Journal of Clinical and Preventive Cardiology

: 2021  |  Volume : 10  |  Issue : 3  |  Page : 80--84

Clopidogrel resistance in cardiovascular disease patients and its association with gene polymorphisms: A pilot study

S Parameshwara1, B Manjula2, Geetha Bhaktha2, Gurupadappa Kallaganad3, GK Ranjith Kumar4,  
1 Department of Cardiology, Super-Speciality Hospital, SIMS, Shimoga, Karnataka, India
2 Multidisciplinary Research Unit (MRU), Shimoga Institute of Medical Sciences, Shimoga, Karnataka, India
3 Department of Biochemistry, Shimoga Institute of Medical Sciences, Shimoga, Karnataka, India
4 General Medicine, McGann Teaching District Hospital, SIMS-Shimoga, Karnataka, India

Correspondence Address:
Dr. S Parameshwara
Department of Cardiology, Super-Speciality Hospital, SIMS, Shimoga, Karnataka


Aims: To determine the genotypic and allelic frequencies of multidrug resistance protein 1 (MDR1), CYP2C19, and P2Y12 gene and their polymorphisms in the Shimoga population who admitted for treatment in McGann teaching district hospital-Shimoga Institute of Medical Sciences (SIMS), Shimoga. Materials and Methods: It is a cross-sectional study that was conducted in McGann teaching district hospital, SIMS, Shimoga. Patients who were admitted to the Intensive coronary care unit due to cardiovascular disease (CVD) were recruited for the study. Sociodemographic data with venous blood samples (5 ml) were collected along with informed consent. Genotyping of the MDR1, CYP2C19, and P2Y12 polymorphisms were done using the polymerase chain reaction (PCR) restriction fragment length polymorphism method. Descriptive and inferential statistics were done using the Statistical Package for the Social Sciences software. The genotypic and allelic frequency was calculated using the Hardy-Weinberg equilibrium. Results: Out of 40 CVD cases, 60% were male and 40% were female and the mean age was found to be 57.13 ± 11.57 years. The percentage of the obese group was more compared to other groups of body mass index. Risk factors such as diabetes, hypertension, food custom, smoking, and tobacco consumption were not significant while alcohol consumption was seen to be significant among the study population at a 5% level of significance. The genotypic frequencies for a heterozygous and mutant type of MDR1 (C3435T) and CYP2C19*2 (G681A) were found to be 35%, 2.5%, 57.5%, and 5%, respectively. Whereas the gene polymorphism of CYP2C19*3 and P2Y12 was not observed in the present study population. Conclusions: This is the first gene polymorphism study with respect to clopidogrel resistance in the Shimoga population. We have demonstrated the presence of polymorphism in the MDR1 and CYP2C19 genes in this study population. A further elaborate study should be conducted for a better understanding of genetic with nongenetic factors involved in poor response toward clopidogrel drug using a large population.

How to cite this article:
Parameshwara S, Manjula B, Bhaktha G, Kallaganad G, Ranjith Kumar G K. Clopidogrel resistance in cardiovascular disease patients and its association with gene polymorphisms: A pilot study.J Clin Prev Cardiol 2021;10:80-84

How to cite this URL:
Parameshwara S, Manjula B, Bhaktha G, Kallaganad G, Ranjith Kumar G K. Clopidogrel resistance in cardiovascular disease patients and its association with gene polymorphisms: A pilot study. J Clin Prev Cardiol [serial online] 2021 [cited 2022 Jan 22 ];10:80-84
Available from:

Full Text


Cardiovascular disease (CVD) is an important cause of mortality and morbidity in the world. Nowadays, these diseases are epidemic in both urban and rural areas. To treat CVD many antiplatelet drugs such as clopidogrel, prasugrel, or ticagrelor, were used in different parts of India. Compared to other antiplatelet drugs, clopidogrel was generally used to treat CVD, due to its efficiency, cost, and limited side effects. These events are associated with poor response to the clopidogrel known as clopidogrel resistance. The precise mechanism for the resistance of clopidogrel is not very well understood/established but is likely multifactorial such as poor absorption, drug–drug interaction, platelet function, single-nucleotide polymorphism (SNP), and many more.

Clopidogrel is an oral thienopyridine that acts as a potent antiplatelet drug. It irreversibly inhibits platelet aggregation induced by adenosine diphosphate (ADP), low concentrations of thrombin, or by collagen.[1] For inhibition of platelet aggregation, the clopidogrel first absorbs and transported through the small intestine by multidrug resistance protein 1 (MDR1) and was converted into its active form in the liver by the Cytochrome P450 (CYP2C19) enzymes system and then acts via irreversible antagonism of the platelet purinergic (P2Y12) ADP receptor. By blocking this receptor, Clopidogrel prevents platelet degranulation and inhibits the action of the glycoprotein IIb/IIIa (GP IIb/IIIa) receptor which is responsible for the binding of fibrinogen and platelet aggregation. Therefore, clopidogrel can be considered as a precursor of an active metabolite.[2] Clopidogrel resistance has been documented in the range of 5%–44% worldwide including India.[3] The percentage varies from state to state and place to place due to regional variation in the Indian population. More work was conducted in the north region of India,[4],[5],[6] whereas in the south region of India only diminutive work[7] was done with a small sample size in Kerala,[8] Hyderabad,[9] and Tamil Nadu,[10] but we do not have any type of information/data about clopidogrel resistance related to gene polymorphism in Karnataka (Shimoga).

For clopidogrel resistance, only one gene polymorphism was not a reason, so in this study, we are going to detect/screen for SNPs of MDR1, CYP2C19, and P2Y12 genes are proposed which influence the response to clopidogrel.

 Materials and Methods

Study population

This is a cross-sectional study conducted in the McGann teaching district hospital-SIMS Shimoga, India. Patients with CVDs were enrolled prospectively for genotyping of the MDR1 (C3435T), CYP2C19*2 (G681A), CYP2C19*3 (G636A), and P2Y12 (i-T744C) polymorphisms. The patients were enrolled according to the inclusion criteria which include patients admitted to the intensive coronary care unit and on the clopidogrel therapy with the age of >18 years and exclusion criteria which includes active neoplasm or history of neoplasm, severe renal and hepatic insufficiency, hemorrhagic diathesis, hematocrit <35% or >50%, pregnant women, cancer patients with treatment and concomitant GPIIb/IIIa inhibitor administration such as abciximab and eptifibatide. Informed written consent was obtained from all the subjects before enrolment.

Patient information was documented in a study pro forma which included demographic data of individuals such as age, gender, body mass index (BMI), systolic blood pressure (SBP), diastolic blood pressure (DBP), smoking, alcohol, and tobacco consumption, hypertension, type of occupation, diabetes mellitus, and family history of CVD.

Blood sampling

Venous blood samples (5 ml) were collected in ethylene-diamine tetra-acetic acid vacutainer from each patient for genetic analysis.

Genetic studies

DNA was extracted from peripheral blood using the spin column kit method (HimediaHiPurA™ Blood Genomic DNA Mini Purification) and DNA yield was estimated by measuring absorbance at 260 nm using Nanodrop (Eppendorf BioSpectrometer® basic). Primers were designed using Primer 3 and BLAST from the National Center for Biotechnology Information and purchased from bioengineering (India). Amplification of the gene was carried out using polymerase chain reaction (PCR) using ×2 PCR Taq Mixture (Himedia) in a thermal cycler (Biorad). The primers used for PCR of each polymorphism are given in [Table 1] and PCR conditions are depicted in [Table 2]. Restriction fragment length polymorphism (RFLP) analysis was done using Sau3A1, SmaI, BamHI, and HpyCH4IV restriction enzymes (New England Biolabs) to identifying the genotypes of MDR1 (C3435T), CYP2C19*2 (G681A), CYP2C19*3 (G636A), and P2Y12 (i-T744C), respectively and bands were analyzed by 1.5% Agarose gel electrophoresis which was stained with ethidium bromide and the banding pattern was documented using Bio-Rad gel doc instrument.{Table 1}{Table 2}

Statistical analysis

The data obtained were coded and entered into Microsoft Excel and analyzed using the Statistical Package for the Social Sciences IBM SPSS software version 22, Armonk, NY: IBM Corp. Descriptive statistics such as frequencies, mean, and standard deviation were calculated. Inferential statistics like analysis of variance and Chi-square test were applied. The statistical significance was evaluated at a 5% level of significance. Allelic frequencies were calculated from genotype frequencies. Genotypes were tested for deviations from the Hardy-Weinberg equilibrium.


Demographic characteristics

In this study, there were 24 males and 16 females with a mean age of 57.13 ± 11.57 years. The mean and standard deviation of SBP, DBP, and BMI of the study population was found to be 131.0 ± 23.18 mmHg, 81.65 ± 12.64 mmHg, and 25.2 ± 3.65 Kg/m2.

In our study population, the percentage of nonvegetarians, nontobacco users, nonalcoholic and nonsmokers, nondiabetic were more when compared to vegetarians, tobacco users, alcoholics, smokers, and diabetic. Risk factors such as diabetes, hypertension, food habit, smoking, and tobacco consumption were not significant, while alcohol consumption was seen to be significant among the study population at a 5% level of significance [Table 3].{Table 3}

The percentage of CVD patients was more in ≥61 years age group when compared to other groups [Figure 1]a.{Figure 1}

The percentage of CVD patients in underweight, normal weight, overweight, obese I, and obese II was found to be 5%, 22.5%, 10%, 57.5%, and 5% [Figure 1]c. Another risk factor of CVD is the lifestyle/type of work of the individuals. In this study, heavy type (52.5%) lifestyle was significantly higher compared to moderate (7.5%) and sedentary (40%) type of occupation [Figure 1]b.

Genetic analysis

PCR-RFLP for the MDR1 (C3435T), CYP2C19*2 (G681A), CYP2C19*3 (G636A), and P2Y12 (i-T744C) was done using Sau3A1, SmaI, BamHI, and HpyCH4IV restriction enzymes, and the banding pattern is presented in [Figure 2] and [Supplementary Figure 1].{Figure 2}[INLINE:1]

MDR1 (C3435T) polymorphism was found to be in 37.5% which included 35% heterozygous (Loss of function allele) and 2.5% homozygous mutant (total loss of function). CYP2C19*2 (G681A) polymorphism was found to be in 62.5% which included 57.5% heterozygous and 5% homozygous mutant. Whereas in the case of CYP2C19*3 (G636A) and P2Y12 (i-T744C) gene polymorphism was not observed in the present study population [Table 4].{Table 4}

Among 15 patients with MDR1 (C3435T) polymorphisms, eight (20%) were female and 7 (17.5%) were male, whereas in CYP2C19*2 (G681A) polymorphism, eight (20%) were female and 17 (42.5%) were male among 25 patients. Females had a significantly higher incidence of MDR1 (C3435T) polymorphisms whereas males had a significantly higher incidence of CYP2C19*2 (G681A) polymorphisms [Table 5].{Table 5}


This is a pilot study that demonstrated the polymorphism of drug absorption (MDR1), and clopidogrel metabolizing enzyme (CYP2C19*2) genes in our study population.

The human MDR1 gene is positioned in chromosome 7 which consists of 28 exons and core promoter regions. C3435T SNP is a silent mutation in exon 26 of the MDR1 gene. It affects the expression and function of P-glycoprotein which is associated with drug absorption in the intestine.[11],[12],[13] In our study population, the frequency of variant genotype of MDR1 (C3435T) which affects the intestinal absorption of clopidogrel was found to be 37.5% (35% heterozygous and 2.5% mutant) which was similar to the other studies conducted in different ethnic groups.[14],[15],[16]

The human CYP2C19 gene plays a significant role in clopidogrel metabolism by converting into its active pro-drug form. There will be variability in the concentration of active metabolites due to polymorphism in the CYP2C19 gene. CYP2C19*1(wild type), CYP2C19*2-G681A (with splicing defect) and CYP2C19*3-G636A (with stop codon) were some genetic variants of the CYP2C19 gene. Many studies show that polymorphism of the CYP2C19*2 is the key marker of low responsiveness to clopidogrel when compared to other alleles of the CYP2C19 gene.[17] Similar to CYP2C19*2 other alleles such as CYP2C19*3, CYP2C19*4, CYP2C19*5, CYP2C19*6, CYP2C19*7, and CYP2C19*8 will also diminish the clopidogrel metabolism, but their frequency is less when compared to CYP2C19*2.[18],[19] The relative risk for major cardiac events among patients who were treated with clopidogrel was found to be 1.53 to 3.69 times higher for carriers of CYP2C19*2 and CYP2C19*3 when compared with noncarriers.[20] In our study population, the frequency of variant genotype of CYP2C19*2 (G681A) was found to be 62.5% (57.5% heterozygous and 5% mutant) which was similar to the other studies in the Indian population.[15],[21],[22],[23] In the present study population, CYP2C19*3 (G636A) and P2Y12 (i-T744C) polymorphisms were absent as in other studies.[21],[24]


It is a pilot study with a small sample sizeIt was a single-center study and hence the results cannot be generalized to the total population.


Compared to other studies worldwide our present study also supports the importance of MDR1 and CYP2C19*2 gene variants as a potential marker of clopidogrel resistance. Further, an elaborate study should be conducted in a larger population with follow-up cases and healthy controls for a better understanding of the phenomenon of clopidogrel heterogeneity of response toward clopidogrel drug and also we should look into the effect of nongenetic components on clopidogrel resistance along with genetic components.


Authors acknowledge the Department of Health Research, New Delhi, India for providing the facilities at Multidisciplinary Research Unit for the study. Also thank Mrs. Revathy, Biostatician for helping through the statistics.

Financial support and sponsorship

Multidisciplinary Research Unit, Department of Health Research, New Delhi, India.

Conflicts of interest

There are no conflicts of interest.


1CAPRIE Steering Committee. A randomised, blinded, trial of Clopidogrel versus Aspirin in Patients at Risk of Ischaemic Events (CAPRIE). CAPRIE Steering Committee. Lancet 1996;348:1329-39.
2Sabatine MS, Cannon CP, Gibson CM, López-Sendón JL, Montalescot G, Theroux P, et al. Addition of clopidogrel to aspirin and fibrinolytic therapy for myocardial infarction with ST-segment elevation. N Engl J Med 2005;352:1179-89.
3Ray S. Clopidogrel resistance: The way forward. Indian Heart J 2014;66:530-4.
4Kumar S, Saran RK, Puri A, Gupta N, Sethi R, Surin WR, et al. Profile and prevalence of clopidogrel resistance in patients of acute coronary syndrome. Indian Heart J 2007;59:152-6.
5Guha S, Sardar P, Guha P, Roy S, Mookerjee S, Chakrabarti P, et al. Dual antiplatelet drug resistance in patients with acute coronary syndrome. Indian Heart J 2009;61:68-73.
6Kar R, Meena A, Yadav BK, Yadav R, Kar SS, Saxena R. Clopidogrel resistance in North Indian patients of coronary artery disease and lack of its association with platelet ADP receptors P2Y1 and P2Y12 gene polymorphisms. Platelets 2013;24:297-302.
7Jose R, Chandrasekaran A, Sam SS, Gerard N, Chanolean S, Abraham BK, et al. CYP2C9 and CYP2C19 genetic polymorphisms: Frequencies in the south Indian population. Fundam Clin Pharmacol 2005;19:101-5.
8Koshy SK, Salahuddin S, Karunakaran B, Nalakath SY, Bhaskaran J, Haridas PV, et al. Aspirin and clopidogrel resistance using the cone and plate (let) analyser in Indian patients with coronary artery disease. Heart Asia 2014;6:159-62.
9Rath PC, Chidambaram S, Rath P, Dikshit B, Naik S, Sahoo PK, et al. A study on the impact of CYP2C19 genotype and platelet reactivity assay on patients undergoing PCI. Indian Heart J 2015;67:114-21.
10Subraja K, Dkhar SA, Priyadharsini R, Ravindra BK, Shewade DG, Satheesh S, et al. Genetic polymorphisms of CYP2C19 influences the response to clopidogrel in ischemic heart disease patientsin the South Indian Tamilian population. Eur J Clin Pharmacol 2013;69:415-22.
11Taubert D, von Beckerath N, Grimberg G, Lazar A, Jung N, Goeser T, et al. Impact of P-glycoprotein on clopidogrel absorption. Clin Pharmacol Ther 2006;80:486-501.
12Tang K, Wong LP, Lee EJ, Chong SS, Lee CG. Genomic evidence for recent positive selection at the human MDR1 gene locus. Hum Mol Genet 2004;13:783-97.
13Fung KL, Gottesman MM. A synonymous polymorphism in a common MDR1 (ABCB1) haplotype shapes protein function. Biochim Biophys Acta 2009;1794:860-71.
14Salem AH, Ali M, Ibrahim A, Ibrahim M. Genotype and allele frequencies of MDR-1 gene polymorphism in Jordanian and Sudanese populations. Am J Med Stud 2014;2:19-23.
15Raju S, Shah VK, Shalia K. Genetic aspect of clopidogrel resistance: An Indian scenario. Indian J Cardio Biol Clin Sci 2015;3:107.
16Idrissi HH, Hmimech W, Khorb NE, Akoudad H, Habbal R, Nadifi S. Association of the C3435T Multi-Drug Resistance Gene-1 (MDR-1) Polymorphism with Clopidogrel Resistance among Moroccan Acute Coronary Syndromes (ACS) Patients. J Thrombo Cir 2016;2:2-9.
17Nguyen TA, Diodati JG, Pharand C. Resistance to clopidogrel: A review of the evidence. J Am Coll Cardiol 2005;45:1157-64.
18Gladding P, Webster M, Zeng I, Farrell H, Stewart J, Ruygrok P, et al. The pharmacogenetics and pharmacodynamics of clopidogrel response: An analysis from the PRINC (Plavix Response in Coronary Intervention) trial. JACC Cardiovasc Interv 2008;1:620-7.
19Shuldiner AR, O'Connell JR, Bliden KP, Gandhi A, Ryan K, Horenstein RB, et al. Association of cytochrome P450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy. JAMA 2009;302:849-57.
20Paré G, Mehta SR, Yusuf S, Anand SS, Connolly SJ, Hirsh J, et al. Effects of CYP2C19 genotype on outcomes of clopidogrel treatment. N Engl J Med 2010;363:1704-14.
21Lamba JK, Dhiman RK, Kohli KK. CYP2C19 genetic mutations in North Indians. Clin Pharmacol Ther 2000;68:328-35.
22Adithan C, Gerard N, Vasu S, Rosemary J, Shashindran CH, Krishnamoorthy R. Allele and genotype frequency of CYP2C19 in a Tamilian population. Br J Clin Pharmacol 2003;56:331-3.
23Gairolla J, Ahluwalia J, Khullar M, Kler R, Kishore K, Medhi B, et al. Clopidogrel response in ischemic stroke patients: Is polymorphism or gender more important? Results of the CRISP study. J Clin Neurosci 2020;76:81-6.
24Cui G, Zhang S, Zou J, Chen Y, Chen H. P2Y12 receptor gene polymorphism and the risk of resistance to clopidogrel: A meta-analysis and review of the literature. Adv Clin Exp Med 2017;26:343-9.