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Comparison of Aspirin Response Measured by Urinary 11-Dehydrothromboxane B2 and VerifyNow Aspirin Assay in Patients with Ischemic Stroke
Comparison of Aspirin Response Measured by Urinary 11-Dehydrothromboxane B2 and VerifyNow Aspirin Assay in Patients with Ischemic Stroke Pornpatr A. Dharmasaroja, MD,* and Suvaraporn Sae-Lim†
Background: We looked for the prevalence of aspirin nonresponders, compared the results of 2 tests assessing aspirin responses—measurement of urinary 11dehydrothromboxane B2 (dTXB2) and VerifyNow Aspirin assay—in patients with ischemic stroke, and examined the relationship of aspirin nonresponse and the outcomes of the patients. Methods: One hundred one patients with ischemic stroke were prospectively included. Aspirin response was assessed by urinary dTXB2 measurement and VerifyNow Aspirin assay. The Spearman correlation coefficients and kappa statistics were calculated to assess correlation and agreement between the 2 tests. The measured outcome was the occurrence of cardiovascular events and death. Results: Prevalence of aspirin nonresponders was 40% and 6%, if they were measured by urinary dTXB2 and VerifyNow Aspirin assay, respectively. Poor correlation in the results between the 2 tests was found (r 5 .135, P 5 .190). The degree of agreement between the 2 tests in relation to resistance status was weak (kappa 5 .032, P 5 .590). With a mean follow-up time of 17 months, the outcomes occurred significantly higher in aspirin nonresponders who were diagnosed by urinary dTXB2 measurement as compared with patients with aspirin response (18% versus 2%, odds ratio 8.8, 95% confidence interval 1.18-65.4, P 5 .037). Conclusions: Our research confirmed poor correlation and lack of agreement between the 2 tests. Only aspirin nonresponders who were diagnosed by dTXB2 measurement were related to having cardiovascular events and death. Further research is still needed to identify the best method of diagnosis of aspirin nonresponders. Key Words: Stroke—aspirin—aspirin nonresponder— VerifyNow—11-dehydrothromboxane B2—Asia. Ó 2013 by National Stroke Association
From the *Division of Neurology, Department of Internal Medicine, Faculty of Medicine, Thammasat University, Pathumthani; and †Research Unit, Faculty of Medicine, Thammasat University, Pathumthani, Thailand. Received July 24, 2013; revision received August 1, 2013; accepted August 8, 2013. Grant support: This research was funded by the National Research University Project of Thailand Office of Higher Education Commission. Competing interests: The authors declare no competing interests. Address correspondence to Pornpatr A. Dharmasaroja, MD, Division of Neurology, Faculty of Medicine, Thammasat University, Klong 1, Klong Luang, Pathumthani 12120, Thailand. E-mail: pornpatr1@ hotmail.com. 1052-3057/$ - see front matter Ó 2013 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.08.001
Aspirin therapy for secondary stroke prevention reduces risk of recurrent stroke by 15%.1 American Stroke Association recommends the use of aspirin 50-325 mg/ d in patients with noncardioembolic stroke or transient ischemic attack.2 However, approximately a third to half of patients have recurrent strokes while on antiplatelet therapy.3 ‘‘Aspirin resistance’’ has been defined as the inability of aspirin to protect individuals from thrombotic complications or to produce an anticipated effect on one or more in vitro tests of platelet function.4,5 However, different terms—‘‘aspirin nonresponders’’ or ‘‘low response’’ or ‘‘high residual platelet reactivity’’—have been used to describe patients who had low or no anticipated response after antiplatelet treatment by different laboratory methods/criteria. Systemic reviews
Journal of Stroke and Cerebrovascular Diseases, Vol. -, No. - (---), 2013: pp 1-5
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P.A. DHARMASAROJA AND S. SAE-LIM
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and meta-analysis showed that patients who were aspirin nonresponders had greater risk of clinically cardiovascular events than aspirin-sensitive patients.6,7 There are many laboratory methods used to measure aspirin responses. However, poor correlation among methods is usually found. It was proposed that methods that directly measure the capacity of platelets to synthesize thromboxane A2 (TXA2) may be preferable.8 Thromboxane B2 is a stable metabolite of TXA2. The results from measurement of urinary 11-dehydrothromboxane B2 (dTXB2) have been shown to correlate with future cardiovascular events in patients at high vascular risk who were treated with aspirin.9 VerifyNow is a fully automated point-of-care platelet aggregometer to measure antiplatelet therapy. VerifyNow Aspirin assay has US Food and Drug Administration approval for monitoring aspirin therapy.10 There is less data about aspirin nonresponders in patients with ischemic stroke as compared with those with coronary artery diseases, especially in Asian patients. The purpose of this research is to look for the prevalence of aspirin nonresponders, compare the results of the tests assessing aspirin responses between urinary dTXB2 measurement and VerifyNow Aspirin assay in patients with ischemic stroke, and compare those results with the clinical outcome of the patients.
Methods Patients with ischemic stroke who were treated with aspirin at Thammasat University Hospital during April 2011 to August 2011 were prospectively included. Patients who were on other antiplatelets, besides aspirin, having contraindications to aspirin, noncompliance or poor compliance, allergy to aspirin, renal failure (required dialysis), or who declined to participate in the study were excluded. Noncompliance or poor compliance was defined by inability to take aspirin every day for at least 7 days before the enrollment in patients with stable ischemic stroke. For the included patients with acute ischemic stroke, uncoated aspirin 325 mg was prescribed at the stroke unit and aspirin 81 or 325 mg was used to treat patients with stable ischemic stroke at the outpatient clinic. All patients were followed up as outpatients at the clinic or by telephone calls or mail in case of patients who were unable to come to the hospital. Information about baseline characteristics, stroke subtypes, stroke severity, compliance of medications, doses of aspirin, side effects of aspirin, and concomitant medications, especially NSAIDS, were studied. The outcomes of the study were cardiovascular events, including recurrent ischemic stroke, transient ischemic attack, myocardial infarction, unstable angina, cardiac interventions, cardiovascular death, and all-cause mortality. Urine samples and blood samples of the patients were collected at the same time, 2-6 hours after taking aspirin. A study revealed that there was no significant difference
in urine dTXB2 level whether measured on day 3 or day 7 after daily aspirin administration.11 Thus, in patients with acute ischemic stroke, urine and blood samples were collected on day 3 or after 3 doses of 325 mg aspirin. All urine samples were kept at 280 C all the time until analysis. Urine samples were assayed for dTXB2 levels with a commercially available enzyme immunoassay (Cayman Chemical). Urinary dTXB2 was normalized to urine creatinine. Subjects presenting urinary dTXB2 levels of 67.9 ng/mmol of creatinine or more were considered ‘‘aspirin nonresponders.’’9 VerifyNow Aspirin assay is a test to detect platelet dysfunction because of aspirin ingestion in whole blood for the point-of-care laboratory setting. The assay incorporates the agonist arachidonic acid to activate platelets, and it measures platelet function based on the ability of activated platelets to bind to fibrinogen. Besides a platelet activator, the cartridges contain fibrinogen-coated beads. Blood sample tubes are mixed before insertion onto the cartridge that has been premounted onto the instrument. Aggregation in response to the agonist is monitored by light transmission through chambers in each cartridge. The assay reports the extent of platelet aggregation as aspirin reaction units (ARUs). ARU values of 550 ARUs or more are considered inadequate antiplatelet effect.10 Continuous variables were presented as mean 6 standard deviation, and categorical variables were presented as frequencies and percentages. Correlation between results from the 2 tests was analyzed using Spearman correlation coefficient. The agreement between the aspirin resistance status assessed by the 2 platelet function tests was evaluated with kappa statistics. The demographics and vascular risk factors were compared between patients with and without aspirin nonresponse using Student t test (for the continuous variables) and the chi-square test (for the proportions). The study was approved by the Faculty of Medicine, Thammasat University’s ethical review committee. All patients read an information sheet and gave their consent to participate in the study.
Results There were 101 patients with ischemic stroke who had both of the antiplatelet function tests during the study period. Baseline characteristics of the patients are presented in Table 1. One hundred one urine and blood samples were studied. Prevalence of aspirin nonresponders was 40% and 6%, if they were measured by urinary dTXB2 and VerifyNow Aspirin assay, respectively. The Spearman correlation coefficients and kappa statistics were calculated to assess correlation and agreement between the 2 tests. Poor correlation in results between the 2 test was found (r 5 .135, P 5 .190). The degree of agreement between the 2 tests in relation to resistance status was weak (kappa 5 .032, P 5 .590) (Fig 1).
COMPARISON OF ASPIRIN RESPONSE TESTS
Table 1. Baseline characteristics of the patients in the study Baseline characteristics Mean age (y) Mean NIHSS Female, n (%) Hypertension, n (%) Diabetes mellitus, n (%) Hyperlipidemia, n (%) Coronary artery disease, n (%) History of ischemic stroke, n (%) History of intracerebral hemorrhage, n (%) History of transient ischemic attack, n (%) Smoking, n (%) Alteplase treatment, n (%) Presentation, n (%) Acute ischemic stroke Stable ischemic stroke Stroke subtype, n (%) Transient ischemic attack Large-artery atherosclerosis Small-artery occlusion Cardioembolic Other determined cause Undetermined cause Mean urine dTXB2 (ng/mmol of creatinine) Mean aspirin reaction units
N 5 101 61 6 13 665 40 (40%) 65 (64%) 15 (15%) 43 (43%) 2 (2%) 5 (5%) 2 (2%) 3 (3%) 28 (28%) 21 (21%) 66 (65%) 35 (35%) 7 (7%) 28 (28%) 58 (57%) 2 (2%) 2 (2%) 4 (4%) 78.55 6 61.2 446 6 50
Abbreviations: dTXB2, 11-dehydrothromboxane B2; NIHSS, National Institute of Health Stroke Scale.
With a mean follow-up time of 17 months, 8 patients had cardiovascular events or death (myocardial infarct in 3 patients, coronary angioplasty in 1 patient, and death in 4 patients). The causes of death were unknown in 2 patients, renal failure in 1 patient, and intracerebral hemorrhage in 1 patient. The outcomes occurred more
Figure 1. Scatter plot of the results of the 2 tests in the patients. (A) Patients with aspirin responders from urine 11-dehydrothromboxane B2 measurement. (B) Patients with aspirin nonresponders from urine 11-dehydrothromboxane B2 measurement (‘‘C’’ represents aspirin nonresponders from VerifyNow Aspirin assay). Abbreviation: ARU, aspirin reaction unit.
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frequently in aspirin nonresponders who were diagnosed by urinary dTXB2 measurement as compared with patients with aspirin response (18% versus 2%, odds ratio 8.8, 95% confidence interval 1.18-65.4, P 5.037). However, the aspirin nonresponse which was evaluated by VerifyNow Aspirin assay did not correlate with the outcomes (Table 2).
Discussion Prevalence of aspirin resistance in patients with various cardiovascular diseases ranges from 3% to 82% from previous studies.12,13 The high variance can be partly attributed to different methods used to assess antiplatelet function of the aspirin. Lordkipanize et al14 studied 201 patients with stable coronary artery disease and found that prevalence of aspirin resistance varied according to the assay used: 10.3%-51.7% for light transmission aggregometry (LTA) using adenosine diphosphate as the agonist, 18% for whole blood aggregometry, 59.5% for PFA-100, 6.7% for VerifyNow Aspirin, and 22.9% by measuring urinary dTXB2. Dosage of aspirin may affect the prevalence of aspirin nonresponder. Helgason et al15 showed that escalation of dosage of aspirin resulted in more complete platelet inhibition in aspirin nonresponders while taking normal to low dose (#325 mg) and suggested that the ability of aspirin and the dose required to inhibit platelet aggregation may depend on the individual. As compared with studies about aspirin resistance in patients with coronary artery disease, the studies in patients with stroke were fewer. Because stroke can be caused by many mechanisms besides atherothrombosis, such as cardioemboli, arterial dissection, or hypoperfusion, thus recurrent ischemic stroke while having antiplatelet therapy may not solely represent resistance to the antiplatelet. Prevalence of aspirin resistance in patients with coronary artery disease and in those with stroke may be different. Poor correlation among platelet function tests in evaluation of antiplatelet resistance is known. Each test has different advantages and disadvantages. Which test is the best method to evaluate aspirin resistance is still debated. LTA is still used as the gold standard of platelet function testing in many centers; however, LTA has been reported to have poorly standardized technique and is sensitive to several variables. Results from this technique may overestimate the prevalence of aspirin resistance.8,10 In evaluation of aspirin resistance, some experts suggested that methods to directly measure the capacity of platelets to synthesize TXA2 were preferable. In vivo, TXA2 is rapidly converted into a more stable and inert metabolite, TXB2, which is further metabolized to dTXB2.10 However, approximately 30% of urinary metabolite derives from extraplatelet sources (eg, monocytes/ macrophages), and this fraction can increase in pathologic conditions such as inflammatory diseases.16,17 Thus, the results from dTXB2 measurement may represent both
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Table 2. Baseline characteristics of patients with and without aspirin resistance in the study Urine dTXB2 measurement
Baseline characteristics
Aspirin responders (n 5 61)
Aspirin nonresponders (n 5 40)
P value
Aspirin responders (n 5 95)
Aspirin nonresponders (n 5 6)
59 5.6 20 (32%) 40 (66%) 7 (11%) 26 (43%) 2 (3%) 1 (2%) 1 (2%) 18 (30%) 15 (24%)
65 6 21 (53%) 25 (63%) 8 (21%) 17 (42%) — 4 (11%) 2 (5%) 10 (24%) 9 (22%)
.026 .786 .047 .771 .167 .942 .513 .154 .563 .478 .794
61 6 40 (42%) 62 (65%) 13 (14%) 41 (43%) 1 (1%) 6 (6%) 3 (3%) 26 (27%) 22 (23%)
65 5 1 (17%) 4 (67%) 2 (33%) 2 (33%) 1 (17%) — — 2 (33%) 1 (17%)
34 (56%) 27 (44%) 264
32 (81%) 8 (19%) 277
.014 .430
62 (65%) 34 (35%) 270
5 (83%) 1 (17%) 264
.660 .846
1 (2%)
7 (18%)
.037
6 (6%)
2 (33%)
.064
P value .484 .687 .396 .925 .218 .964 .124
.667 .580
Abbreviations: CE, cardioembolism; LAA, large-artery atherosclerosis; OC, other determined etiology; rtPA, recombinant tissue plasminogen activator; SAO, small-artery occlusion; UND, undetermined etiology.
P.A. DHARMASAROJA AND S. SAE-LIM
Mean age (y) Mean NIHSS Female, n (%) Hypertension, n (%) Diabetes mellitus, n (%) Hyperlipidemia, n (%) Coronary artery disease, n (%) History of ischemic stroke, n (%) History of transient ischemic attack, n (%) Smoking, n (%) rtPA treatment, n (%) Presentation, n (%) Acute ischemic stroke Stable ischemic stroke Mean doses of aspirin (mg) Outcomes Cardiovascular events and death, n (%)
VerifyNow aspirin assay
COMPARISON OF ASPIRIN RESPONSE TESTS
platelet function and inflammatory status of the patients, which may be better used to predict the occurrence of future cardiovascular events. Our study also showed varied prevalence of aspirin nonresponders in patients with ischemic stroke, with 40% from urinary dTXB2 measurement and 6% from VerifyNow Aspirin assay. Such a big difference in the prevalence of aspirin nonresponders assessed by 2 methods at the same time may be explained by the difference in the methods. VerifyNow Aspirin assay directly measures platelet aggregation induced by the agonist, arachidonic acid, in the whole blood of the patients, whereas dTXB2 level represents TXA2 production in the body, both by platelets and extraplatelet sources. We hypothesized that aspirin may be able to inhibit platelet aggregation in most of the patients but may not be enough to both inhibit platelet aggregation and inflammation that occurred more during acute atherothrombosis because almost two thirds of the patients were included in the acute phase of stroke. Our study found that aspirin nonresponders, diagnosed by dTXB2 measurement, were associated with older age, female, and acute stroke presentation. Previous studies also showed the sex difference in platelet function in response to aspirin. Women demonstrated increased baseline platelet reactivity and also have greater residual platelet activity after taking aspirin compared with men.11,18,19 Linden et al20 suggested that platelet hyperreactivity may explain higher prevalence of aspirin nonresponders during acute atherothrombosis. Several studies revealed that patients with aspirin nonresponse had greater risk of clinically cardiovascular events than aspirin-sensitive patients.6,7,21 In our study, only aspirin nonresponders who were diagnosed by dTXB2 measurement, not by VerifyNow Aspirin assays, were related to having cardiovascular events and death. This may be explained in part by the smaller numbers of aspirin nonresponders that were diagnosed by VerifyNow Aspirin assays. This study is a prospective study with follow-up results of cardiovascular events and death. However, the small number of patients included in the study is the limitation of the study. Further research is still needed to identify the best method of diagnosis of aspirin resistance, its clinical importance, and how to manage these patients. Our research confirmed the poor correlation and agreement between the 2 tests. Only aspirin nonresponders diagnosed by dTXB2 measurement were related to having cardiovascular events and death.
References 1. Johnson ES, Lanes SF, Wentworth CE, et al. A metaregression analysis of the dose-response effect of aspirin on stroke. Arch Intern Med 1999;159:1248-1253. 2. Furie KL, Kasner SE, Adams RJ, et al. Guidelines for the prevention of stroke in patients with stroke or transient ischemic attack. A guideline for healthcare professionals
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from the American Heart Association/American Stroke Association. Stroke 2011;42:227-276. Lloyd-Jones D, Adams RJ, Brown TM, et al. Heart disease and stroke statistics—2010 update: a report from the American Heart Association. Circulation 2010;121:948-954. Sztriha LK, Sas K, Vessei L. Aspirin resistance in stroke: 2004. J Neurol Sci 2005;229-230:163-169. Pamukcu B. A review of aspirin resistance; definition, possible mechanisms, detection with platelet function tests, and its clinical outcomes. J Thromb Thrombolysis 2007;23:213-222. Snoep JD, Hovens MC, Eikenboom JC, et al. Association of laboratory-defined aspirin resistance with a higher risk of recurrent cardiovascular events. A systemic review and meta-analysis. Arch Intern Med 2007;167:1593-1599. Krasopoulos G, Brister SJ, Beattie WS, et al. Aspirin ‘resistance’ and risk of cardiovascular morbidity: systemic review and meta-analysis. BMJ 2008;336:195-198. Cattaneo M. The clinical relevance of response variability to antiplatelet therapy. Hematology Am Soc Hematol Educ Program 2011;2011:70-75. Eikelboom JW, Hirsh J, Weitz JL, et al. Aspirin resistance and the risk of myocardial infarction, stroke or cardiovascular death in patients at high risk of cardiovascular outcomes. Circulation 2002;105:1650-1655. Harrison P, Frelinger AL III, Furman MI, et al. Measuring antiplatelet drug effects in the laboratory. Thrombosis Res 2007;120:323-336. Dharmasaroja PA, Sae-Lim S. Effects of different doses, enteric-coated preparation of aspirin, and sex on urinary 11-dehydrothromboxane B2 in healthy volunteers. Blood Coagul Fibrinolysis 2010;21:649-652. Topcuglu MA, Arsava EM, Ay H. Antiplatelet resistance in stroke. Expert Rev Neurother 2011;11:251-263. Bennett D, Yan B, Macqreqor L, et al. A pilot study of resistance to aspirin in stroke patients. J Clin Neurosci 2008; 15:1204-1209. Lordkipanidze M, Pharand C, Schamper E, et al. 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;28:1702-1708. Helgason CM, Tortorice KL, Winkler SR, et al. Aspirin response and failure in cerebral infarction. Stroke 1993; 24:345-350. Cattaneo M. Aspirin and clopidogrel. Efficacy, safety and the issue of drug resistance. Arterioscler Thromb Vasc Biol 2004;24:1980-1987. Frelinger AL III, Furman MI, Linden MD, et al. Residual arachidonic acid-induced platelet activation via an adenosine diphosphate-dependent but cyclooxygenase-1- and cyclooxygenase-2-independent pathway: a 700-patient study of aspirin resistance. Circulation 2006;113:2888-2896. Shen H, Herzog W, Drolet MA, et al. Aspirin resistance in healthy drug-na€ıve men versus women (from the hereditary phenotype intervention heart study). Am J Cardiol 2009;104:606-612. Qayyum R, Becker DM, Yanek LR, et al. Platelet inhibition by aspirin 81 and 325 mg/day in men versus women without clinically apparent cardiovascular disease. Am J Cardiol 2008;101:1359-1363. Linden MD, Tran H, Woods R, et al. High platelet reactivity and antiplatelet therapy resistance. Semin Thromb Hemost 2012;38:200-212. Grotemeyer KH, Scharafinski HW, Husstedt IW. Twoyear follow-up of aspirin responder and aspirin nonresponders. A pilot-study including 180 post-stroke patients. Thromb Res 1993;71:397-403.
Background: We looked for the prevalence of aspirin nonresponders, compared the results of 2 tests assessing aspirin responses—measurement of urinary 11dehydrothromboxane B2 (dTXB2) and VerifyNow Aspirin assay—in patients with ischemic stroke, and examined the relationship of aspirin nonresponse and the outcomes of the patients. Methods: One hundred one patients with ischemic stroke were prospectively included. Aspirin response was assessed by urinary dTXB2 measurement and VerifyNow Aspirin assay. The Spearman correlation coefficients and kappa statistics were calculated to assess correlation and agreement between the 2 tests. The measured outcome was the occurrence of cardiovascular events and death. Results: Prevalence of aspirin nonresponders was 40% and 6%, if they were measured by urinary dTXB2 and VerifyNow Aspirin assay, respectively. Poor correlation in the results between the 2 tests was found (r 5 .135, P 5 .190). The degree of agreement between the 2 tests in relation to resistance status was weak (kappa 5 .032, P 5 .590). With a mean follow-up time of 17 months, the outcomes occurred significantly higher in aspirin nonresponders who were diagnosed by urinary dTXB2 measurement as compared with patients with aspirin response (18% versus 2%, odds ratio 8.8, 95% confidence interval 1.18-65.4, P 5 .037). Conclusions: Our research confirmed poor correlation and lack of agreement between the 2 tests. Only aspirin nonresponders who were diagnosed by dTXB2 measurement were related to having cardiovascular events and death. Further research is still needed to identify the best method of diagnosis of aspirin nonresponders. Key Words: Stroke—aspirin—aspirin nonresponder— VerifyNow—11-dehydrothromboxane B2—Asia. Ó 2013 by National Stroke Association
From the *Division of Neurology, Department of Internal Medicine, Faculty of Medicine, Thammasat University, Pathumthani; and †Research Unit, Faculty of Medicine, Thammasat University, Pathumthani, Thailand. Received July 24, 2013; revision received August 1, 2013; accepted August 8, 2013. Grant support: This research was funded by the National Research University Project of Thailand Office of Higher Education Commission. Competing interests: The authors declare no competing interests. Address correspondence to Pornpatr A. Dharmasaroja, MD, Division of Neurology, Faculty of Medicine, Thammasat University, Klong 1, Klong Luang, Pathumthani 12120, Thailand. E-mail: pornpatr1@ hotmail.com. 1052-3057/$ - see front matter Ó 2013 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.08.001
Aspirin therapy for secondary stroke prevention reduces risk of recurrent stroke by 15%.1 American Stroke Association recommends the use of aspirin 50-325 mg/ d in patients with noncardioembolic stroke or transient ischemic attack.2 However, approximately a third to half of patients have recurrent strokes while on antiplatelet therapy.3 ‘‘Aspirin resistance’’ has been defined as the inability of aspirin to protect individuals from thrombotic complications or to produce an anticipated effect on one or more in vitro tests of platelet function.4,5 However, different terms—‘‘aspirin nonresponders’’ or ‘‘low response’’ or ‘‘high residual platelet reactivity’’—have been used to describe patients who had low or no anticipated response after antiplatelet treatment by different laboratory methods/criteria. Systemic reviews
Journal of Stroke and Cerebrovascular Diseases, Vol. -, No. - (---), 2013: pp 1-5
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P.A. DHARMASAROJA AND S. SAE-LIM
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and meta-analysis showed that patients who were aspirin nonresponders had greater risk of clinically cardiovascular events than aspirin-sensitive patients.6,7 There are many laboratory methods used to measure aspirin responses. However, poor correlation among methods is usually found. It was proposed that methods that directly measure the capacity of platelets to synthesize thromboxane A2 (TXA2) may be preferable.8 Thromboxane B2 is a stable metabolite of TXA2. The results from measurement of urinary 11-dehydrothromboxane B2 (dTXB2) have been shown to correlate with future cardiovascular events in patients at high vascular risk who were treated with aspirin.9 VerifyNow is a fully automated point-of-care platelet aggregometer to measure antiplatelet therapy. VerifyNow Aspirin assay has US Food and Drug Administration approval for monitoring aspirin therapy.10 There is less data about aspirin nonresponders in patients with ischemic stroke as compared with those with coronary artery diseases, especially in Asian patients. The purpose of this research is to look for the prevalence of aspirin nonresponders, compare the results of the tests assessing aspirin responses between urinary dTXB2 measurement and VerifyNow Aspirin assay in patients with ischemic stroke, and compare those results with the clinical outcome of the patients.
Methods Patients with ischemic stroke who were treated with aspirin at Thammasat University Hospital during April 2011 to August 2011 were prospectively included. Patients who were on other antiplatelets, besides aspirin, having contraindications to aspirin, noncompliance or poor compliance, allergy to aspirin, renal failure (required dialysis), or who declined to participate in the study were excluded. Noncompliance or poor compliance was defined by inability to take aspirin every day for at least 7 days before the enrollment in patients with stable ischemic stroke. For the included patients with acute ischemic stroke, uncoated aspirin 325 mg was prescribed at the stroke unit and aspirin 81 or 325 mg was used to treat patients with stable ischemic stroke at the outpatient clinic. All patients were followed up as outpatients at the clinic or by telephone calls or mail in case of patients who were unable to come to the hospital. Information about baseline characteristics, stroke subtypes, stroke severity, compliance of medications, doses of aspirin, side effects of aspirin, and concomitant medications, especially NSAIDS, were studied. The outcomes of the study were cardiovascular events, including recurrent ischemic stroke, transient ischemic attack, myocardial infarction, unstable angina, cardiac interventions, cardiovascular death, and all-cause mortality. Urine samples and blood samples of the patients were collected at the same time, 2-6 hours after taking aspirin. A study revealed that there was no significant difference
in urine dTXB2 level whether measured on day 3 or day 7 after daily aspirin administration.11 Thus, in patients with acute ischemic stroke, urine and blood samples were collected on day 3 or after 3 doses of 325 mg aspirin. All urine samples were kept at 280 C all the time until analysis. Urine samples were assayed for dTXB2 levels with a commercially available enzyme immunoassay (Cayman Chemical). Urinary dTXB2 was normalized to urine creatinine. Subjects presenting urinary dTXB2 levels of 67.9 ng/mmol of creatinine or more were considered ‘‘aspirin nonresponders.’’9 VerifyNow Aspirin assay is a test to detect platelet dysfunction because of aspirin ingestion in whole blood for the point-of-care laboratory setting. The assay incorporates the agonist arachidonic acid to activate platelets, and it measures platelet function based on the ability of activated platelets to bind to fibrinogen. Besides a platelet activator, the cartridges contain fibrinogen-coated beads. Blood sample tubes are mixed before insertion onto the cartridge that has been premounted onto the instrument. Aggregation in response to the agonist is monitored by light transmission through chambers in each cartridge. The assay reports the extent of platelet aggregation as aspirin reaction units (ARUs). ARU values of 550 ARUs or more are considered inadequate antiplatelet effect.10 Continuous variables were presented as mean 6 standard deviation, and categorical variables were presented as frequencies and percentages. Correlation between results from the 2 tests was analyzed using Spearman correlation coefficient. The agreement between the aspirin resistance status assessed by the 2 platelet function tests was evaluated with kappa statistics. The demographics and vascular risk factors were compared between patients with and without aspirin nonresponse using Student t test (for the continuous variables) and the chi-square test (for the proportions). The study was approved by the Faculty of Medicine, Thammasat University’s ethical review committee. All patients read an information sheet and gave their consent to participate in the study.
Results There were 101 patients with ischemic stroke who had both of the antiplatelet function tests during the study period. Baseline characteristics of the patients are presented in Table 1. One hundred one urine and blood samples were studied. Prevalence of aspirin nonresponders was 40% and 6%, if they were measured by urinary dTXB2 and VerifyNow Aspirin assay, respectively. The Spearman correlation coefficients and kappa statistics were calculated to assess correlation and agreement between the 2 tests. Poor correlation in results between the 2 test was found (r 5 .135, P 5 .190). The degree of agreement between the 2 tests in relation to resistance status was weak (kappa 5 .032, P 5 .590) (Fig 1).
COMPARISON OF ASPIRIN RESPONSE TESTS
Table 1. Baseline characteristics of the patients in the study Baseline characteristics Mean age (y) Mean NIHSS Female, n (%) Hypertension, n (%) Diabetes mellitus, n (%) Hyperlipidemia, n (%) Coronary artery disease, n (%) History of ischemic stroke, n (%) History of intracerebral hemorrhage, n (%) History of transient ischemic attack, n (%) Smoking, n (%) Alteplase treatment, n (%) Presentation, n (%) Acute ischemic stroke Stable ischemic stroke Stroke subtype, n (%) Transient ischemic attack Large-artery atherosclerosis Small-artery occlusion Cardioembolic Other determined cause Undetermined cause Mean urine dTXB2 (ng/mmol of creatinine) Mean aspirin reaction units
N 5 101 61 6 13 665 40 (40%) 65 (64%) 15 (15%) 43 (43%) 2 (2%) 5 (5%) 2 (2%) 3 (3%) 28 (28%) 21 (21%) 66 (65%) 35 (35%) 7 (7%) 28 (28%) 58 (57%) 2 (2%) 2 (2%) 4 (4%) 78.55 6 61.2 446 6 50
Abbreviations: dTXB2, 11-dehydrothromboxane B2; NIHSS, National Institute of Health Stroke Scale.
With a mean follow-up time of 17 months, 8 patients had cardiovascular events or death (myocardial infarct in 3 patients, coronary angioplasty in 1 patient, and death in 4 patients). The causes of death were unknown in 2 patients, renal failure in 1 patient, and intracerebral hemorrhage in 1 patient. The outcomes occurred more
Figure 1. Scatter plot of the results of the 2 tests in the patients. (A) Patients with aspirin responders from urine 11-dehydrothromboxane B2 measurement. (B) Patients with aspirin nonresponders from urine 11-dehydrothromboxane B2 measurement (‘‘C’’ represents aspirin nonresponders from VerifyNow Aspirin assay). Abbreviation: ARU, aspirin reaction unit.
3
frequently in aspirin nonresponders who were diagnosed by urinary dTXB2 measurement as compared with patients with aspirin response (18% versus 2%, odds ratio 8.8, 95% confidence interval 1.18-65.4, P 5.037). However, the aspirin nonresponse which was evaluated by VerifyNow Aspirin assay did not correlate with the outcomes (Table 2).
Discussion Prevalence of aspirin resistance in patients with various cardiovascular diseases ranges from 3% to 82% from previous studies.12,13 The high variance can be partly attributed to different methods used to assess antiplatelet function of the aspirin. Lordkipanize et al14 studied 201 patients with stable coronary artery disease and found that prevalence of aspirin resistance varied according to the assay used: 10.3%-51.7% for light transmission aggregometry (LTA) using adenosine diphosphate as the agonist, 18% for whole blood aggregometry, 59.5% for PFA-100, 6.7% for VerifyNow Aspirin, and 22.9% by measuring urinary dTXB2. Dosage of aspirin may affect the prevalence of aspirin nonresponder. Helgason et al15 showed that escalation of dosage of aspirin resulted in more complete platelet inhibition in aspirin nonresponders while taking normal to low dose (#325 mg) and suggested that the ability of aspirin and the dose required to inhibit platelet aggregation may depend on the individual. As compared with studies about aspirin resistance in patients with coronary artery disease, the studies in patients with stroke were fewer. Because stroke can be caused by many mechanisms besides atherothrombosis, such as cardioemboli, arterial dissection, or hypoperfusion, thus recurrent ischemic stroke while having antiplatelet therapy may not solely represent resistance to the antiplatelet. Prevalence of aspirin resistance in patients with coronary artery disease and in those with stroke may be different. Poor correlation among platelet function tests in evaluation of antiplatelet resistance is known. Each test has different advantages and disadvantages. Which test is the best method to evaluate aspirin resistance is still debated. LTA is still used as the gold standard of platelet function testing in many centers; however, LTA has been reported to have poorly standardized technique and is sensitive to several variables. Results from this technique may overestimate the prevalence of aspirin resistance.8,10 In evaluation of aspirin resistance, some experts suggested that methods to directly measure the capacity of platelets to synthesize TXA2 were preferable. In vivo, TXA2 is rapidly converted into a more stable and inert metabolite, TXB2, which is further metabolized to dTXB2.10 However, approximately 30% of urinary metabolite derives from extraplatelet sources (eg, monocytes/ macrophages), and this fraction can increase in pathologic conditions such as inflammatory diseases.16,17 Thus, the results from dTXB2 measurement may represent both
4
Table 2. Baseline characteristics of patients with and without aspirin resistance in the study Urine dTXB2 measurement
Baseline characteristics
Aspirin responders (n 5 61)
Aspirin nonresponders (n 5 40)
P value
Aspirin responders (n 5 95)
Aspirin nonresponders (n 5 6)
59 5.6 20 (32%) 40 (66%) 7 (11%) 26 (43%) 2 (3%) 1 (2%) 1 (2%) 18 (30%) 15 (24%)
65 6 21 (53%) 25 (63%) 8 (21%) 17 (42%) — 4 (11%) 2 (5%) 10 (24%) 9 (22%)
.026 .786 .047 .771 .167 .942 .513 .154 .563 .478 .794
61 6 40 (42%) 62 (65%) 13 (14%) 41 (43%) 1 (1%) 6 (6%) 3 (3%) 26 (27%) 22 (23%)
65 5 1 (17%) 4 (67%) 2 (33%) 2 (33%) 1 (17%) — — 2 (33%) 1 (17%)
34 (56%) 27 (44%) 264
32 (81%) 8 (19%) 277
.014 .430
62 (65%) 34 (35%) 270
5 (83%) 1 (17%) 264
.660 .846
1 (2%)
7 (18%)
.037
6 (6%)
2 (33%)
.064
P value .484 .687 .396 .925 .218 .964 .124
.667 .580
Abbreviations: CE, cardioembolism; LAA, large-artery atherosclerosis; OC, other determined etiology; rtPA, recombinant tissue plasminogen activator; SAO, small-artery occlusion; UND, undetermined etiology.
P.A. DHARMASAROJA AND S. SAE-LIM
Mean age (y) Mean NIHSS Female, n (%) Hypertension, n (%) Diabetes mellitus, n (%) Hyperlipidemia, n (%) Coronary artery disease, n (%) History of ischemic stroke, n (%) History of transient ischemic attack, n (%) Smoking, n (%) rtPA treatment, n (%) Presentation, n (%) Acute ischemic stroke Stable ischemic stroke Mean doses of aspirin (mg) Outcomes Cardiovascular events and death, n (%)
VerifyNow aspirin assay
COMPARISON OF ASPIRIN RESPONSE TESTS
platelet function and inflammatory status of the patients, which may be better used to predict the occurrence of future cardiovascular events. Our study also showed varied prevalence of aspirin nonresponders in patients with ischemic stroke, with 40% from urinary dTXB2 measurement and 6% from VerifyNow Aspirin assay. Such a big difference in the prevalence of aspirin nonresponders assessed by 2 methods at the same time may be explained by the difference in the methods. VerifyNow Aspirin assay directly measures platelet aggregation induced by the agonist, arachidonic acid, in the whole blood of the patients, whereas dTXB2 level represents TXA2 production in the body, both by platelets and extraplatelet sources. We hypothesized that aspirin may be able to inhibit platelet aggregation in most of the patients but may not be enough to both inhibit platelet aggregation and inflammation that occurred more during acute atherothrombosis because almost two thirds of the patients were included in the acute phase of stroke. Our study found that aspirin nonresponders, diagnosed by dTXB2 measurement, were associated with older age, female, and acute stroke presentation. Previous studies also showed the sex difference in platelet function in response to aspirin. Women demonstrated increased baseline platelet reactivity and also have greater residual platelet activity after taking aspirin compared with men.11,18,19 Linden et al20 suggested that platelet hyperreactivity may explain higher prevalence of aspirin nonresponders during acute atherothrombosis. Several studies revealed that patients with aspirin nonresponse had greater risk of clinically cardiovascular events than aspirin-sensitive patients.6,7,21 In our study, only aspirin nonresponders who were diagnosed by dTXB2 measurement, not by VerifyNow Aspirin assays, were related to having cardiovascular events and death. This may be explained in part by the smaller numbers of aspirin nonresponders that were diagnosed by VerifyNow Aspirin assays. This study is a prospective study with follow-up results of cardiovascular events and death. However, the small number of patients included in the study is the limitation of the study. Further research is still needed to identify the best method of diagnosis of aspirin resistance, its clinical importance, and how to manage these patients. Our research confirmed the poor correlation and agreement between the 2 tests. Only aspirin nonresponders diagnosed by dTXB2 measurement were related to having cardiovascular events and death.
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