Dual Antiplatelet Drug Resistance in Patients with
Acute Coronary Syndrome.

Santanu Guha, Partha Sardar, Pradipta Guha, Sarita Roy, Soura Mookerjee,
Prantar Chakrabarti, P. K. Deb, Utpal Chaudhuri, Suryyani Deb, Rathindranath Karmakar,
Anjan Kr. Dasgupta, Prabir Lahiri
Department of Cardiology, Medical College, Kolkata, India

MATERIALS AND METHODS

Methods: Study group

We prospectively enrolled 144 patients with a diagnosis of ACS between July 2008 and December 2008 after obtaining their written consent. The patients were recruited from consecutive patients admitted to the cardiology ward and ICCU. The study was reviewed and approved by the Institutional Review Board. Patients who were >21 years old and taking both aspirin and clopidogrel (325 mg of aspirin and 300mg clopidogrel as loading dose and 150 mg aspirin and 75 mg clopidogrel as daily maintenance dose) for > 7 days were eligible for enrollment. Exclusion criteria was use of nonsteroidal anti-inflammatory drugs, family or personal history of bleeding disorders, platelet count <150x10³ /L or >450 x10³/ L. Out of a total 144 patients, 106 had ST elevated acute coronary syndrome (STEACS) and 38 had Non ST elevated acute coronary syndrome (NSTEACS).

Blood samples

Blood samples for platelet function assays were collected from an antecubital vein using a 21-gauge needle 2 to 4 hours after the last dose of antiplatelet therapy. The first 2 to 4 mL of blood was discarded to avoid spontaneous platelet activation. Blood samples were collected in 3.2% citrated plasma.

Platelet Function Analysis

Platelet Aggregation (PA) with 2mg/ml collagen and 10mM ADP was performed with light transmittance aggregometry in all patients according to the standard protocol^8,9. Tests were done within 3 hours of sampling with platelet-rich plasma (PRP) by the turbidimetric method in a 2-channel aggregometer (Chrono-Log 490 Model, Chrono-Log Corp, Havertown, Pa). PRP was obtained as a supernatant after centrifugation of citrated blood at 800 rpm for 10 minutes. The isolated PRP was kept at 37°C before use. Curves were recorded for 6 minutes. For optical platelet aggregation, optical density changes were detected photoelectrically as platelets began to aggregate. ADP promotes the release of endogenous ADP and thromboxane A2 when added to platelet-rich plasma causing irreversible aggregation. Collagen is used to evaluate the degree of inhibition of platelet aggregation by aspirin. The addition of collagen to platelet-rich plasma enhances platelet aggregation by producing thromboxane A2.

Definition of low response

Platelet aggregation studies were done 7 days after the loading dose. Patients with >70% aggregation with collagen(2 mg/ml) but <70% aggregation with ADP (10mM) were labeled as aspirin resistant and >70% aggregation with ADP (10mM ) but < 70% aggregation with collagen (2 mg/ml) were labeled as resistant to clopidogrel. Clopidogrel and aspirin dual resistant were defined as > 70% aggregation with both collagen (2 mg/ml) and ADP(10mM ), as previously published in literatures⁹. Patients showing <70% but >30% aggregation were defined as hyporesponders and <30% aggregation as resistant.

Patients with drug resistance

The maintenance dose of aspirin was doubled (300mg instead of 150 mg) in cases of aspirin resistance. Similarly the maintenance dose of clopidogrel was doubled (150mg instead of 75 mg) in cases of clopidogrel resistance. In cases of dual drug resistance, maintenance dose of both aspirin and clopidogrel was doubled (300mg aspirin and 150 mg clopidogrel). Repeat aggregation studies were performed 7 days after dose doubling.

Statistical Analysis

Normally distributed continuous variables are presented as mean+SD. Variables were analyzed for a normal distribution with the Kolmogorov-Smirnov test. Categorical variables are expressed as frequencies and percentages. Differences between groups were assessed with the Fisher exact test for categorical variables. Unpaired t tests were used for comparison of normally distributed continuous variables between the two groups. p<0.05 was considered statistically significant. Statistical analysis was performed with SPSSv10.0 software (SPSS Inc. Chicago),

RESULTS

The baseline characteristics of patients in the 3 groups based on aspirin and clopidogrel responsiveness are presented in Table 1. Among 144 patients, 22 (15.3%) patients showed resistance to aspirin (group A), 28 (19.4 %) patients showed resistance to clopidogrel (group B) and 18 patients (12.5%) showed a primary resistance to both the antiplatelet agents (group C). In aspirin resistant patients (group A, n =22), mean platelet aggregation with 2mg/ml collagen was 76+11.5 % and in clopidogrel resistants (group B, n=28), mean platelet aggregation with 10mM/L ADP was 80+5.3 %,while in the dual drug resistant patients (group C, n=18), it was

When response to aspirin was analysed, it revealed 66.7% of patients were responders, 18.0% of patients were semiresponders (<70% but ³ 30% aggregation) and 15.3% patients were found to be resistant (figure 2).

Similarly, clopidogrel response pattern analysis revealed 26.4% of patients were responders, 54.2% semiresponders (<70% but ³ 30% aggregation) and 19.4% resistant (figure 3).

Dual aspirin and clopidogrel response pattern analysis showed 12.5% of patients were resistant to both the drugs (Figure 4).

Aspirin and clopidogrel responsiveness in patients with different risk factors is shown in figures 5, 6, 7 and 8. Aspirin and clopidogrel resistance were higher (though it did not reach statistical significance) in diabetic patients and smokers (figures 5 & 6). Aspirin resistance was higher in hypertensive patients and patients with elevated LDL (>100mg/dl)-(figures 7 & 8). All other parameters like obesity, ejection fraction and other drug intake did not show any significant difference among single or dual drug resistant groups.

All the six aspirin resistant patients showed adequate response after doubling the maintenance dose. However, among the twelve clopidogrel resistant patients, 8 adequately responded, 2 remained semi responders and no change was found in the remaining 2 patients after doubling the maintenance dose. (Table 2).

DISCUSSION

Aspirin and clopidogrel dual resistance may occur in 5-30% patients depending on the study and the method of determining therapeutic failure as reported in world literature⁷. Clinical trials have shown the efficacy of aspirin and clopidogrel in prevention of myocardial infarction, stroke and cardiovascular death. The Antithrombotic Trialists' Collaboration found an approximately 25% reduction in stroke, myocardial infarction or cardiovascular death with aspirin^2,6. Clopidogrel is a thienopyridine which inhibits ADP-mediated platelet activation⁶. The Clopidogrel vs. Aspirin in Patients at Risk of Ischaemic Events (CAPRIE) study revealed a statistically significant 8.7% (p = 0.043) relative risk reduction in stroke, myocardial infarction or ischaemic death in patients treated with clopidogrel in comparison to aspirin¹⁰.

A study by Gum et al using optical platelet aggregometry revealed 5% patients to be aspirin-resistant ¹². Aspirin and clopidogrel dual resistance in Indian population has not been studied extensively. The most commonly used dose of aspirin prescribed in India is 150 mg OD, which is different from the doses used in western trials on aspirin resistance. Sadiq et al prospectively evaluate the prevalence of aspirin resistance in Indian patients with stable coronary artery disease (CAD) on 150 mg daily dose of aspirin with 0.5 mg/ml of arachidonic acid and 10 µM ADP. In their study 2.08% patients were found to be aspirin-resistant, 39.58% were aspirin semi-responders and 58.33% were aspirin responders¹³. H Mardikar et al prospectively studied high risk patients with either established CAD or stroke or transient ischemic attack (TIA) or peripheral vascular disease or with multiple atherothrombotic risk factors and receiving single 150 mg dose of aspirin daily. Patients with PA values more than two S.D. of the mean were termed as hypo-responders to aspirin in this study, 3.1% patients were reported hypo-responders¹⁴. Separate criteria was used for defining aspirin resistance in these studies and they assessed only aspirin resistance in patients with stable CAD and those with various cardiovascular diseases or risk factors. In our study, we assessed both aspirin and clopidogrel resistance in patients with ACS in Indian population.

Dual drug resistance to both aspirin and clopidogrel was found in 12% patients, 17% patients were aspirin resistant and 19% patients were clopidogrel resistant. 64% patients showed adequate response to both aspirin and clopidogrel.

A study by AA Desai et al has shown enhanced platelet aggregation induced by various agonists including collagen and ADP in hypertensive patients as compared to normal subjects¹⁵. Another study by Joseph Hung revealed that smoking significantly increase platelet aggregation and smoking-enhanced platelet thrombosis may be an important contributory mechanism for acute coronary events in smokers. They also suggest that catecholamine release and enhanced platelet aggregation

response to in vivo agonists may contribute to the prothrombotic effects of smoking in ACS patients¹⁶. In a study by Angiolillo et al, increased platelet aggregation and activation were observed in diabetics as compared to non-diabetic subjects who were on long-term therapy (>1 month) of combined aspirin (100 mg/day) and clopidogrel (75 mg/day) treatment (p < 0.05 for all platelet function assays). Diabetic subjects had a higher number of clopidogrel nonresponders ( p < 0.04)¹⁷.

In our study, both aspirin and clopidogrel resistance was higher in diabetic patients and smokers. Aspirin resistance was higher in hypertensive population and patients with elevated LDL. The mechanism of resistance remains incompletely understood, although specific clinical, cellular and genetic factors have been shown to influence therapeutic failure. Causes of aspirin resistance are multifactorial ranging from clinical factors like noncompliance, failure to prescribe appropriate doses, nonabsorption and others; cellular factors like insufficient suppression of CoxI, genetic factors like COXI, Glycoprotein (Gp) IIb/IIIa receptor polymorphism etc. Variability in clopidogrel response can be due to extrinsic or intrinsic mechanisms such as drug-drug interactions involving CYP3A4, or genetic polymorphisms of the P2Y12 receptor and CYP3As^{18, 19}.

Several case-control studies have demonstrated that higher post treatment platelet reactivity is associated with adverse consequences. Chen et al showed adverse consequences of aspirin-resistance²⁰ and Matetzky et al have shown a 25%increase in the recurrence of cardiovascular events during a 6-month follow-up in ST Elevation Myocardial Infarction (STEMI) patients with a reduced pharmacological response to clopidogrel²¹. Clopidogrel resistance is also associated with late stent thrombosis in post-PCI patients²². A recent study also reported that high platelet reactivity in Type 2 diabetes patients with coronary artery disease while on chronic dual antiplatelet therapy is associated with a higher risk of long-term adverse cardiovascular events²³. Taken together, these studies support the hypothesis that platelet responsiveness carries direct clinical relevance. The treatment for failed antiplatelet therapy is as yet undefined. While in some of our patients after doubling the dose of the antiplatelet drugs, we observed satisfactory inhibition of platelet aggregation. Larger trials from India are required to find out the consequences of dual drug resistance and to advocate proper therapeutic options.

Acknowledgments

The authors acknowledge the technical support rendered by Sri Biswajit Bhar during the experiments.

The present work is sponsored by DST, Govt. of India (SR\SO\HS-84\2004).

Conflict of Interest Disclosures

None
References

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