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Annals of Clinical & Laboratory Science 39:289-294 (2009)
© 2009 Association of Clinical Scientists

Prevalence and Risk Factors for Aspirin and Clopidogrel Resistance in Patients with Coronary Artery Disease or Ischemic Cerebrovascular Disease

Hyunjung Kim1, Hae Kyung Lee1, Kyungja Han1 and Hui-Kyung Jeon2
1 Departments of Laboratory Medicine and 2 Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea.

Address correspondence to Hae Kyung Lee, M.D., Dept. of Laboratory Medicine, College of Medicine, Catholic University, Uijeongbu St. Mary’s Hospital, 65-1 Kumoh-dong, Uijeongbu, Kyunggi-do, 480-130, South Korea; tel 82 31 820 3159; fax 82 31 847 6266; e-mail hkl{at}catholic.ac.kr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The objective of this study was to identify possible risk factors associated with a lack of response to aspirin and clopidogrel treatments in patients with coronary or cerebral ischemic artery disease. A point-of-care analyzer, VerifyNow (Accumetrics, San Diego, CA), was used to measure adenosine-5-diphosphate and platelet P2YI2 receptor blockage to investigate the responses of a group of 197 patients to aspirin and/ or clopidogrel therapies (aspirin therapy, 178; clopidogrel therapy, 139; both drugs, 144). Of these 197 patients, 135 (68.5%) had coronary artery disease and 72 (31.5%) had ischemic cerebrovascular disease. Aspirin resistance was defined as an ARU (aspirin reaction units) ≥550, and clopidogrel resistance was defined as platelet inhibition <20%. Twenty-five of 178 aspirin users (14.0%) were resistant to aspirin, and 54 of 139 (38.8%) clopidogrel users were resistant to clopidogrel. The data indicate that low hemoglobin (Hb) level in aspirin users and high systolic and diastolic blood pressures in clopidogrel users are significantly related to treatment resistance (p < 0.05). The latter finding is possibly due to the greater adhesiveness and increased aggregability of platelets in hypertensive patients.

Keywords: aspirin, clopidogrel, drug resistance, hypertension, hemoglobin, VerifyNow analyzer


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Platelet activation plays a crucial role in the pathogenesis of coronary artery disease (CAD) and ischemic stroke. Aspirin and clopidogrel are considered to be standard care for the prevention of ischemic events in these diseases. A meta-analysis by the Antithrombotic Trialists’ Collaboration showed that such antiplatelet treatments produce 25% reduction in serious vascular events in high-risk patients with CAD or ischemic stroke [1]. However, the antiplatelet effects of the treatments are not observed in all patients, and some patients experience thromboembolic events despite regular antiplatelet treatments. Those patients are clinically designated as aspirin or clopidogrel resistant or nonresponders. A number of studies have shown that aspirin or clopidogrel resistance, proven by laboratory testing, is associated with increased risk of recurrent cardiovascular events [2,3].

Light transmittance aggregometry (LTA) is a classic method for studying platelet function, but platelet aggregometry has major disadvantages, including poor reproducibility, large sample volume, slow assay time, and the need for sample preparation and a skilled technician [4]. Recently, two point-of-care devices that evaluate platelet function have become available: the PFA-100 analyzer (Dade-Behring, Marburg, Germany) and the VerifyNow system (Accumetrics, San Diego, CA). The VerifyNow assay is a simple, rapid method that has become widely used in daily practice instead of LTA [4]. Although correlations between the results of the VerifyNow assay and those of LTA vary according to the studies, VerifyNow has shown greater sensitivity in detecting resistance to aspirin or clopidogrel [5–7]. Many studies have reported antiplatelet treatment responses, but because various methods have been used in different patients, no consistent estimates of the prevalence of antiplatelet treatment resistance or its clinically significant predictors have been produced. Few studies have evaluated the prevalence of resistance in both CAD and stroke patients. Therefore, using the VerifyNow assay, we investigated the prevalence of aspirin and clopidogrel resistance and we identified the predictors of reduced anti-platelet response among patients with CAD or ischemic cerebrovascular disease.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients.  The study was approved by the Institutional Review Board of Uijeongbu St Mary’s Hospital. During the period from January 2007 to June 2008, 197 consecutive patients who had received aspirin and/or clopidogrel for CAD or ischemic cerebrovascular disease at our institution were enrolled (aspirin therapy, 178 patients; clopidogrel therapy, 139 patients; both drugs, 144 patients). The inclusion criterion was treatment with aspirin and/or clopidogrel for at least one mo before enrolment for the prevention of vascular thrombotic events. The exclusion criteria were discontinuation of the drug before testing (for ≥3 consecutive days for aspirin and for ≥5 consecutive days for clopidogrel), use of nonsteroidal anti-inflammatory drugs, thrombocytopenia (platelet count <100 x 103/µl), serum creatinine ≥3 mg/dl, or use of an oral anti-coagulant (warfarin), GPIIb/IIIa inhibitors, or fibrinolytic drugs within 30 days before testing. Clinical, treatment, and laboratory data for the patients were reviewed retrospectively.

VerifyNow assay.  A venous blood sample was collected in a tube containing 3.2% sodium citrate. The VerifyNow system mimics LTA because it is a turbidimetric-based optical detection system that measures platelet-induced aggregation. The VerifyNow aspirin assay and VerifyNow P2Y12 assay were used to test the effects of aspirin and clopidogrel treatments, respectively.

The VerifyNow aspirin assay contains fibrinogen-coated microparticles and is designed to measure platelet function based upon the ability of activated platelets to bind fibrinogen. Light transmittance increases as the activated platelets bind and aggregate with fibrinogen-coated beads. The instrument measures the change of the optical signal caused by aggregation. The assay results are reported in aspirin reaction units (ARU), calculated as a function of the rate of aggregation.

The VerifyNow P2Y12 assay is designed to measure platelet P2Y12 receptor blockade, since clopidogrel specifically blocks the P2Y12 receptor. In one channel of this assay, the reagent adenosine-5-diphosphate/prostaglandin E1 (ADP/ PGE1) is incorporated and is formulated specifically to measure P2Y12-mediated platelet aggregation. In a similar manner, two other activators, iso-TRAP (thrombin-receptor-activating peptide) and PAR4-AP (PAR4-activating peptide) are incorporated with fibrinogen-coated microparticles into a second channel (base channel) of the assay device. The light transmittance in the second channel indicates the baseline platelet function of the sample. The assay results are reported as the percentage change in the transmittance of the ADP/ PGE1 channel relative to the baseline channel transmittance.

Aspirin resistance was defined as ARU ≥550, and clopidogrel resistance was defined as platelet inhibition <20% [7,8]. We performed electronic quality control (EQC) and wet quality control (WQC) according to the manufacturer’s recommendations. EQC was run once each day to confirm the integrity of the instrument. WQC measures two levels of the turbidimetric signal, which verify the dynamic range of the instrument (lower range and higher range of values). We performed WQC before the first use of each new lot of assay device kits or for troubleshooting.

Statistics.  Associations between demographic, clinical, laboratory, and treatment variables were tested vs the antiplatelet treatment responses. Comparisons between continuous variables were made with the independent-sample t test. Comparisons between categorical variables were made with the {chi}2 or Fisher’s exact tests, as appropriate, using MedCalc statistical software (Mariakerke, Belgium). A 2- sided p value of <0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Twenty-five of 178 aspirin users (14.0%) were resistant to aspirin, and 54 of 139 (38.8%) clopidogrel users were resistant to clopidogrel. The frequency of aspirin-resistant patients was 13.5% (18/133) of those with CAD and 15.6% (7/45) of those with ischemic cerebrovascular disease. The frequency of clopidogrel resistant patients was 39.8% (49/123) of those with CAD and 31.3% (5/16) of those with ischemic cerebrovascular disease. Resistance to aspirin or clopidogrel did not differ significantly between patients with CAD vs ischemic cerebrovascular disease (p >0.05).

The age (mean ± SD) of the 197 patients treated with aspirin and/or clopidogrel was 61.9 ± 11.3 yr, and the male:female ratio was 125:72 (1.7:1). The diagnoses were 72 (36.5%) myocardial infarction, 63 (32.0%) angina pectoris, 59 (29.9%) ischemic stroke, and 3 (1.5%) transient ischemic attack (Table 1Go). At the time of testing, 178 (90.4%) of the 197 patients were taking aspirin, 139 (70.6%) were taking clopidogrel, and 144 (73.1%) were taking both aspirin and clopidogrel.


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  		Table 1. Clinical characteristics and treatments.
 
Patients who were resistant to aspirin showed significantly lower blood hemoglobin (Hb) levels than those of aspirin-sensitive patients (resistant 12.6 ± 1.5 g/dl, sensitive 13.4 ± 1.7 g/dl, p <0.05). Clopidogrel-resistant patients showed significantly higher systolic (SBP) and diastolic (DBP) blood pressures than those of clopidogrel-sensitive patients (SBP: resistant 128.2 ± 18.2 vs sensitive 117.2 ± 14.0 mmHg, p< 0.05; DBP: resistant 78.6 ± 11.2 vs sensitive 74.8 ± 8.7 mmHg, p <0.05). Other clinical, treatment, and laboratory factors are listed in Tables 2Go and 3Go. No factors other than Hb, SBP, and DBP differed significantly between the aspirin-or clopidogrel-resistant and -sensitive patients.


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  		Table 2. Clinical characteristics and medication use among aspirin- and clopidogrel-resistant versus -sensitive patients.
 

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  		Table 3. Laboratory data from aspirin- and clopidogrel-resistant versus -sensitive patients.
 
The duration of aspirin use averaged 18.6 ± 30.8 mo, and the duration of clopidogrel use averaged 10.9 ± 9.1 mo. The daily dose of aspirin was 100 mg (n = 154, 86.5%), 200 mg (n = 8, 4.5%), 300 mg (n = 2, 1.1%), or 400 mg (n = 14, 7.9%), and the daily dose of clopidogrel was 75 mg in all users. Neither the dose nor duration of aspirin use in aspirin users, nor the duration of clopidogrel use in clopidogrel users, differed significantly between resistant and sensitive patients (p <0.05).

Acute disease (first or subsequent attack) was recorded in 71 of 178 aspirin users (39.9%) and in 37 of 139 clopidogrel users (26.6%). Of 178 aspirin users, the mean ARU value was 440.9 ± 46.4 in aspirin-sensitive patients and 585.0 ± 35.5 in aspirin-resistant patients. Of 139 clopidogrel users, the percentage inhibition was 44.0 ± 20.0% in clopidogrel-sensitive patients and 6.3 ± 6.1% in clopidogrel-resistant patients.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, the prevalence of aspirin resistance was 14.0% and that of clopidogrel resistance was 38.8% in patients with CAD or ischemic cerebrovascular disease. The reported incidence of aspirin resistance varies widely (5–60%), and the incidence of clopidogrel resistance is 4–30% [9]. In other studies of Korean subjects, the incidence of aspirin resistance was estimated to be 2% using LTA arachidonic acid, 4% using PFA-100, and 10.1% using the VerifyNow assay [5,10]. The incidence of clopidogrel resistance was 18.5% in Korean patients with acute ischemic stroke using a whole-blood aggregometer (Chrono-log) [11]. This variation may be due to the different test methods and definitions of resistance that were used. Our study revealed that hemoglobin levels are associated with aspirin resistance in patients treated with aspirin for CAD or ischemic cerebrovascular disease. This finding is consistent with those of earlier studies, which reported that aspirin-resistant patients showed lower Hb and hematocrit values than aspirin-sensitive patients evaluated with VerifyNow [10,12–13]. An interesting finding of our study is that SBP and DBP are associated with clopidogrel resistance. The lack of any relationship between a history of hypertension and the platelet response to this antiplatelet treatment may arise because hypertensive patients usually receive antihypertensive treatments. The mechanism by which SBP and DBP are related to the platelet response to clopidogrel treatment is unclear. However, platelets from patients with hypertension show increased aggregability and adhesiveness compared to those of normotensive subjects. It is well accepted that platelets from patients with hypertension are in a state of activation for a variety of reasons [14,15].

Other reported factors associated with aspirin resistance are female sex, increased age, diabetes, and high plasma triglycerides [12,16,17]. Other factors associated with clopidogrel resistance are length of stenting, stent location, blood glucose, and diabetes [18,19]. However, these factors were not significant predictors of resistance to aspirin or clopidogrel in our study.

Many tests are available to monitor antiplatelet therapies [9]. Some studies have reported poor correlations between platelet function tests for aspirin, including LTA, PFA-100, VerifyNow, whole-blood aggregometry, and urinary 11-dehydrothromboxane B2 concentrations [5,20,21]. However, one study reported that LTA using arachidonic acid and PFA-100 correlated well and both gave accurate assessments of the response to aspirin [22]. In another study, PFA-100 was deemed useful for the detection of aspirin effects but not for the detection of clopidogrel effects, whereas a multiplate analyzer was useful for monitoring both aspirin and clopidogrel treatments [23]. It is reported that the results of the VerifyNow assay for clopidogrel resistance correlated significantly with those of ADP-induced LTA [8]. None of the currently available platelet function assays, including the VerifyNow system, has been sufficiently validated and standardized to monitor antiplatelet therapies. Therefore, clear definitions of aspirin and clopidogrel resistance must be established.

We could not evaluate the effects of platelet resistance to aspirin or clopidogrel on clinical outcome because the follow-up period was too short. Only one patient, who had received both aspirin and clopidogrel and showed resistance to only aspirin on VerifyNow tests, experienced a secondary ischemic cardiac event during the study. There have been many reports of clopidogrel and aspirin resistance in previous studies of coronary disease, and it has been reported that high post-stent platelet reactivity may be a predictor of recurrent coronary events and stent thrombosis [24,25]. Prospective long-term monitoring of these patients should be performed to determine the effects of resistance to antiplatelet treatments on clinical outcomes.

In conclusion, a point-of-care platelet function test in patients with CAD or ischemic cerebrovascular disease is a useful tool with which to monitor the effects of antiplatelet treatments. The results of this study suggest that blood Hb level in aspirin users and SBP and DBP in clopidogrel users affect the platelet response to these treatments. We intend to perform a further study to determine whether these factors affect the response of platelets to antiplatelet treatments and the clinical outcomes, and to identify those persons at risk of experiencing recurrent cardiovascular events. Larger, long-term follow-up clinical trials are needed to standardize these tests, to determine their clinical relevance, to identify significant predictors, and to establish treatment strategies for resistant patients.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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