinical outcome compared to normal responders and whether the hypo-responsiveness can be ameliorated by either increased dose of aspirin or use of an addition antiplatelet agent.

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