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

Transcription

1 Available online at Annals of Clinical & Laboratory Science, vol. 39, no. 3, Prevalence and Risk Factors for Aspirin and Clopidogrel Resistance in Patients with Coronary Artery Disease or Ischemic Cerebrovascular Disease Hyunjung Kim, 1 Hae Kyung Lee, 1 Kyungja Han, 1 and Hui-Kyung Jeon 2 Departments of 1 Laboratory Medicine and 2 Internal Medicine, College of Medicine, The Catholic University of Korea, Seoul, Korea. Abstract. 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 pointof-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 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 highrisk 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 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, , South Korea; tel ; fax ; hkl@catholic.ac.kr 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 /09/ $ by the Association of Clinical Scientists, Inc.

2 290 Annals of Clinical & Laboratory Science, vol. 39, no. 3, 2009 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 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 antiinflammatory drugs, thrombocytopenia (platelet count < /μl), serum creatinine 3 mg/dl, or use of an oral anticoagulant (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-receptoractivating 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 χ 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 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 Table 1. Clinical characteristics and treatments. All patients Aspirin Clopidogrel (n = 197) (n = 178) (n = 139) Age (yr) 61.9 ± ± ± 11.0 Diagnosis MI 72 (36.5%) 70 (39.3%) 65 (46.8%) Angina 63 (32.0%) 63 (35.4%) 58 (41.7%) Stroke 59 (29.9%) 43 (24.2%) 15 (10.8%) TIA 3 (1.5%) 2 (1.1%) 1 (0.7%) Values are presented as means ± SD or n (%). MI: myocardial infarction. TIA: transient ischemic attack.

3 Table 2. Clinical characteristics and medication use among aspirin- and clopidogrel-resistant versus -sensitive patients. All patients Aspirin- Aspirin- p Clopidogrel- Clopidogrel- p resistant sensitive resistant sensitive (n = 197) (n = 25) (n = 153) (n = 54) (n = 85) Age (yr) 61.9 ± ± ± ± ± M:F 125:72 (1.7:1) 14:11 (1.3:1) 102:51 (2.0:1) :18 (1.7:1) 58:27 (2.2:1) Acute disease 86 (43.9%) 12 (48.0%) 59 (38.6%) (33.3%) 19 (22.4%) Hypertension 112 (56.8%) 13 (52.0%) 86 (56.2%) (50.0%) 43 (50.6%) SBP ± ± ± ± ± DBP 78.0 ± ± ± ± ± Diabetes 78 (40.4%) 12 (48.0%) 55 (35.9%) (38.9%) 27 (31.8%) Smoking 68 (34.5%) 6 (24.0%) 56 (36.6%) (40.7%) 28 (32.9%) PCI 134(68.0%) 17 (68.0%) 115 (75.2%) (87.0%) 73 (85.9%) Surgery 60 (31.3%) 11 (44.0%) 43 (28.1%) (33.3%) 21 (24.7%) BMI 25.6 ± ± ± ± ± Duration (mo)* 18.3 ± ± ± ± Dosage (mg) ± ± ACE-I use (%) 20 (80.0%) 99 (64.7%) (72.2%) 64 (75.3%) Statin use 145 (73.6%) 16 (64.0%) 120 (78.4%) (77.8%) 70 (82.4%) Beta blocker use 77 (39.1%) 8 (32.0%) 66 (43.1%) (40.7%) 44 (51.8%) Ca channel blocker use 50 (25.4%) 3 (12.0%) 41 (26.8%) (25.9%) 21 (24.7%) Aspirin use 178 (90.4%) 50 (92.6%) 71 (83.5%) Clopidogrel use 139 (70.6%) 20 (80.0%) 121 (79.1%) K + opener 78 (39.6%) 10 (40.0%) 68 (44.4%) (92.6%) 40 (47.1%) Aspirin results (ARU) ± ± ± ± Clopidogrel results (% inhibition) 27.9 ± ± ± ± 20.0 Values are presented as means ± SD or n (%). SBP: systolic blood pressure; DBP: diastolic blood pressure; PCI: percutaneous coronary intervention; BMI: body mass index; ACE-I: angiotensin-converting enzyme inhibitor; ARU: aspirin reaction unit. * Duration: duration of aspirin in aspirin users, of clopidogrel in clopidogrel users; Dosage: dosage of aspirin in aspirin users, of clopidogrel in clopidogrel users; Aspirin use: aspirin factor in clopidogrel users; Clopidogrel use: clopidogrel factor in aspirin users. Table 3. Laboratory data from aspirin- and clopidogrel-resistant versus -sensitive patients. All patients Aspirin- Aspirin- p Clopidogrel- Clopidogrel- p resistant sensitive resistant sensitive (n = 197) (n = 25) (n = 153) (n = 54) (n = 85) Hb (g/dl) 13.2 ± ± ± ± ± WBC (10 3 /μl) 7.0 ± ± ± ± ± Platelets (10 3 /μl) ± ± ± ± ± Creatinine (mg/dl) 1.0 ± ± ± ± ± Glucose (mg/dl)* ± ± ± ± ± Cholesterol (mg/dl) ± ± ± ± ± Triglyceride (mg/dl) ± ± ± ± ± LDL (mg/dl) 84.2 ± ± ± ± ± HDL(mg/dl) 39.0 ± ± ± ± ± Values are presented as means ± SD or n (%). * Fasting serum glucose; Serum total cholesterol. LDL: low-density lipoprotein. HDL: high-density lipoprotein. Aspirin- and clopidogrel-resistance in coronary and cerebrovascular disease 291

4 292 Annals of Clinical & Laboratory Science, vol. 39, no. 3, % (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 1). 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. 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 ± 18.2 vs sensitive ± 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 2 and 3. No factors other than Hb, SBP, and DBP differed significantly between the aspirinor clopidogrel-resistant and -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 ± 46.4 in aspirin-sensitive patients and ± 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 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

5 Aspirin- and clopidogrel-resistance in coronary and cerebrovascular disease 293 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 poststent 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 1 Antithrombotic Trialists Collaboration. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002;324: Krasopoulos G, Brister SJ, Beattie WS, Buchanan MR. Aspirin resistance and risk of cardiovascular morbidity: systematic review and meta-analysis. BMJ 2008;336: Snoep JD, Hovens MM, Eikenboom JC, van der Bom JG, Jukema JW, Huisman, MV. Clopidogrel nonresponsiveness in patients undergoing percutaneous coronary intervention with stenting: a systematic review and meta-analysis. Am Heart J 2007;154: Michelson AD. Platelet function testing in cardiovascular diseases. Circulation 2004;110:e Kim KE, Woo KS, Goh RY, Quan ML, Cha KS, Kim MH, Han JY. Comparison of laboratory detection methods of aspirin resistance in coronary artery disease patients. Int J Lab Hematol 2008 [E-pub ahead of print]. 6 Malinin A, Pokov A, Spergling M, Defranco A, Schwartz K, Schwartz D, Mahmud E, Atar D, Serebruany V. Monitoring platelet inhibition after clopidogrel with the VerifyNow-P2Y12 rapid analyzer: the Verify Thrombosis Risk Assessment (VERITAS) study. Thromb Res 2007; 119: van Werkum JW, Harmsze AM, Elsenberg EH, Bouman HJ, ten Berg JM, Hackeng CM. The use of the VerifyNow system to monitor antiplatelet therapy: a review of the current evidence. Platelets 2008;19: Jeong YH, Kim IS, Choi BR, Kwak CH, Hwang JY. The optimal threshold of high post-treatment platelet reactivity could be defined by a point-of-care VerifyNow P2Y12 assay. Eur Heart J 2008 [E-pub ahead of print]. 9 Harrison P, Frelinger AL, Furman MI, Michelson AD. Measuring antiplatelet drug effects in the laboratory. Thromb Res 2007;120: Lee YK, Kim HS, Park JY, Kang HJ. Incidence of aspirin resistance in the patient group of a university hospital in Korea. Korean J Lab Med 2008;28: Jeon SM, Cha JK, Kim SH, Kim JW. Clopidogrel resistance in acute ischemic stroke. J Korean Neurol Assoc 2006;24: Lev EI, Patel RT, Maresh KJ, Guthikonda S, Granada J, DeLao T, Bray PF, Kleiman NS. Aspirin and clopidogrel drug response in patients undergoing percutaneous coronary intervention: the role of dual drug resistance. J Am Coll Cardiol 2006;47:27 33.

6 294 Annals of Clinical & Laboratory Science, vol. 39, no. 3, Lee PY, Chen WH, Ng W, Cheng X, Kwok JY, Tse HF, Lau CP. Low-dose aspirin increases aspirin resistance in patients with coronary artery disease. Am J Med 2005; 118: Nadar S, Lip GY. The prothrombotic state in hypertension and the effects of antihypertensive treatment. Curr Pharm Des 2003;9: Le Quan Sang KH, Benlian P, Kanawati C, Montenay- Garestier T, Meyer P, Devynck MA. Platelet cytosolic free calcium concentration in primary hypertension. J Hypertens Suppl 1985;3:S Gum PA, Kottke-Marchant K, Welsh PA, White J, Topol EJ. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol 2003;41: Karepov V, Tolpina G, Kuliczkowski W, Serebruany V. Plasma triglycerides as predictors of platelet responsiveness to aspirin in patients after first ischemic stroke. Cerebrovasc Dis 2008;26: Gurbel PA, Bliden KP, Hiatt BL, O Connor CM. Clopidogrel for coronary stenting: response variability, drug resistance, and the effect of pretreatment platelet reactivity. Circulation 2003;107: Prabhakaran S, Wells KR, Lee VH, Flaherty CA, Lopes DK. Prevalence and risk factors for aspirin and clopidogrel resistance in cerebrovascular stenting. AJNR Am J Neuroradiol 2008;29: Frelinger AL, Li Y, Linden MD, Tarnow I, Barnard MR, Fox ML, Michelson AD. Aspirin resistance : role of preexistent platelet reactivity and correlation between tests. J Thromb Haemost 2008;6: Lordkipanidze M, Pharand C, Schampaert E, Turgeon J, Palisaitis DA, Diodati JG. A comparison of six major platelet function tests to determine the prevalence of aspirin resistance in patients with stable coronary artery disease. Eur Heart J 2007;28: Lee YW, Cho YH, Kim YH, Na JS, Shin HB, Ki CS, Choi TY, Lee YK. Aspirin non-responsiveness in Korean subjects on dual anti-platelet treatment determined by two different platelet function assays. Ann Clin Lab Sci 2008;38: Mueller T, Dieplinger B, Poelz W, Haltmayer M. Utility of the PFA-100 instrument and the novel multiplate analyzer for the assessment of aspirin and clopidogrel effects on platelet function in patients with cardiovascular disease. Clin Appl Thromb Hemost 2008;19 [E-pub ahead of print]. 24 Matetzky S, Shenkman B, Guetta V, Shechter M, Bienart R, Goldenberg I, Novikov I, Pres H, Savion N, Varon D, Hod H. Clopidogrel resistance is associated with increased risk of recurrent atherothrombotic events in patients with acute myocardial infarction. Circulation 2004;109: Chen WH, Lee PY, Ng W, Tse HF, Lau CP. Aspirin resistance is associated with a high incidence of myonecrosis after non-urgent percutaneous coronary intervention despite clopidogrel pretreatment. J Am Coll Cardiol 2004;43: