Aspirin and Clopidogrel Resistance in Indian Patients with Ischemic Stroke and its Associations with Gene Polymorphisms: A Pilot Study.

INTRODUCTION: Antiplatelet resistance is one of the urgent issues in current stroke care. One-third to one-half of the patients who experience a recurrent stroke is already on antiplatelet medications. We studied resistance to aspirin and clopidogrel in Indian stroke patients and its association with gene polymorphisms.
METHODS: Platelet function testing by light transmission aggregometry was performed on 65 patients with ischemic stroke who were stable on dual antiplatelet therapy (clopidogrel 75 mg OD and aspirin 75 mg OD) along with 65 age-matched controls. Aspirin resistance was considered as mean platelet aggregation ≥70% with 10 μM adenosine diphosphate (ADP) and ≥20% with 0.75 mM arachidonic acid. Clopidogrel resistance was defined as <10% decrease from the baseline in platelet aggregation in response to ADP 10 μM and semi-response as <30% decrease from the baseline. Polymorphisms CYP2C19 * 2 and GPIIb/IIIa (PLA1/A2) were genotyped by polymerase chain reaction-restriction fragment length polymorphism.
RESULTS: We found 64.6% (42/65) patients with inadequate response to clopidogrel (15.4% [10/65] resistant and 49.2% [32/65] semi-responders) and 4.6% (3/65) patients with inadequate response to aspirin (3.1% [2/65] resistant and 1.5% [1/65] semi-responder). The frequency of CYP2C19*2 mutant genotype was significantly higher in clopidogrel nonresponders compared to responders (P = 0.014). Clopidogrel nonresponsiveness was much higher in small vessel stroke.
CONCLUSION: Unlike aspirin, a high proportion of nonresponders to clopidogrel was identified. In an interim analysis on 65 Indian patients, a significant association was found between CYP2C19*2 and clopidogrel nonresponsiveness.

INTRODUCTION

Stroke recurrence is an important public health concern. Around 14% of stroke survivors have a recurrent stroke within 12 months. The risk of first recurrent stroke is six times higher than the risk of first-ever stroke in the general population of the same age and sex. Almost one-half of the survivors remain disabled, and one-seventh of the survivors require institutional care.[1] After an ischemic stroke or transient ischemic attack (TIA), antiplatelet therapy is currently recommended to reduce the risk of recurrent ischemic events. Antiplatelet agents reduce ischemic events by 22% in patients with prior ischemic stroke or TIA.[2] However, much variability has been observed to the response to antiplatelet therapy with aspirin or clopidogrel among patients. The prevalence of aspirin or clopidogrel nonresponse ranges from 5% to 60% and 8% to 28%, respectively.[34] Platelet function tests have been used to find interpatient response variability to antiplatelet drugs. Light transmission aggregometry (LTA), platelet function analyzer (PFA)-100, and VerifyNow are currently the best-studied tests. There is evidence of a relationship between platelet response variability to antiplatelet therapy and clinical outcomes.[56]

The incidence of antiplatelet resistance varies widely depending on the detection technique used, variable definition of resistance and standardization of methods. Several patient-related factors such as comorbidities and genetics and drug-related factors such as pharmacodynamics and pharmacokinetic have been investigated to explain variable inter-patient response to clopidogrel.[78] Underlying genetic diversity among individuals could account for nonresponsiveness. The mechanisms proposed are polymorphisms of cytochrome P450 (CYP) and platelet-receptor genes. The “loss-of-function” allele, CYP2C19*2, is the most common and the most investigated polymorphism. Carriers of at least one loss-of-function allele have a reduced platelet aggregation in response to clopidogrel as compared with noncarriers.[910] Polymorphisms of gene-encoding glycoprotein GPIIIa have also been associated with low response to antiplatelet drugs.[1112]

Despite antiplatelet nonresponse signifying a risk factor for adverse events, improvement in clinical outcomes by intensifying antiplatelet therapy has not been demonstrated in patients with ischemic stroke or TIA.[13] There are relatively more data available on the link between antiplatelet resistance and clinical outcome in coronary artery disease (CAD). However, there is marked paucity of data in ischemic stroke.[14] The present study was needed to ascertain the prevalence of resistance, its association with selected genetic variants and identify the target population who require screening.

METHODS

Subjects

We enrolled prospectively 65 patients presenting to or on follow-up in neurology outpatient department (OPD) of Sir Ganga Ram Hospital, New Delhi, for ischemic stroke. All patients received dual antiplatelet therapy and were on stable dose of clopidogrel 75 mg once a day and aspirin 75 mg once a day for a minimum period of 15 days. Compliance to aspirin and clopidogrel was controlled by the attending physician.

The patients were enrolled according to the inclusion criteria which include patients of ischemic stroke on dual antiplatelet therapy (aspirin 75 mg+ clopidogrel 75 mg) with age of >18 years and no prior stroke. Patients with coagulopathy or personal or family history of bleeding disorders, patients on oral anticoagulants, and platelet count <100 × 109/L were excluded from the study.

An equal number of age- and sex-matched healthy controls were also studied for defining baseline range for platelet aggregation to different agonists. Controls were also important to determine the cutoff values for responders and semi-responders. The healthy controls were those who accompanied the patients as controls for the platelet aggregation studies. None of the control subjects had hypertension, diabetes mellitus, CAD, dyslipidemia, or history of alcoholism and smoking.

The study protocol was approved by the ethics committee of the Hospital. Informed written consent was obtained from all the subjects before enrolment.

Clinical data collection

Diagnosis of cerebral infarction was confirmed by history, clinical signs and symptoms and cerebral computerized tomography or magnetic resonance imaging. Patient information was documented in a study proforma which included demographic and epidemiological data, clinical background, treatment history, and comorbid conditions (e.g., diabetes mellitus and dyslipidemia) which can modify test results and common nonpharmacogenetic reasons for resistance such as noncompliance and drug interactions.

Blood sampling

Venous blood samples were collected in ethylenediaminetetraacetic acid (EDTA) vacutainer from each patient and control for platelet function tests and genetic analysis. Platelet function test was performed on light transmission platelet aggregometry. It required fasting blood sample. The patient were advised to not consumed tobacco, alcohol or nonsteroidal anti-inflammatory drugs (NSAIDs) at least a week before the test. Controls on antiplatelet drugs were excluded from the study. Platelet aggregation and other hematology parameters were tested within 2–3 h after sampling.

Platelet function test

For platelet function test, 4.5 mL blood was drawn into a vacutainer containing 0.5 mL 3.2% trisodium citrate dihydrate. Platelet-rich plasma (PRP) with platelet count adjusted to 250–300 × 109/L was separated by centrifugation at 1000 rpm for 15 min. Platelet-poor plasma (Platelet count <10 × 109) was prepared from the same sample after separation of the PRP. Platelet aggregation studies were performed with 50 μl of agonists including ADP (10 μM) and arachidonic acid (0.75 nM). Aggregation was monitored with Dual channel Lumi-Aggregometer (Model 400, Chrono-log Corporation, Chicago, USA) using 450-μl aliquots of PRP placed in cuvettes with a stir bar and warmed to 37°C. Maximal (100%) and baseline (0%) responses were set using PPP and PRP from the patient. For each different agonist concentration, the aggregation response was recorded and analyzed in duplicate and averaged.

Criteria of drug resistance

The criteria for aspirin and clopidogrel resistance was utilized as previously published.[15] It was defined as for Aspirin resistance: a mean platelet aggregation ≥70% with 10 μM adenosine diphosphate (ADP) and ≥20% with 0.75 mM of arachidonic acid. Aspirin semiresponder was defined as those meeting only one of the criteria. Clopidogrel resistance was defined as <10% decrease from the baseline in platelet aggregation in response to ADP 10 μM. Clopidogrel semi responders were those with change in platelet reactivity to ADP 10 μM by <30% from baseline. We evaluated the specific cutoffs for clopidogrel resistance in using healthy controls.

Genetic studies

For genetic study, 2.0 ml peripheral blood was drawn into a purple-top EDTA vacutainer. High-molecular-weight DNA was extracted from peripheral blood using the QIAamp blood kit (QIAGEN, Hilden, Germany) and DNA yields estimated by measuring absorbance at 260 nm. The association for CYP2C19 * 2 and GPIIb/IIIa (PLA1/A2) gene polymorphisms on antiplatelet resistance in ischemic stroke patients were determined by genotyping analysis. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for the genotyping of both the genetic variants. Briefly, single-step PCR was performed for CYP2C19*2 with 10 pico mole of each primers forward AAT TAC AAC CAG AGC TTG GC and reverse TAT CAC TTT CCA TAA AAG CAA G. An amplified pcr product was subjected to restriction digestion by using restriction endonuclease SmaI and resulted fragments were resoluted on 2% agarose gel to interpret the presence or absence of polymorphism. Similarly, a flanking region of PLA1/A2 was amplified by using primers forward TTC TGA TTG CTG GAC TTC TCT T and reverse TCT CTC CCC ATG GCA AAG AGT. PCR product was digested with MspI restriction enzyme and analysis was done by fragment separation on agarose gel. The representative gel picture of fragments obtained after PCR-RFLP for CYP219*2 and PLA1/A2 is given in Figure 1.

Figure 1

(a) Representative gel picture showing polymerase chain reaction-restriction fragment length polymorphism products of CYP2C19 * 2 polymorphism. Lane 1: Undigested polymerase chain reaction product, Lane 2: DNA ladder, Lane 3–7 representing different samples, showing wild-type genotype. Lane 8: Heterozygous for CYP2C19 * 2. (b) Representative gel picture of fragments obtained by polymerase chain reaction-restriction fragment length polymorphism of PLA1/A2 polymorphism. Lane 1: Undigested polymerase chain reaction product, Lane 2: DNA Ladder, Lane 3-7: Wildtype genotype of different samples Lane 8: Heterozygous patient

Statistical analysis

Sample size was calculated by keeping the power 80%, and two-sided alpha error 0.05% minimum 64 patients were required to observe resistance pattern as per the previous assumption. Continuous variables are presented as mean ± standard deviation and categorical variables are presented as absolute numbers and percentage. The Fisher's exact test was used to determine the relationship between gene polymorphisms in different groups of patients and to determine the association of these gene variants with comorbidities and clinical outcome. P < 0.05 was considered statistically significant. All the statistical analysis was performed by the SPSS program for Windows (version 20, SPSS, version 20, Chicago, IL, USA).

RESULTS

Demographic data and clinical characteristics of the patients are presented in Table 1. There were 48 males and 17 females ranging in age from 34 to 78 years with the mean age of 59.66 ± 9.65 years. The most common subtypes of acute ischemic stroke as per TOAST criteria were as follows: large vessel, small vessel, and cardioembolic. Hypertension was most commonly observed risk factor for stroke. The other risk factors in decreasing order of frequency included CAD, diabetes, dyslipidemia, smoking, and alcohol.

Table 1

Demographic and clinical characteristics (n=65)

Variable	Total patients, n (%)
Age (years)	59.66±9.65
Sex (male:female)	48:17
Risk factor
Hypertension	42 (64.6)
Diabetes	21 (32.3)
Coronary artery disease	39 (60.0)
Dyslipidemia	39 (60.0)
Atrial fibrillation	1 (1.5)
Smoking	23 (35.4)
Alcohol	22 (33.8)
TOAST classification
Large vessel	25 (38.5)
Lacunar	21 (32.3)
Cardio embolic	13 (20.0)
Stroke of undetermined etiology	4 (6.2)
Stroke of other determined etiology	2 (3.1)
Duration of stable antiplatelet agents
<6 months	31 (47.7)
6 months–1 year	22 (33.8)
>1 year–3 years	12 (18.5)
Concomitant drugs
PPI	49 (75.4)
Statin	64 (98.5)
NSAIDS	3 (4.6)

NSAIDS=Nonsteroidal anti-inflammatory drugs, PPI=Proton pump inhibitors, TOAST=Trail of ORG 10172 in acute stroke treatment

Drug response

Out of 65 patients, inadequate response to aspirin was observed in 3 patients (4.6%). Two (3.1%) patients showed complete resistance to aspirin whereas only one patient (1.5%) was semi responder. However; remarkably higher number of patients 42 (64.6%) had shown inadequate response to clopidogrel. Among these, resistance to clopidogrel was found in 10 (15.4%) patients, and 32 (49.2%) patients represent semi-response to the drug [Figure 2]. Aspirin and clopidogrel dual-resistant was seen in 2 (3.1%) patients.

Figure 2

The prevalence of aspirin and clopidogrel resistance in stroke patients

Genetic analysis

CYP2C19*2 and GPIIb/IIIa (PLA1/A2) gene polymorphisms were carried out in 57 out of 65 patients. CYP2C19*2 polymorphism was observed in 36 (63.2%) patients which included 29 (50.9%) heterozygous and 7 (12.3%) homozygous. PLA1/A2 polymorphism was observed in 17 (29.8%) patients which included 16 (28.1%) heterozygous and 1 (1.7%) homozygous. There was significant association observed between CYP2C19* 2 polymorphism and clopidogrel responsiveness (P = 0.014) [Table 2]. This association gets highly significant when we merged the patients of clopidogrel resistant and semi-responder group (P = 0.006). No significant association was seen for PLA1/A2 gene polymorphism among clopidogrel resistant, clopidogrel semi responder, and responders (P = 0.863) [Table 2]. Although we did not observed any association between CYP2C19*2 and PLA1/A2 polymorphism with aspirin resistance (P = 0.171 and P = 0.960, respectively).

Table 2

Genotype frequencies of CYP2C19*2 and PLA1/A2 polymorphisms and clopidogrel responsiveness

Gene polymorphisms	Genotype	Number of patients (%)			P
		Responder	Semi responder	Resistant
CYP2C19*2	Heterozygous (*1/*2) + homozygous (*2/*2)	9 (40.9)	22 (81.5)	5 (62.5)	0.014
	Wild type (*1/*1)	13 (59.1)	5 (18.5)	3 (37.5)
PLA1/A2	Heterozygous (A1/A2) + homozygous (A2/A2)	6 (27.3)	8 (29.6)	3 (37.5)	0.863
	Wild type (A1/A1)	16 (72.7)	19 (70.4)	5 (62.5)

The mean level of ADP was found to be significantly higher (P = 0.05) in patients with mutant CYP2C19 * 2 genotypes as compared to wildtype genotype. However, no significant difference was found in the mean levels of ADP in patients with mutant PLA1/A2 genotypes than to patients with wild-type genotype (P = 0.621).

Clinical correlation

No significant correlation was found between clopidogrel nonresponsiveness in relation to age group (P = 0.92), sex (P = 0.211), concomitant drugs (PPI [P = 0.455], statin [P = 0.396], NSAIDS [P = 0.374]), and duration of antiplatelet agents (P = 0.653). Clopidogrel nonresponsiveness was much higher in small vessel subtypes (76.2%); however, there was no statistically significant difference observed between stroke subtypes and clopidogrel responsiveness (P = 0.495) [Figure 3]. Although no statistically significant correlation was found between stroke risk factors and clopidogrel nonresponsiveness, higher clopidogrel nonresponsiveness was found in CAD (P = 0.09). In addition, clopidogrel nonresponsiveness (69.5%) was found more commonly in smokers [Figure 4].

Figure 3

Clopidogrel responsiveness in stroke subtypes as per TOAST classification

Figure 4

The prevalence of stroke risk factors among stroke patients group of clopidogrel responder, semiresponder, and resistant

DISCUSSION

It is well recognized that antiplatelet effects of clopidogrel are nonuniform in different patients.[16] Even though the biological and clinical resistance to antiplatelet are well established in literature, there is currently no universally acknowledged laboratory definition and no consensus on the best management strategy for these individuals.[5] The present study was initiated to prospectively evaluate the prevalence of aspirin and clopidogrel resistance in patients with ischemic stroke on dual antiplatelet therapy by measuring platelet aggregation on Light Transmission Platelet Aggregometry and to find its possible association with selected genetic polymorphisms such as CYP2C19*2 and GPIIb/IIIa (PLA1/A2). The data on Indian ethnic groups in this regard are scarce.

In 65 patients studied, 15.4% were resistant, and 49.2% were semi-responder to clopidogrel on platelet aggregation analysis. In sharp contrast, only 3.1% patients were resistant to aspirin and 1.5% was semi-responder. There is a wide variation in the figures reported for aspirin and clopidogrel resistance both in the Western and Indian literature, particularly the latter. This is mainly due to the different methodology used to study antiplatelet resistance as well as to the ethnic groups studied from the Indian subcontinent. The heterogeneity in antiplatelet resistance is partly attributed to the lack of correlation among different measurement techniques.[17]

The prevalence of antiplatelet resistance is highly assay and agonist-dependent. In our study, we used LTA as the method of choice to assess platelet aggregation. The assay is most widely used and considered as the gold standard. It provides us with ample information; however, it is time-consuming and needs technical expertise especially with regard to sample preparation. The other methods which have been used to assess response to these drugs are Verify Now, VASP phosphorylation, PFA-100, and electrical impedance.[17]

The frequency of clopidogrel resistance has been reported to vary from 2.5% to 72.5% in such studies.[18] Yi et al. in their recent study of 426 acute minor ischemic stroke Chinese patients found that 24.4% exhibit aspirin resistance, 35.9% exhibit clopidogrel resistance, and 19.2% display concomitant aspirin and clopidogrel resistance. They also found that antiplatelet resistance was associated with early neurological deterioration and recurrent ischemic stroke.[19] In another study of 375 ischemic stroke patients by Yi et al., clopidogrel resistance was seen in 153 patients (40.8%). They found that clopidogrel-resistant patients were more likely to have poor outcomes (mRS >2 points) compared with clopidogrel-sensitive patients.[20] Liu et al. found 36% clopidogrel resistance by LTA in 191 Chinese patients with ischemic stroke.[21] The high frequency of clopidogrel resistance in our study group is comparable to the Chinese population. Since our study was single-point study to determine the prevalence of aspirin and clopidogrel resistance; we did not do the follow-up of the patients to see the impact of aspirin and clopidogrel resistance on adverse clinical outcome which includes stroke recurrence.

In the present study, only 3.1% patients were resistant to aspirin, and 1.5% was semi-responder. Global incidence of aspirin resistance has ranged between 5.5% and 60%.[5] In a recent study conducted by Vališ et al., the prevalence of aspirin resistance in neurovascular patients with clinical nonresponsiveness to aspirin treatment determined by the PFA-100 and propyl gallate-induced platelet aggregation techniques were 50.6% and 17.7%, respectively.[22] Such high frequency of aspirin resistance in their study could be due to the targeted selection of patients who were nonresponsive to aspirin.

In our study, 64.6% patients were hypertensive, and there was no significant difference between responders and semi-responders to clopidogrel. Contrary, Liu et al. found a significant association between hypertension and clopidogrel resistance in ischemic stroke patients (P = 0.022).[20] No significant correlation was observed between clopidogrel nonresponsiveness and patient's age group, sex, duration of antiplatelet agents, and concomitant drugs. Clopidogrel unresponsiveness was much higher in small vessel subtypes (76.2%); however, there was no statistically significant difference between stroke subtypes and clopidogrel responsiveness.

In the present study, CYP2C19*2 loss of function allele (*2) frequency was 38.5% which is in the same line of frequencies reported from the other regions like Asians (~30%), Japanese (~28%), and Chinese (~45%). Although Caucasians (13%) and African-Americans (~18%) have relatively low frequencies.[23]

We observed in our patient group clopidogrel response was significantly associated with the CYP2C19*2 polymorphism. We found CYP2C19*2 polymorphism was significantly higher in patients with clopidogrel nonresponders than responders (P = 0.014). Frequency of loss-of-function genotypes (*2/*2 or *2/*1) of CYP2C19*2 was 81.5% in clopidogrel semi-responders and 62.5% in resistant compared to responders (40.9%). In recent study of 189 clopidogrel-treated patients with acute coronary syndromes and noncardiogenic ischemic stroke, the distribution of CYP2C19 polymorphisms was as follows: 61.1% of patients were CYP2C19 wild-type homozygotes, 27.7% of patients were CYP2C19*2 heterozygotes, 1.1% of patients were CYP2C19*3 heterozygotes, and 10% of patients were CYP2C19*2 homozygotes. Similar to our results, they observed that clopidogrel response was significantly influenced by the presence of CYP2C19 polymorphisms and concomitant use of aspirin had a significant impact on platelet response to clopidogrel, indicating a synergic interaction between these drugs.[24] We observed significantly high platelet aggregation induced by ADP in the patients with at least one loss of function allele (*2) of CYP2C19*2 (homozygous *2/*2 or heterozygous *1/*2) as compared to the those with wildtype allele (*1).

We did not observe a significant association of mutant genotype (A2/A2 or A2/A1) with nonresponsiveness to clopidogrel (P = 0.171). In addition, no difference was noticed in the rate of ADP-induced platelet aggregation among the patients with mutant PLA1/A2 genotypes and wild-type genotype. The role of PLA1/A2 gene polymorphism in aspirin hyporesponsiveness has been shown in earlier studies.[1112] No association was found between PLA1/A2 polymorphism and aspirin resistance in our study (P = 0.960). The probable explanation could be the small number of patients who showed aspirin resistant in our study. CYP2C19*2 polymorphism was present in two out of three patients who showed aspirin resistance, but we could not draw any statistical conclusion due to less number of samples who showed poor response to aspirin.

In the recent studies, Yi et al. and Sun et al. have reported CYP2C19 loss-of-function allele (*2) carriers were observed with higher risk of subsequent vascular events compared with noncarriers.[1925]

The present study has few limitations. A sample size of the present study was small, and therefore, it may limit statistical inferences specially association of gene polymorphisms. It was a time-bound study; patients coming to neurology OPD and fulfilling inclusion criteria over the period of 18 months were included in the study. We performed an analysis of only two polymorphism; some rare polymorphisms may have been left undetected in this population. Thus, we could not exclude their role in regulation of clopidogrel and aspirin resistance. Despite the prospective nature of the study and detailed interrogation of patients, it is not entirely possible to be sure of medication compliance, which might inadvertently affect the results of our analyses. This was a one point study and follow-up of patients with antiplatelet resistance has not been done. However, to the best of our knowledge, it is the first Indian study to address the aspirin and clopidogrel resistance in ischemic stroke and its connection with genetic polymorphism.

CONCLUSION

Unlike aspirin, a high proportion of nonresponders to clopidogrel was identified. A significant association was found between CYP2C19*2 and clopidogrel nonresponsiveness, suggesting the role of genetic polymorphism in individual variability in response to clopidogrel in Indian patients. Similar studies of larger sample size and of longer follow-up may strengthen our view.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.