Aspirin resistance in Indian patients with coronary artery disease and cardiovascular eventsVS Thomson, B John, P George, G Joseph, J Jose
Department of Cardiology, Christian Medical College and Hospital, Vellore, India
Background : Aspirin resistance is a major problem and its incidence and clinical significance in Indian patients with documented coronary artery disease are not known. Aim : We sought to study the incidence of aspirin resistance and its clinical significance in a cohort of Indian patients with coronary heart disease on therapy with aspirin using urinary 11-Dehydrothromboxane B2 levels as a surrogate marker for antiplatelet efficacy. Setting and Design : Non randomized single center prospective study in cohort of patients with stable cardiovascular disease on chronic aspirin therapy attending the cardiology outpatient clinic of a tertiary care hospital. Materials and Methods : Urinary dehydrothromboxane levels were analyzed in a cohort of 63 patients with stable documented coronary artery disease and in 21 healthy volunteers. The cases were followed up prospectively for a median period of 36 (1-53) months. The clinical endpoint was a composite of acute coronary syndrome, stroke, revascularization and death. Statistical Analysis : Comparison of urinary dehydrothromboxane concentration values between various risk factors was done using Mann Whitney U test, a non parametric alternative of independent t test. All statistical analyses were done using SPSS 11.0 (Chicago, USA) software. Results : The median (range) absolute values of urinary11- dehydrothromboxane B2 levels for the healthy volunteers and cases were 440 (286-2050) pg/ml and 320 (72-2600) pg/ml (P=0.007). The corresponding normalized values were 87.3 (43-143) and 60.8 (16.7-943) ng/mmol of creatinine (P=0.131). Among the various vascular risk factors, patients who were overweight had higher absolute levels of 11- urinary dehydrothomboxane B2 levels (P=0.016). There were significantly more clinical events in patients with absolute urinary 11-dehydrothromboxane B2 levels in the upper two quartiles compared to the lower two quartiles (P=0.04). Conclusion : The incidence of aspirin resistance in the cohort of patients with documented heart disease was 38.1%. Patients with elevated absolute urinary dehydrothomboxane levels (>320 pg/ml) on chronic aspirin therapy constitute a high risk subset for recurrent vascular events.
Keywords: Aspirin resistance, myocardial infarction, platelet aggregation
Long-term aspirin therapy confers significant reduction in risk of subsequent myocardial infarction, stroke, and vascular death among patients with prior manifestations of cardiovascular disease. Despite the demonstrated benefit of aspirin in secondary prevention and its possible beneficial effects in selected individuals for primary prevention, there remain a large proportion of patients at risk, who do not benefit from aspirin.  The principal mechanism proposed for the antithrombotic effect of aspirin is inhibition of platelet cyclooxygenase, thus decreasing the production of the potent platelet aggregation promoter thromboxane. Urinary levels of the stable thromboxane metabolite, 11- dehydrothromboxane B2, reflect in vivo platelet activation;  and provides a surrogate marker of aspirin efficacy in patients on aspirin therapy. Ethnic variability leading to different treatment outcomes was illustrated by Mak et al., in the Charisma study.  Indian studies on aspirin resistance are sparse and none of the studies have correlated the incidence of aspirin resistance to clinical outcomes. Hence we studied the levels of urinary dehydrothromboxane B2 in healthy volunteers and patients with stable coronary artery disease on aspirin therapy, and the correlation of aspirin resistance determined in this fashion with subsequent adverse cardiovascular events; to our knowledge this is the first such study from India.
A prospective study was undertaken on a cohort of consecutive patients with documented coronary artery disease attending cardiology outpatient clinic of Christian Medical College, Vellore; a tertiary care referral hospital located in South India. The study was conducted between August 2003 and February 2008. The study, including the informed consent procedure, was approved by the institutional ethics committee.
Inclusion and exclusion criteria
Patients over 30 years of age with history of chronic stable angina and documented coronary artery disease (Q waves on ECG with echocardiographic evidence of regional wall motion abnormalities, evidence of inducible ischemia on standard Bruce protocol exercise testing, angiographic evidence of coronary artery disease, or prior revascularization for obstructive coronary artery disease) who gave informed consent for study participation were included in the study. All patients were on regular therapy with enteric coated aspirin at a dose of 75 mg daily for a minimum of seven days prior to urinary sample collection.
Exclusion criteria were history of acute coronary syndrome during the previous seven days, and therapy with other antiplatelet agents, nonsteroidal anti-inflammatory drugs, unfractionated or low molecular weight heparin. Patients were required to follow up once in six months. Clinical events were ascertained by auditing the medical records.
The baseline levels of urinary 11 dehydrothromboxane (DTB2) in the normal population was determined from 21 normal healthy volunteers (drawn from patient bystanders) over the age of 30 years who were free of coronary risk factors and coronary artery disease, and not on any antiplatelet drugs two weeks prior to sample collection and who gave informed consent to participate in the study.
Measurement of urinary 11-dehydrothromboxane levels
Thromboxane B2 (TXB2) was quantitatively measured using a commercially available competitive immunoassay (Assay Designs). TXB2 in the standards or in the urine specimen competes with TXB2 labeled with alkaline phosphatase for binding sites on rabbit polyclonal antibody to TXB2. The wells in the micro liter plate are coated with goat antibody specific to the Fc portion of rabbit IgG to capture the immune complexes. An early morning sample of urine was used for the analysis.
The samples were divided into aliquots, stored at -30oC within 60 min of collection and analyzed in batches within 5 months. After an incubation period of 2 h at room temperature the excess reagents is washed away and the substrate p-nitro phenyl phosphate added and incubated for 45 min. The enzyme reaction is stopped and the intensity of yellow color generated is read at 405 nm on a micro plate reader. The intensity of the yellow color is inversely proportional to the concentration of TXB2 in the sample and the set of standards (13.7-10000 pg/ ml). The intra- assay precision as determined by coefficient of variation for low, medium and high concentrations of DTB2 were 13.2,15.3, and 8.1%, respectively and the inter- assay precision for the samples were 17.5, 12.6, and 16.3%, respectively.
Definition of aspirin resistance and composite cardiac events
Aspirin resistance was defined as normalized urinary DTB2 level $ 67.9 ng/mmol of creatinine.  The composite cardiac events considered for analysis on follow up was documented cardiac death, acute coronary syndromes, revascularization procedures or stroke.
Sample size was calculated to be 65 subjects assuming an incidence of aspirin resistance of 42% from prior studies, with 12% precision and 95% confidence interval. All continuous variables were summarized using mean and standard deviation, and categorical variables were summarized using frequencies and percentages. The distribution of the levels of DTB2 was found to be skewed and hence median concentrations were used as a summary measure. Comparison of concentration values between various risk factors were done using MannWhitney U test, a non parametric alternative of independent t test. A 'P' value of less than 0.05 was considered to be statistically significant. All statistical analyses were done using SPSS 11.0 (Chicago, USA).
Sixty three subjects and 21 healthy volunteers were screened for urinary dehydrothromboxane levels. The baseline characteristics of the patients with documented coronary artery disease are presented in [Table 1]. The healthy volunteers had a mean age of 46.5 ± 8.6 years, with a body - mass index of 23.9 ± 3.0 kg/ m 2 ; 71% of them were males. The median values and range of absolute and normalized values of urinary DTB2 levels for the healthy volunteers were 440 (286 to 2050) pg/ ml, and 87.3 (43 to 143) ng/mmol of creatinine, respectively. The corresponding values for patients with documented coronary artery disease were 320 (72 to 2600) pg/ml and 60.8 (16.7 to 943) ng/mmol of creatinine. Absolute urinary DTB2 levels were significantly lower in patients on aspirin compared to healthy controls (P = 0.007); normalized DTB2 levels were also lower in patients compared to healthy controls, but the difference was not statistically significant (P = 0.131).
Normalized values of urinary DTB2 did not differ significantly between the subsets of patients with different vascular risk factors. Absolute values of urinary DTB2 were also comparable between the various vascular risk subsets except for overweight individuals who had significantly higher levels [Table 2]. The median follow up in months (range) for patients was 36 months (1-53 months). Eleven patients were lost to follow up. There were seven documented events in the entire cohort: One patient developed unstable angina, two patients developed non - ST segment elevation myocardial infarction, two developed ST- segment elevation myocardial infarction and two underwent revascularization procedures. There were no strokes or death during the period of follow up. There were significantly more adverse clinical events in patients with absolute urinary DTB2 level greater than the median value of 320 pg/ml compared to those below (six clinical events vs. one) (P = 0.04); however, no such clustering of events could be demonstrated when considering the normalized urinary DTB2 levels. Among patients with documented coronary artery disease, 38.1% had normalized levels of urinary DTB2 value greater than 67.9 ng/ mmol of creatinine, and were considered to be aspirin resistant.
Clinical events (composite of acute coronary syndromes, stroke, death and revascularization) mostly occurred in patients with absolute levels of urinary DTB2 levels in third and fourth quartiles, compared to the first and second quartile and this difference was statistically significant (P = 0.04); however, the difference was not present when normalized levels of urinary DTB2 were considered [Figure 1].
Our study demonstrated a small reduction (27.2 and 30.3%) in absolute and normalized urinary DTB2 values in patients with documented coronary artery disease on aspirin compared to healthy controls. Other studies had shown reductions varying between 42 and 91%. , In a study by Bruno et al., in African-American stroke patients given aspirin, there was a 44% reduction in normalized levels of urinary DTB2 with no difference in DTB2 levels between patients given low or high dose of aspirin.  In another study by Tohgi et al., in post stroke patients, the reduction in absolute urinary DTB2 levels was 42,78,91% at dosages of 40,320 and 1280 mg/day of aspirin, respectively.  The milder reduction in DTB2 in our study may be due to the presence of atherosclerosis and multiple risk factors, ,,,,, and is unlikely to be due to dose, , or preparation of aspirin used in the study.  Aspirin-insensitive TXA2 biosynthesis can occur as a result of COX-2 induction in non - platelet cells (monocytes/macrophages or endothelial cells) resulting from local inflammatory stimuli which can contribute to higher levels of DTB2. ,
In our study, patients who were overweight had significantly higher absolute levels of urinary DTB2 implying that low - dose aspirin at 75 mg per day may not be optimal in overweight Indian patients for secondary prevention [Table 2]. Maree et al.,  in their study of 131 patients with documented coronary artery disease treated with 75 mg per day of enteric coated aspirin demonstrated that younger and heavier patients had persistent platelet activity as evidenced by elevated serum thromboxane levels.
Our study failed to identify any particular vascular risk subset with significantly elevated levels of urinary DTB2 with the exception of overweight individuals, unlike some studies which suggested persistent increased platelet activity in diabetics,  smokers,  and females.  Eikelboom et al.,  in a well defined cohort of aspirin-treated patients at high risk of cardiovascular events, demonstrated that increasing baseline urinary concentrations of 11-dehydrothromboxane B2 were associated with an increasing risk of cardiovascular events, particularly myocardial infarction and cardiovascular death. External validation of the above finding was done in a cohort of the Charisma study patients which confirmed the association between adverse cardiovascular events and elevated urinary DTB2 levels.  In the study by Gum et al., patients who were aspirin resistant, 4 of 17 (24%) experienced death, MI, or CVA, compared with 30 of 309 (10%) patients who were not aspirin resistant. 
These findings suggest that patients with persistent platelet activity on low dose aspirin might require higher doses or additional antiplatelet therapy with Clopidogrel or Ticlopidine and constitute a subset of patients at high risk for recurrent vascular events.
In our study, the incidence of aspirin resistance was 38.1% compared to 22.9% as described by Lordkipanidze et al., in a similar study.  The higher percentage of aspirin resistance in Indian patients is in line with the lower degree of urinary dehydrothromboxane level inhibition achieved with standard doses of aspirin. Sadiq et al., in their study of 48 Indian patients with coronary artery disease found 41.66% of patients to have inadequate response to aspirin (2.08% aspirin resistant and 39.58% to be semi-responders). They described aspirin resistance and semi-responsiveness based on optical platelet aggregation using ADP and arachidonic acid as agonists. This gives added credibility to the findings of the our study in which the incidence of aspirin resistance was similar (38.1 vs. 41.66%) to that of the study by Sadiq et al., though a different method was used to determine aspirin responsiveness in the latter.  Several other studies in literature have reported incidence of aspirin resistance to be between 5 and 45% in patients on chronic aspirin therapy. , However, there has been poor correlation between the major platelet function tests in determining the prevalence of aspirin resistance as illustrated by Lordkipanidze et al., in their study. 
In our study, absolute levels of urinary DTB2 levels demonstrated better correlation with clinical events than normalized levels. Normalized values of DTB2 are used in most current studies and our study did not find any correlation between clinical events and normalized values of Urinary DTB2. However, this may be because our study may have been underpowered to detect a significant association between normalized urinary levels of DTB2 and clinical events even though the median levels of normalized DTB2 levels was lower in patients with no clinical events than in patients with clinical events.
The present study is vulnerable to biases since aspirin therapy was not supervised and compliance was not verified by assaying salicylate levels. The findings of our study need to be validated in larger studies using multiple platelet function assays to determine the prevalence of aspirin resistance and its bearing on subsequent cardiac events. The numbers of clinical events in the cohort of patients were few and subgroup analyses may not always be representative of the real picture; hence, further studies are warranted with a larger sample size with clinical events as primary end points.
We conclude that incidence of aspirin resistance in Indian patients with documented coronary artery disease is comparable to or higher than that of their western counterparts. Urinary DTB2 levels can be used to identify patients at increased risk of recurrent cardiovascular events, and in such patient's therapy with higher doses of aspirin or additional antiplatelet may be necessary to prevent further cardiovascular events, after compliance to aspirin therapy has been ascertained.
[Table 1], [Table 2]