HM Medical Clinic

Frequency of Clopidogrel Resistance
in Patients of Ischemic Heart Disease

Syed Khizar Abbas Rizvi1, Shahida Mohsin1, Tahir Saeed2, Saeed Ahmad3,
Shabbir Hussain1 Muhammad Ikram Ullah2

Received: 14/3/13, Reviewed 10/4/13, Accepted: 16/4/13
Key words: Clopidogrel, Ischemic Heart Disease, platelet Aggregation, Platelet Aggregation Inhibitors
DOI: 10.5083/ejcm.20424884.93

1. Department of Haematology,
University of Health Sciences Clopidogrel and Aspirin are widely used antiplatelet agents in the prevention and treatment of isch-
2. Department of Biochemistry,
emic heart disease (IHD). Many patients have been noticed with recurrence of major ischemic events,
University of Health Sciences due to resistance of these drugs. Different platelet function tests can be used to evaluate the de-
gree of achieved platelet inhibition in patients treated with clopidogrel. The objective of this study
3. Department of Pathology,
was to determine frequency of clopidogrel resistance in patients of ischemic heart disease. Seventy
Sharif Medical and Dental College one patients of IHD were selected from out-patient department of Punjab Institute of Cardiology
Lahore. Platelet aggregation studies were performed on Diamed Impact R. Clopidogrel response as-
4. Hussani Blood Transfusion
Centre Karachi say was performed with DiaAdin(ADP 110µmol/L). Chi-square test was applied to measure statistical
significance. Resistance to Clopidogrel was observed in 17% (12 out of 71). Clopidogrel resistance
was significantly associated with female gender (p=0.046). In our study no statistically significant as-
sociation was observed between clopidogrel resistance and risk factors like diabetes mellitus, family
history ischemic heart disease, hypertension and smoking. We concluded that resistance to Clopido-
Syed Khizar Abbas Rizvi, grel therapy is seen in significant number of patients and female patients are at high risk of develop-
ing the resistance to clopidogrel therapy. These patients can be identified by performing platelet
Department of Haematology, aggregation studies on Impact R.
University of Health Sciences Contact No. 0092-322-6686637 A number of studies have shown that aspirin or clopidogrel resistance is associated with in- [email protected] Ischemic heart disease is a major public health creased risk of recurrent cardiovascular events problem and a leading cause of death in the [6,7].The term resistance is used to indicate fail-
world. Its risk increases with age, smoking, hy- LIMITATIONS OF STUDY
ure of an agent to prevent the clinical condi- Our study was a descriptive percholesterolemia, diabetes and hypertension tion for which it is used or failure of the agent study with its limitations. The [1]. Currently, cardiovascular diseases accounts
to achieve the biochemical (pharmacokinetic exact prevalence of clopidogrel for 16.7 million deaths worldwide each year and and/or pharmacodynamic) effect [8]. Different
resistance may be different from strategies for improving prevention and man- platelet function tests have been used to mea- the one which is observed in the agement of thrombotic conditions are required sure the degree of platelet inhibition in patients present study.
[2]. The most commonly used antiplatelet drugs
treated with clopidogrel. Light transmission ag- are Acetylsalicylic acid (Aspirin) and thienopyri- gregometry with ADP as an agonist, is the most We acknowledge University used method, but the test is time consuming of Health Sciences, Lahore, and not practical for routine use. Pakistan for technically Clopidogrel is a prodrug, which needs to be me- supporting this research project. tabolized in the liver to active metabolites. Clop- New point of care devices like PFA100, Veri- We also extend our gratitude to idogrel inhibits the ADP receptor P2Y12, and fyNow and The cone and plate(let) analyser the participants of the study.
thereby inhibiting the ADP mediated platelet are being utilised for the determination of an- activation. In vivo transformation of clopidogrel CONFLICTS OF INTEREST
tiplatelet therapy effectiveness. The PFA-100® to active metabolites is an important and criti- We are grateful to measures the fall in flow rate as platelets within cal step for the drug effect. This metabolisation University of Health Sciences, citrated whole blood are aspirated through a Lahore, Pakistan for providing is dependent on the hepatic cytochrome P450 capillary and begin to seal a 150μm aperture financial support to pursue this isoenzymes like CYP2C19, CYP1A2, CYP2B6, CY- within a collagen coated membrane. The disad- research project. There are no P2C9 and CYP3A4 [3]. One of the key enzymes
vantages of PFA 100 are: This is inflexible, VWF conflicts of interest.
in clopidogrel metabolism is CYP2C19, which is dependent, Haematocrit dependent and insen- involved in both stages of clopidogrel biotrans- sitive to clopidogrel [9].

VerifyNow is fully automated platelet aggregometer which can be used to monitor the therapy of aspirin, clopidogrel and GpIIbIIIa in- Figure 1: Illustration of test report of clopidogrel resistance in a hibitors but this assay is expensive and Cartridges can only be used patient. (Processed on Diamed Impact-R, Software Version: 1.28). for single purpose [9]. The cone and plate(let) analyser, originally
developed by Varon and Savion, monitors platelet adhesion and aggregation to a plate coated with collagen or extracellular matrix (ECM) under high shear conditions of 1800 s− 1[10]. In the Com-
mercial version of the device, the Impact® (DiaMed), a plastic plate is utilized instead of a collagen or an ECM-coated surface.
In this study we used Diamed Impact R to determine the frequency of resistance of clopidogrel in patients of IHD. Diamed Impact R is the instrument which provides the physiological condition for platelet adhesion and aggregation. Platelet adhesion and aggrega- tion on the polystyrene surface is evaluated using an image analy- sis system. The results are expressed as the percentage of surface covered (%SC) by platelets and the average particle size (AS; micron m2). Normal value of % SC is >7.5 and AS is >25 micron m2 for physiological platelet function.
It was a descriptive cross sectional study conducted at the depart- ment of Haematology, University of Health Sciences Lahore in col- *SC: Surface Covered; *AS: Average Size; *Ob: Object Number laboration with Punjab Institute of Cardiology, Lahore Pakistan. The study was approved by ethical committee of University of health sciences Lahore. Patients were enrolled after informed written consent from April 2011- March 2012. Diagnosed patients of IHD who were more than 21 years of age and on Clopidogrel 75 mg for at least more than 07 days were included in study and patients who have Family or personal history of bleeding disorders, Platelet All data was entered and analysed with the help of SPSS version count <150 or >450 × 109/L, Hemoglobin <8 g/dl, Major surgical 18.0. The quantitative variables were expressed as mean+SD. Chi- procedure within one week before enrollment and Administration square test was applied to measure the statistical significance of of un-fractionated or low molecular weight heparin within 24 hours frequency of drug resistance between groups e.g. Diabetics vs. before enrollment were excluded from study.
Non-diabetics, Males vs. Females. A p value of less than 0.05 was considered for statistical significance.
Laboratory Techniques
5ml of venous blood was collected using aseptic methods. Citrated whole blood (3ml) was used for platelet studies (platelet adhe- sion and aggregation) using Impact R (Diamed, Israel). DiaAdin Out of 71 patients 37 patients had IHD without PCI (Percutaneous (ADP110µmol/L) was used as platelet agonist. 2ml blood (in EDTA) Coronay Intervention) or CABG (Coronary Arterial Bypass Graft- was used to determine Haemoglobin, Haematocrit and platelet ing). Nineteen patients had IHD with Angioplasty and 15 patients count using Sysmex XI-1800 (manufact). Resistance to Clopidogrel were treated with CABG. Mean Age in the study population in years therapy was assessed by Impact R on the basis of software generat- was 52.85+ 1.14 (95% CI 50.56-55.13) Median duration of illness in ed results: Surface covered (SC) > 2.8% was considered as response the study population was 24+ 39 (Tukeys's Hinges: 9-48) months. to clopidogrel and SC < 2.8 % was considered as no response (or Demographic Characteristics of study population are shown in Resistance) to Clopidogrel. Resistance to clopidogrel therapy in a Table 1.
patient is illustrated in Figure 1.
Hemoglobin, Hematocrit and platelet count were measured for each subject before performing the platelet aggregation studies to fulfill exclusion criteria for Hb, HCT% and platelet count Data about patient's demographic features (age, sex, and address), (Table 2).
clinical diagnosis, duration of illness, drug intake history, history of recurrent ischemic events and relevant clinical history was ob- tained on a specially designed proforma. The data about platelet aggregation studies was entered after performing the laboratory tests in the specified columns. EUROPEAN JOURNAL OF CARDIOVASCULAR MEDICINE VOL II ISSUE III

Table 1: Demographic Data of the Subjects included in the Study Family H/o IHD
Rec. ischemia
*DM: Diabetes Mellitus *HTN: Hypertension *IHD: Ischemic Heart Disease *Rec: Recurrent Table 2: Demographic Data of the Subjects included in the Study Figure 2: Frequency of Clopidpgrel Resistance Hb of the patients (g/dl)
Hct. of the patients (%)
Platelets count of the patient (109/L)
*Hb: Haemoglobin *Hct: Hematocrit *SD:Standard Deviation Figure 3: Comparison of % SC (Surface Covered) of Clopidogrel Response Assay between male and female patients Clopidogrel Response Assay
Out of 71 patients12 (17%) were found resistant to clopidogrel. The frequency of resistance to clopidogrel is shown in Figure 2. Samples of resistant patients were retested, without DiaAdin to assess any baseline platelet functional aggregation defect. No case was found to have any platelet functional defect.
There was significant association (P value of 0.046) between resis- tance to Clopidogrel and patients with female gender. It was also observed that variance in % SC (surface covered) was higher in fe- male patients as compare to male patients. Variance of %SC of male and female patients is shown in Figure 3. Significant association of Clopidogrel resistance and other risk factors like hypertension, dia- betes mellitus, smoking and family history of ischemic heart dis- ease was not observed (p value of >0.05) (Table 1). EUROPEAN JOURNAL OF CARDIOVASCULAR MEDICINE VOL II ISSUE III FREQUENCY OF CLOPIDOGREL RESISTANCE IN PATIENTS OF ISCHEMIC HEART DISEASE
In our study no significant association was observed between clop- idogrel resistance and risk factors like diabetes mellitus, family H/O Antiplatelet therapy is effective in primary and secondary preven- ischemic heart disease, hypertension and smoking. However re- tion of atherothrombotic events in patients of ischemic heart dis- sistance to clopidogrel therapy was noted more in female patients ease[11].The concept of clopidogrel resistance has emerged in the
with a statistically significant P value (P = 0.046). In a study by Boris medical literature to reflect the failure to inhibit platelet function et al. (2006) it was reported that female are predisposed to clopi- in vitro, although its existence and definition remain to be estab- dogrel resistance (P = 0.0002) This marked sex-related difference in lished. It has been proposed that the term resistance encompasses clopidogrel responsiveness needs to be confirmed in a larger num- patients for whom clopidogrel does not achieve its pharmacologi- ber of patients matched for other potential confounders. Similarly cal effect, and failure of therapy reflects patients who have recurrent various risk factors like age, smoking, diabetes, hypertension, obe- events on therapy [12]. Despite intensified antiplatelet treatment,
sity, cholesterol, did not show any statistically significant difference up to 4.7% of the patients undergoing coronary stenting develop among the groups in a study by Kumar et al. (2007). thrombotic stent occlusion, suggesting incomplete platelet inhibi- tion due to clopidogrel resistance [13].
Class IIB recommendations from the American College of Cardi- ology (ACC)/American Heart Association (AHA) have stated that The prevalence of clopidogrel non-response in patients is estimat- platelet aggregation studies are warranted in patients undergoing ed 4% to 30%[14].The reported rates vary between studies because
PCI who are at risk of sub-acute stent thrombosis, with the option of the technique used to measure the extent of platelet aggrega- of increasing their maintenance dose of clopidogrel from 75 to tion and the presence of factors contributing to platelet reactivity 150mg/day in order to suppress platelet reactivity below 50% [18].
[15]. In our study non responders to clopidogrel were 12 (17%) and
Recent studies have evaluated the effect of modifying therapy on all patients were on standard dose of 75mg OD. Clopidogrel assay clinical outcome for patients deemed non-responsive as measured was measured with DiaAdin (ADP110 µmol/L) on Diamed Impact by platelet function tests.
R. In another study 16% of Coronary arterial disease (CAD) patients were classified as non-responders and the electrical impedance ag- Clopidogrel response can also vary because of inter-individual dif- gregometry was performed in diluted whole blood in the presence ferences in drug absorption, resulting in lower levels of the active of 5 and 20 μmol/L ADP [16].
metabolite. Medications such as statins and certain proton pump inhibitors have been proposed to affect the metabolisation of In a study by Mobley et al. (2004) the prevalence of clopidogrel re- clopidogrel by the CYP isozyme 3A4, although data is controver- sistance was estimated 30%. These patients were on 75 mg daily sial [19,20]. Individuals with low baseline metabolic activity of the
maintenance dose and the method used was Optical aggregome- CYP3A4 enzyme have also been shown to have poor clopidogrel try 1 µmol/L ADP. A study conducted in India, 2.54% patients were responsiveness and this aspect should be evaluated on large ran- found to be clopidogrel resistant, 12.7% were clopidogrel semi- domised trials [21].
responders and 84.7% were clopidogrelresponders and the opti- cal aggregometry was used to define clopidogrel resistance, semi- It is concluded that resistance to clopidogrel therapy is seen in sig- responders and responders [17]. Estimated frequency of clopidogrel
nificant proportion of patients. These patients can be identified by resistance in different studies is summarised in Table 3.
performing platelet aggregation studies on Diamed Impact R and female patients are at high risk for developing the resistance to Table 3: Clopidogrel resistance in different studies Müller et al.
Optical aggregometry Jaremo et al.
Optical Aggregometry Mobley et al.
Optical Aggregometry Boris et al.
Optical Aggregometry Kumar et al.
Optical aggregometry Boris et al.
Impedence aggregometry Our study
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