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Comparison between urinary 11-dehydrothromboxane B2 detection and platelet Light Transmission Aggregometry (LTA) assays for evaluating aspirin response in elderly patients with coronary artery disease
Gene 571 (2015) 23–27
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Comparison between urinary 11-dehydrothromboxane B2 detection and platelet Light Transmission Aggregometry (LTA) assays for evaluating aspirin response in elderly patients with coronary artery disease Tengfei Liu a, Jingwei Zhang a, Xiahuan Chen a, Xueru Feng a, Sidney W. Fu b, Timothy A. McCaffrey b, Meilin Liu a,⁎ a b
Department of Geriatrics, Peking University First Hospital, Beijing 100034, China Department of Medicine (Division of Genomic Medicine), The George Washington University School of Medicine and Health Sciences, Washington DC 20037, USA
a r t i c l e
i n f o
Article history: Received 18 March 2015 Received in revised form 19 May 2015 Accepted 16 June 2015 Available online 19 June 2015 Keywords: Coronary artery disease (CAD) Aspirin 11-Dehydrothromboxane B2 (11dhTxB2) Light Transmission Aggregometry (LTA) High-on aspirin platelet reactivity (HAPR)
a b s t r a c t Aspirin is widely used in the primary and secondary prevention of cardiovascular diseases. The aim of our study was to compare between two established methods of aspirin response, urinary 11-dehydrothromboxane B2 (11dhTXB2) and platelet Light Transmission Aggregometry (LTA) assays in elderly Chinese patients with coronary artery disease (CAD), and to investigate the clinical significance of both methods in predicting cardiovascular events. Urinary 11dhTxB2 assay and arachidonic acid-induced (AA, 0.5 mg/ml) platelet aggregation by Light Transmission Aggregometry (LTAAA) assay were measured to evaluate aspirin responses. High-on aspirin platelet reactivity (HAPR) was defined as urinary 11dhTxB2 N 1500 pg/mg or AA-induced platelet aggregation ≥ 15.22%—the upper quartile of our enrolled population. The two tests showed a poor correlation for aspirin inhibition (r = 0.063) and a poor agreement in classifying HAPR (kappa = 0.053). With a mean follow-up time of 12 months, cardiovascular events occurred more frequently in HAPR patients who were diagnosed by LTA assay as compared with no-HAPR patients (22.5% versus 10.6%, P = 0.039, OR = 2.45, 95% CI = 1.06–5.63). However, the HAPR status, as determined by urinary 11dTXB2 measurement, did not show a significant correlation with outcomes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Aspirin is a widely prescribed antiplatelet drug in the primary and secondary prevention of coronary artery disease (CAD) (Jack, 1997). It exerts an antithrombotic effect by irreversibly acetylating platelet cyclo-oxygenase-1 (COX1), thereby inhibiting thromboxane A2 (TXA2) synthesis (Patrono et al., 2004; Tantry et al., 2005). However, a small proportion of patients on regular aspirin therapy experienced thromboembolic events, which are closely associated with the laboratory observations of high-on aspirin platelet reactivity (HAPR) (Patrono et al., 2005). Currently, a myriad of tests are available to assess inhibition of platelet aggregation function in clinical practice (Michelson, 2004). The occurrence of high platelet reactivity on-treatment by aspirin is variable and the reported range varies broadly, from 0.4% to 35%, mainly depending on the methods used for platelet function tests and the population studied (Tantry et al., 2005).
Abbreviations: CAD, coronary artery disease; 11dhTxB2, 11-dehydrothromboxane B2; LTA, Light Transmission Aggregometry; HAPR, high-on aspirin platelet reactivity. ⁎ Corresponding author at: Department of Geriatrics, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China. E-mail address: [email protected] (M. Liu).
http://dx.doi.org/10.1016/j.gene.2015.06.045 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Light Transmission Aggregometry (LTA) assay, the widely assumed gold standard, has been used for over 40 years to assess platelet reactivity and was shown to predict clinical outcomes in patients with HAPR (Michelson, 2004; Harrison, 2000; Nicholson et al., 1998; Gum et al., 2003). But poor standardization and requirement for manipulation by skilled technicians limits its use to specialized laboratories. Because the responses to aspirin vary from one patient to another, there is a need for reliable methods that correlate well with clinical outcomes. Methods that directly measure the capacity of platelets to synthesize TXA2 might be preferable (Cattaneo, 2011). However, TXA2 is unstable and easily degraded. The 11-dehydrothromboxane B2 (11dhTxB2), down-stream metabolite of TXA2, is biologically inactive, with a long circulating half-life (Catella et al., 1986; Harrison et al., 2007; Cattaneo, 2011), which is readily excreted in urine and relatively unaffected by ex vivo platelet activation and other pre-analytical variables. Hence, 11dhTxB2 is considered as a reliable biomarker in evaluating platelet activation (Eikelboom et al., 2008). Some studies have investigated the correlation between various platelet function tests and clinical outcomes (Michelson, 2004; Faraday et al., 2006; Dharmasaroja and Sae-Lim, 2014). However, there is little data concerning the Asian elderly population. Furthermore, the poor agreement and correlation among different tests result in fierce debate on the
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T. Liu et al. / Gene 571 (2015) 23–27
reliability of various platelet function tests. The purpose of this study was to compare between two well established methods of aspirin responses: urinary 11dhTxB2 and platelet LTA assays in the elderly Chinese patients with CAD, and to investigate the clinical significance of both methods in predicting future cardiovascular events. To the best of our knowledge, this is the first study to compare results simultaneously from two major platelet function tests in the assessment of the prevalence of HAPR in elderly Chinese CAD patients and to investigate their clinical significance. 2. Materials and methods
diabetes mellitus (DM) or stroke, compliance to medications, dosages and duration of aspirin treatment, hemorrhagic complications, and concomitant medications, especially non-steroidal anti-inflammatory drugs (NSAIDS). Clinical outcomes were defined as the occurrence of cardiovascular events, including unstable angina pectoris, non-fatal myocardial infarction, non-fatal stroke, recurrent ischemia requiring urgent revascularization, cardiovascular death, or confirmed progression of CAD through coronary angiography. Progression of CAD is defined as increased number of segment lesions or significantly increased degree of stenosis, confirmed by coronary angiography, meaning the degree of stenosis N20%, or N30% stenosis in original normal segments.
2.1. Characteristics of the patients Patients on regular aspirin treatment (100 mg/d) for at least 6 months, who met the inclusion criteria, were consecutively recruited from the inpatient Department of Geriatrics, First Hospital of Peking University from January to July 2013. Inclusion criteria was as follows: older than 60 years with clinical diagnosis of: a) stable angina pectoris confirmed by coronary angiography; or b) acute coronary syndrome (unstable angina, ST-elevated myocardial infarction, and non-ST-elevated myocardial infarction) by percutaneous coronary intervention (PCI); or c) coronary artery bypass grafting (CABG). Patients were excluded from the study if they had one of the following: contraindication for aspirin treatment; platelet count ≤100 × 109/l; hematological disorders, serious liver disease, or malignancies; co-therapy with glycoprotein IIb/IIIa inhibitor, warfarin/coumadin, or non-steroid anti-inflammatory drug; peptic ulcer or history of gastrointestinal hemorrhage; or low clopidogrel response. Initially, 355 patients were enrolled, but 32 were excluded (10 for co-administrating warfarin and non-steroid anti-inflammatory drug, and 22 for incomplete clinical or laboratory data) (Fig. 1). This study conformed to the ethical guidelines of the Helsinki declaration. The ethics approvals were obtained from Peking University First Hospital Ethical Review Committee, and written informed consents were obtained from all enrolled patients or their immediate family members. All patients were contacted directly in the outpatient clinic or by telephone calls. Demographic information collected includes the following: CAD subtypes, histories of PCI or CABG, co-morbidities such as
2.2. Urine and blood sample collection Urine and blood samples of the patients were collected simultaneously after taking aspirin (100 mg/d) for at least 6 months. Urine samples were kept at −20 °C until analysis. A total of 3 ml peripheral venous blood for each patient was drawn into a 3.2% sodium citrate vacutainer tube for LTA analysis. AA-induced (0.5 mg/ml) platelet aggregation via LTA assay was completed to assess aspirin response. ADP-induced (20 μmol/l) platelet aggregation was also measured in patients under dual antiplatelet medications to exclude those with low clopidogrel response. 2.3. Urinary 11dhTxB2 assay Urine samples were thawed and assayed for 11dhTxB2 with a commercially available enzyme-linked immunoassay (ELISA) kit that has a coefficient of variation of 6.9% for replicate measurements (Corgenix Inc., USA). Urinary 11dhTxB2 levels are normalized against urine creatinine concentration. The measurements of 11dhTxB2 are reported as pg/mg (pg 11dhTxB2 per mg creatinine). It is important to point out that this assay measures the systemic production of thromboxane (COX-1 and COX-2-derived), and directly reflects global COX inhibition by aspirin. Patients with urinary 11dTXB2 N 1500 pg/mg were classified into HAPR group.
Fig. 1. The flow chart of the study.
T. Liu et al. / Gene 571 (2015) 23–27
25
2.4. Light Transmission Aggregometry (LTA) assay The blood-citrate tubes were centrifuged at 120 g for 5 min to prepare platelet-rich plasma (PRP) and further centrifuged at 850 g for 10 min to prepare platelet-poor plasma (PPP). The PRP and PPP plasma were stored at room temperature to be used within 30 min. Platelet aggregation was measured by LTA method to assess 0.5 mg/ml AA-mediated platelet aggregation (LTAAA) as described (Angiolillo et al., 2005, 2006, 2007). Aggregation was expressed as maximum percentage change in light transmittance from baseline, while PPP were used as a reference (Gurbel et al., 2007). A 20 μmol/l ADP-induced platelet aggregation (LTAADP) was used to evaluate clopidogrel response, and the low clopidogrel response was defined as LTAADP ≥70%. 2.5. Statistical analysis Continuous variables are shown as mean ± standard deviation (SD). Categorical variables were presented as frequencies and percentages. Correlations between measurements of two tests were assessed by Spearman correlation coefficient. The agreement between the HAPR status assessed by the two platelet function tests was evaluated with kappa statistics. The demographics and cardiovascular risk factors were compared between HAPR and no-HAPR patients using the Student t-test (for the continuous variables) and the chi-square test (for the proportions). A two-tailed P b 0.05 was considered significant. SPSS version 17.0 (Chicago, Illinois) was used for statistical analysis. 3. Results During the study period, 323 elderly CAD patients with complete records of the antiplatelet function tests, urinary 11dhTxB2 and platelet aggregation by LTA assay, were enrolled. Baseline characteristics including age, gender, BMI, CV risk factors/past medical history and essential medicines are presented in Table 1. Of the 323 elderly subjects studied, 239 (74.0%) were male, with mean age of 75.8 ± 10.8 years (ranging from 60–97 years). Mean urinary 11dhTxB2 level and mean platelet aggregation were 1371.8 ± 769 pg/mg and 13.07% ± 4.9%, respectively. Among the 323 patients, 105 patients were under dual anti-platelet treatment, and no patient with low clopidogrel response status was observed according to LTAADP assay (Fig. 2).
Fig. 2. Scatter plot of LTAAA and LTAADP assays in patients under dual anti-platelet treatment. (“△” represents patients from LTAAA assay and “▽” represents patients from LTAADP assay). Horizontal dotted lines indicate test-specific cut-off values for HAPR on left, low clopidogrel response on right. The arrow indicates the zone within which patients are considered HAPR or low clopidogrel response.
The measurement of platelet aggregation by LTAAA and urinary 11dhTxB2 segregated patients into two distinct groups according to their HAPR status (Fig. 3). In Table 1, 104 patients were classified as HAPR based on urinary 11dhTxB2 levels (prevalence of 32.2%), while only forty patients were classified as HAPR according to LTAAA assay (prevalence of 12.4%). In terms of general medications, no obvious differences were observed between HAPR and no-HAPR groups, regardless of the assay methods. The Spearman correlation coefficients and kappa statistics were calculated to assess the correlation and agreement between LTAAA and urinary 11dhTxB2 assays. Although measurement of urinary 11dhTxB2 has been considered a specific indicator of detecting platelet inhibition by aspirin, the results demonstrated a poor correlation with the gold standard LTA assay (r = 0.063, P = 0.2612). Likewise, the agreement between the two assays in relation to the classification of HAPR status was weak (kappa = 0.053, P = 0.259). With a mean follow-up time of 12 months, cardiovascular events occurred in 39 patients (unstable angina pectoris in 7, non-fatal myocardial infarction in 1, recurrent ischemic stroke in 2, PCI in 5, and progression of coronary artery confirmed by coronary angiography in 31). The outcomes occurred more frequently in HAPR patients who were diagnosed by LTA assay as compared with no-HAPR patients (22.5% versus 10.6%, P = 0.039, OR = 2.45, 95% CI = 1.06–5.63). However, the HAPR
Table 1 Baseline characteristic of patients with and without HAPR status in the study. Variables
Mean age, y Male, n (%) BMI, kg/m2 Risk factors/past medical history Hypertension g, n (%) Diabetes, n (%) Current smoking, n (%) Hyperlipidemia, n (%) Prior MI, n (%) Prior PCI, n (%) Prior CVA, n (%) Essential medicines Clopidogrel, n (%) ACEI/ARB, n (%) β-Blocker, n (%) Calcium channel blocker, n (%) Nitrates, n (%) Statin, n (%) Outcomes n = 39 Cardiovascular events and death, n (%)
Urine 11dhTXb2
LTA analysis
HAPR (n = 104)
No-AR (n = 219)
P value
HAPR (n = 40)
No-AR (n = 283)
P value
76.0 ± 10.7 78 (75.0) 25.21 ± 3.4
75.5 ± 10.9 161 (73.5) 23.88 ± 3.5
0.008 0.892 0.383
76.1 ± 11.0 29 (72.5) 25.9 ± 3.5
75.3 ± 9.9 210 (74.2) 23.7 ± 3.6
0. 003 0.848 0.204
84 (80.8) 60 (57.7) 32 (30.8) 89 (85.6) 25 (24.0) 42 (40.4) 9 (8.7)
153 (70.8) 101 (46.1) 48 (21.9) 173 (79.0) 48 (21.9) 74 (33.8) 23 (10.5)
0.059 0.057 0.098 0.174 0.672 0.265 0.693
33 (82.5) 26 (65.0) 15 (37.5) 36 (90.0) 12 (30.0) 19 (47.5) 3 (7.5)
206 (72.8) 135 (47.7) 65 (23.0) 226 (79.9) 61 (21.6) 97 (34.3) 29 (10.2)
0.248 0.044 0.052 0.193 0.231 0.115 0.780
37 (35.6) 53 (51.0) 69 (66.3) 46 (44.2) 38 (36.5) 85 (81.7)
68 (31.1) 89 (40.6) 126 (57.0) 93 (42.5) 70 (32.0) 161 (73.5)
0.447 0.093 0.116 0.810 0.450 0.125
15 (37.5) 20 (50.0) 29 (72.5) 18 (45.0) 15 (37.5) 31 (62.0)
90 (31.8) 122 (43.1) 166 (58.7) 121 (42.8) 93 (32.9) 215 (76.0)
0.475 0.497 0.120 0.865 0.593 0.053
16 (15.4)
23 (10.5%)
0.207
9 (22.5)
30 (10.6)
0.039
HAPR, high-on aspirin platelet response; BMI, body mass index; MI, myocardial infarction; PCI, percutaneous coronary intervention; CVA: cardiovascular accident; ACEI/ARB, angiotensin antagonist inhibitor/ angiotensin receptor blocker.
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T. Liu et al. / Gene 571 (2015) 23–27
Fig. 3. Scatter plot of the results of LTAAA and urine 11dTxB2 assays in the patients. X axis presets patients with HAPR from LTAAA platelet aggregation assay, Y axis indices patients with HAPR by urinary 11-dehydrothromboxane B2 (11dTxB2) measurement. Horizontal and vertical dotted lines indicate test-specific cut-off values for HAPR by LTAAA and 11dTxB2 assay respectively. The arrow indicates the zone within which patients are considered HAPR. Abbreviation: HAPR, High on aspirin platelet response; LTA, Light Transmission Aggregometry.
status, as determined by urinary 11dTXB2 measurement, did not show significant correlation with the outcomes (Table 1). 4. Discussion To the best of our knowledge, our study is the first to compare simultaneously two different assays in the elderly Chinese population, in order to evaluate the prevalence of HAPR. Using LTAAA assay, we observed that the elderly CAD patients (mean age 75.8) had a high prevalence of HAPR (12.4%), while Lordkipanize et al. study showed that the prevalence of high platelet reactivity was 4% by LTAAA assay in the population with the mean age of 66.5 years[19]. Further, Nauder et al. found a 0.4% prevalence of high platelet reactivity in their study population with an average age of 45 years (Faraday et al., 2006). Moreover, Hovens et al. in a recent systematic review showed a pooled unadjusted prevalence of 6% using LTAAA assay (Hovens et al., 2007). Several factors might be responsible for the heterogeneity among these studies (Bennett et al., 2008; Topcuoglu et al., 2011). On one hand, factors such as aspirin dosage and different racial backgrounds would affect the estimated prevalence of HAPR. On the other hand, because age was considered as an important risk factor in HAPR, the relatively advanced age in our enrolled population might explain the high prevalence. Among the assays available to estimate the antiplatelet effect of aspirin, LTAAA assay is considered as a gold standard because of its relatively high specificity for platelets, and AA is used as the agonist to exploit the specific pathways affected by aspirin (Tantry et al., 2005). However, its requirement for highly skilled measurement and poor standardization of precise method restricts the wide applications of this technology. In our study, the LTAAA assays were all carried out by professional technicians at the First Hospital of Peking University, thus ensuring the accuracy of the measurements, which is another advantage of this study. It is well established that TxB2 is the major metabolite of platelet TxA2 in plasma. The presence of its metabolite, 11dhTxB2, is believed to be predominantly attributable to platelet activation, and 11dhTxB2 levels decrease after aspirin treatment (Catella et al., 1986). Some studies suggest that methods directly measuring urinary 11dhTxB2 might be better in assessing platelet reactivity and predicting the risk of cardiovascular events (Eikelboom et al., 2002). Using the 11dhTxB2 method, Dharmasaroja et al. enrolled 101 CAD patients and found prevalence of HAPR of 40% (Dharmasaroja and Sae-Lim, 2014). Similarly, in Lordkipanize et al. study, a 22.9% prevalence of HAPR was observed (Lordkipanidze et al., 2007). Thus, the 32.2% prevalence of HAPR found
in the current study is generally in agreement with earlier studies by others. However, the weak agreement among different platelet function tests and poor correlation with clinical outcomes raise questions about which method is reliable in assessing aspirin responses (Faraday et al., 2006; Dharmasaroja and Sae-Lim, 2014; Lordkipanidze et al., 2007). Lordkipanize et al. compared six methods to assess platelet function and found poor correlations and low agreements among the various assays (Lordkipanidze et al., 2007). Pornpatr et al. found a poor correlation and a low agreement when comparing VerifyNow with urinary 11dhTxB2 assays (Dharmasaroja and Sae-Lim, 2014). Likewise, Fallahi et al. observed a 6.8% rate of HAPR by VerifyNow assay and a poor correlation with urinary 11dhTxB2 (Fallahi et al., 2013). Similarly, we found a poor correlation between urinary 11dhTxB2 measurement and LTAAA assay. Our study found that HAPR patients, diagnosed by both urinary 11dhTxB2 measurement and LTAAA assay, were prone to be older, with higher BMI, and have more co-morbidities, such as hypertension, diabetes, current smoking and hyperlipidemia. In previous studies, females demonstrated an increased baseline platelet reactivity and a greater residual platelet activity after taking aspirin when compared with men, while in our study these differences were not observed. Furthermore, it was widely acknowledged in previous studies that patients with poor aspirin response might suffer from higher risk of cardiovascular events than those with normal aspirin response (Krasopoulos et al., 2008). In our study, patients with HAPR tend to experience a higher risk of cardiovascular events, and HAPR status based on LTAAA was significantly related to clinical outcomes. Nevertheless, in Pornpatr's study, patients with poor aspirin response diagnosed by urinary 11dhTxB2 measurement display a more close correlation with clinical outcomes than LTAAA assay. The variance in the results may be due to the relatively small scale of their study, and the definition of poor aspirin response. Although the measurement of urinary 11dhTxB2 is platelet specific, it was observed that at times of stress such as inflammation, non-platelet derived TXA2 might be produced, while the LTAAA assay reflects the current activity of the platelets toward a defined stimulus. Based on the previous studies, our research confirmed poor correlation and low agreement between the two tests, and revealed that LTAAA assay might be more reliable in predicting risks of cardiovascular events. This study has several strengths and limitations. Firstly, this is the first study to compare two methods on platelet function in the Chinese elderly population. Meanwhile, in patients under dual antiplatelet medications, ADP-induced platelet aggregation was used to exclude patients with low clopidogrel response. Secondly, the LTAAA assays in our study were all carried out by the same professional technicians, which ensured the accuracy and reproducibility of the measurement. With regard to limitations, other detection methods such as PFA-100, VerifyNow assay, or Thrombelastogram were not included in this study. In addition, variations in lifestyle factors, such as diet, smoking history, and physical activity, can affect the outcomes in any study. Therefore, more patients should be included in further studies to confirm our conclusions, and further research is still needed to identify the best method to evaluate aspirin response. In conclusion, we performed a prospective study with follow-up results of cardiovascular events and mortality. Our data showed a poor correlation and a low agreement between the two tests in evaluating aspirin response. A significant correlation between LTAAA assay and clinical outcomes was observed. Therefore, the findings of our study can be added to the growing evidences that consider LTAAA assay as a more stable and accurate method to assess platelet function on antiplatelet treatment.
Competing financial interests There are no conflicts of interest.
T. Liu et al. / Gene 571 (2015) 23–27
Acknowledgments The work was supported by grants from the International Science & Technology Cooperation Projects of China (2013DFA30860), the National Science & Technology Pillar Program of China (2012BAI 37B00), and Beijing Huikangyisheng Sci-Tech Co., Ltd. (00020140616003). References Angiolillo, D.J., Fernandez-Ortiz, A., Bernardo, E., Ramirez, C., Sabate, M., et al., 2005. Platelet function profiles in patients with type 2 diabetes and coronary artery disease on combined aspirin and clopidogrel treatment. Diabetes 54, 2430–2435. Angiolillo, D.J., Bernardo, E., Ramirez, C., Costa, M.A., Sabate, M., et al., 2006. Insulin therapy is associated with platelet dysfunction in patients with type 2 diabetes mellitus on dual oral antiplatelet treatment. J. Am. Coll. Cardiol. 48, 298–304. Angiolillo, D.J., Bernardo, E., Sabate, M., Jimenez-Quevedo, P., Costa, M.A., et al., 2007. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease. J. Am. Coll. Cardiol. 50, 1541–1547. Bennett, D., Yan, B., Macgregor, L., Eccleston, D., Davis, S.M., 2008. A pilot study of resistance to aspirin in stroke patients. J. Clin. Neurosci. 15, 1204–1209. Catella, F., Healy, D., Lawson, J.A., FitzGerald, G.A., 1986. 11-Dehydrothromboxane B2: a quantitative index of thromboxane A2 formation in the human circulation. Proc. Natl. Acad. Sci. U. S. A. 83, 5861–5865. Cattaneo, M., 2011. The clinical relevance of response variability to antiplatelet therapy. Hematol. Am. Soc. Hematol. Educ. Prog. 2011, 70–75. Dharmasaroja, P.A., Sae-Lim, S., 2014. Comparison of aspirin response measured by urinary 11-dehydrothromboxane B2 and VerifyNow aspirin assay in patients with ischemic stroke. J. Stroke Cerebrovasc. Dis. 23, 953–957. Eikelboom, J.W., Hirsh, J., Weitz, J.I., Johnston, M., Yi, Q., et al., 2002. Aspirin-resistant thromboxane biosynthesis and the risk of myocardial infarction, stroke, or cardiovascular death in patients at high risk for cardiovascular events. Circulation 105, 1650–1655. Eikelboom, J.W., Hankey, G.J., Thom, J., Bhatt, D.L., Steg, P.G., et al., 2008. Incomplete inhibition of thromboxane biosynthesis by acetylsalicylic acid: determinants and effect on cardiovascular risk. Circulation 118, 1705–1712. Fallahi, P., Katz, R., Toma, I., Li, R., Reiner, J., et al., 2013. Aspirin insensitive thrombophilia: transcript profiling of blood identifies platelet abnormalities and HLA restriction. Gene 520, 131–138.
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Faraday, N., Becker, D.M., Yanek, L.R., Herrera-Galeano, J.E., Segal, J.B., et al., 2006. Relation between atherosclerosis risk factors and aspirin resistance in a primary prevention population. Am. J. Cardiol. 98, 774–779. Gum, P.A., Kottke-Marchant, K., Welsh, P.A., White, J., Topol, E.J., 2003. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J. Am. Coll. Cardiol. 41, 961–965. Gurbel, P.A., Bliden, K.P., DiChiara, J., Newcomer, J., Weng, W., et al., 2007. Evaluation of dose-related effects of aspirin on platelet function: results from the Aspirin-Induced Platelet Effect (ASPECT) study. Circulation 115, 3156–3164. Harrison, P., 2000. Progress in the assessment of platelet function. Br. J. Haematol. 111, 733–744. Harrison, P., Frelinger, A.L., Furman, M.I., Michelson, A.D., 2007. Measuring antiplatelet drug effects in the laboratory. Thromb. Res. 120, 323–336. Hovens, M.M., Snoep, J.D., Eikenboom, J.C., van der Bom, J.G., Mertens, B.J., et al., 2007. Prevalence of persistent platelet reactivity despite use of aspirin: a systematic review. Am. Heart J. 153, 175–181. Jack, D.B., 1997. One hundred years of aspirin. Lancet 350, 437–439. Krasopoulos, G., Brister, S.J., Beattie, W.S., Buchanan, M.R., 2008. Aspirin “resistance” and risk of cardiovascular morbidity: systematic review and meta-analysis. BMJ 336, 195–198. Lordkipanidze, M., Pharand, C., Schampaert, E., Turgeon, J., Palisaitis, D.A., et al., 2007. 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. 28, 1702–1708. Michelson, A.D., 2004. Platelet function testing in cardiovascular diseases. Circulation 110, e489–e493. Nicholson, N.S., Panzer-Knodle, S.G., Haas, N.F., Taite, B.B., Szalony, J.A., et al., 1998. Assessment of platelet function assays. Am. Heart J. 135, S170–S178. Patrono, C., Coller, B., FitzGerald, G.A., Hirsh, J., Roth, G., 2004. Platelet-active drugs: the relationships among dose, effectiveness, and side effects: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 126, 234S–264S. Patrono, C., Garcia Rodriguez, L.A., Landolfi, R., aigent, C., 2005. Low-dose aspirin for the prevention of atherothrombosis. N. Engl. J. Med. 353, 2373–2383. Tantry, U.S., Bliden, K.P., Gurbel, P.A., 2005. Resistance to antiplatelet drugs: current status and future research. Expert. Opin. Pharmacother. 6, 2027–2045. Topcuoglu, M.A., Arsava, E.M., Ay, H., 2011. Antiplatelet resistance in stroke. Expert. Rev. Neurother. 11, 251–263.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Comparison between urinary 11-dehydrothromboxane B2 detection and platelet Light Transmission Aggregometry (LTA) assays for evaluating aspirin response in elderly patients with coronary artery disease Tengfei Liu a, Jingwei Zhang a, Xiahuan Chen a, Xueru Feng a, Sidney W. Fu b, Timothy A. McCaffrey b, Meilin Liu a,⁎ a b
Department of Geriatrics, Peking University First Hospital, Beijing 100034, China Department of Medicine (Division of Genomic Medicine), The George Washington University School of Medicine and Health Sciences, Washington DC 20037, USA
a r t i c l e
i n f o
Article history: Received 18 March 2015 Received in revised form 19 May 2015 Accepted 16 June 2015 Available online 19 June 2015 Keywords: Coronary artery disease (CAD) Aspirin 11-Dehydrothromboxane B2 (11dhTxB2) Light Transmission Aggregometry (LTA) High-on aspirin platelet reactivity (HAPR)
a b s t r a c t Aspirin is widely used in the primary and secondary prevention of cardiovascular diseases. The aim of our study was to compare between two established methods of aspirin response, urinary 11-dehydrothromboxane B2 (11dhTXB2) and platelet Light Transmission Aggregometry (LTA) assays in elderly Chinese patients with coronary artery disease (CAD), and to investigate the clinical significance of both methods in predicting cardiovascular events. Urinary 11dhTxB2 assay and arachidonic acid-induced (AA, 0.5 mg/ml) platelet aggregation by Light Transmission Aggregometry (LTAAA) assay were measured to evaluate aspirin responses. High-on aspirin platelet reactivity (HAPR) was defined as urinary 11dhTxB2 N 1500 pg/mg or AA-induced platelet aggregation ≥ 15.22%—the upper quartile of our enrolled population. The two tests showed a poor correlation for aspirin inhibition (r = 0.063) and a poor agreement in classifying HAPR (kappa = 0.053). With a mean follow-up time of 12 months, cardiovascular events occurred more frequently in HAPR patients who were diagnosed by LTA assay as compared with no-HAPR patients (22.5% versus 10.6%, P = 0.039, OR = 2.45, 95% CI = 1.06–5.63). However, the HAPR status, as determined by urinary 11dTXB2 measurement, did not show a significant correlation with outcomes. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Aspirin is a widely prescribed antiplatelet drug in the primary and secondary prevention of coronary artery disease (CAD) (Jack, 1997). It exerts an antithrombotic effect by irreversibly acetylating platelet cyclo-oxygenase-1 (COX1), thereby inhibiting thromboxane A2 (TXA2) synthesis (Patrono et al., 2004; Tantry et al., 2005). However, a small proportion of patients on regular aspirin therapy experienced thromboembolic events, which are closely associated with the laboratory observations of high-on aspirin platelet reactivity (HAPR) (Patrono et al., 2005). Currently, a myriad of tests are available to assess inhibition of platelet aggregation function in clinical practice (Michelson, 2004). The occurrence of high platelet reactivity on-treatment by aspirin is variable and the reported range varies broadly, from 0.4% to 35%, mainly depending on the methods used for platelet function tests and the population studied (Tantry et al., 2005).
Abbreviations: CAD, coronary artery disease; 11dhTxB2, 11-dehydrothromboxane B2; LTA, Light Transmission Aggregometry; HAPR, high-on aspirin platelet reactivity. ⁎ Corresponding author at: Department of Geriatrics, Peking University First Hospital, No. 8, Xishiku Street, Xicheng District, Beijing 100034, China. E-mail address: [email protected] (M. Liu).
http://dx.doi.org/10.1016/j.gene.2015.06.045 0378-1119/© 2015 Elsevier B.V. All rights reserved.
Light Transmission Aggregometry (LTA) assay, the widely assumed gold standard, has been used for over 40 years to assess platelet reactivity and was shown to predict clinical outcomes in patients with HAPR (Michelson, 2004; Harrison, 2000; Nicholson et al., 1998; Gum et al., 2003). But poor standardization and requirement for manipulation by skilled technicians limits its use to specialized laboratories. Because the responses to aspirin vary from one patient to another, there is a need for reliable methods that correlate well with clinical outcomes. Methods that directly measure the capacity of platelets to synthesize TXA2 might be preferable (Cattaneo, 2011). However, TXA2 is unstable and easily degraded. The 11-dehydrothromboxane B2 (11dhTxB2), down-stream metabolite of TXA2, is biologically inactive, with a long circulating half-life (Catella et al., 1986; Harrison et al., 2007; Cattaneo, 2011), which is readily excreted in urine and relatively unaffected by ex vivo platelet activation and other pre-analytical variables. Hence, 11dhTxB2 is considered as a reliable biomarker in evaluating platelet activation (Eikelboom et al., 2008). Some studies have investigated the correlation between various platelet function tests and clinical outcomes (Michelson, 2004; Faraday et al., 2006; Dharmasaroja and Sae-Lim, 2014). However, there is little data concerning the Asian elderly population. Furthermore, the poor agreement and correlation among different tests result in fierce debate on the
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reliability of various platelet function tests. The purpose of this study was to compare between two well established methods of aspirin responses: urinary 11dhTxB2 and platelet LTA assays in the elderly Chinese patients with CAD, and to investigate the clinical significance of both methods in predicting future cardiovascular events. To the best of our knowledge, this is the first study to compare results simultaneously from two major platelet function tests in the assessment of the prevalence of HAPR in elderly Chinese CAD patients and to investigate their clinical significance. 2. Materials and methods
diabetes mellitus (DM) or stroke, compliance to medications, dosages and duration of aspirin treatment, hemorrhagic complications, and concomitant medications, especially non-steroidal anti-inflammatory drugs (NSAIDS). Clinical outcomes were defined as the occurrence of cardiovascular events, including unstable angina pectoris, non-fatal myocardial infarction, non-fatal stroke, recurrent ischemia requiring urgent revascularization, cardiovascular death, or confirmed progression of CAD through coronary angiography. Progression of CAD is defined as increased number of segment lesions or significantly increased degree of stenosis, confirmed by coronary angiography, meaning the degree of stenosis N20%, or N30% stenosis in original normal segments.
2.1. Characteristics of the patients Patients on regular aspirin treatment (100 mg/d) for at least 6 months, who met the inclusion criteria, were consecutively recruited from the inpatient Department of Geriatrics, First Hospital of Peking University from January to July 2013. Inclusion criteria was as follows: older than 60 years with clinical diagnosis of: a) stable angina pectoris confirmed by coronary angiography; or b) acute coronary syndrome (unstable angina, ST-elevated myocardial infarction, and non-ST-elevated myocardial infarction) by percutaneous coronary intervention (PCI); or c) coronary artery bypass grafting (CABG). Patients were excluded from the study if they had one of the following: contraindication for aspirin treatment; platelet count ≤100 × 109/l; hematological disorders, serious liver disease, or malignancies; co-therapy with glycoprotein IIb/IIIa inhibitor, warfarin/coumadin, or non-steroid anti-inflammatory drug; peptic ulcer or history of gastrointestinal hemorrhage; or low clopidogrel response. Initially, 355 patients were enrolled, but 32 were excluded (10 for co-administrating warfarin and non-steroid anti-inflammatory drug, and 22 for incomplete clinical or laboratory data) (Fig. 1). This study conformed to the ethical guidelines of the Helsinki declaration. The ethics approvals were obtained from Peking University First Hospital Ethical Review Committee, and written informed consents were obtained from all enrolled patients or their immediate family members. All patients were contacted directly in the outpatient clinic or by telephone calls. Demographic information collected includes the following: CAD subtypes, histories of PCI or CABG, co-morbidities such as
2.2. Urine and blood sample collection Urine and blood samples of the patients were collected simultaneously after taking aspirin (100 mg/d) for at least 6 months. Urine samples were kept at −20 °C until analysis. A total of 3 ml peripheral venous blood for each patient was drawn into a 3.2% sodium citrate vacutainer tube for LTA analysis. AA-induced (0.5 mg/ml) platelet aggregation via LTA assay was completed to assess aspirin response. ADP-induced (20 μmol/l) platelet aggregation was also measured in patients under dual antiplatelet medications to exclude those with low clopidogrel response. 2.3. Urinary 11dhTxB2 assay Urine samples were thawed and assayed for 11dhTxB2 with a commercially available enzyme-linked immunoassay (ELISA) kit that has a coefficient of variation of 6.9% for replicate measurements (Corgenix Inc., USA). Urinary 11dhTxB2 levels are normalized against urine creatinine concentration. The measurements of 11dhTxB2 are reported as pg/mg (pg 11dhTxB2 per mg creatinine). It is important to point out that this assay measures the systemic production of thromboxane (COX-1 and COX-2-derived), and directly reflects global COX inhibition by aspirin. Patients with urinary 11dTXB2 N 1500 pg/mg were classified into HAPR group.
Fig. 1. The flow chart of the study.
T. Liu et al. / Gene 571 (2015) 23–27
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2.4. Light Transmission Aggregometry (LTA) assay The blood-citrate tubes were centrifuged at 120 g for 5 min to prepare platelet-rich plasma (PRP) and further centrifuged at 850 g for 10 min to prepare platelet-poor plasma (PPP). The PRP and PPP plasma were stored at room temperature to be used within 30 min. Platelet aggregation was measured by LTA method to assess 0.5 mg/ml AA-mediated platelet aggregation (LTAAA) as described (Angiolillo et al., 2005, 2006, 2007). Aggregation was expressed as maximum percentage change in light transmittance from baseline, while PPP were used as a reference (Gurbel et al., 2007). A 20 μmol/l ADP-induced platelet aggregation (LTAADP) was used to evaluate clopidogrel response, and the low clopidogrel response was defined as LTAADP ≥70%. 2.5. Statistical analysis Continuous variables are shown as mean ± standard deviation (SD). Categorical variables were presented as frequencies and percentages. Correlations between measurements of two tests were assessed by Spearman correlation coefficient. The agreement between the HAPR status assessed by the two platelet function tests was evaluated with kappa statistics. The demographics and cardiovascular risk factors were compared between HAPR and no-HAPR patients using the Student t-test (for the continuous variables) and the chi-square test (for the proportions). A two-tailed P b 0.05 was considered significant. SPSS version 17.0 (Chicago, Illinois) was used for statistical analysis. 3. Results During the study period, 323 elderly CAD patients with complete records of the antiplatelet function tests, urinary 11dhTxB2 and platelet aggregation by LTA assay, were enrolled. Baseline characteristics including age, gender, BMI, CV risk factors/past medical history and essential medicines are presented in Table 1. Of the 323 elderly subjects studied, 239 (74.0%) were male, with mean age of 75.8 ± 10.8 years (ranging from 60–97 years). Mean urinary 11dhTxB2 level and mean platelet aggregation were 1371.8 ± 769 pg/mg and 13.07% ± 4.9%, respectively. Among the 323 patients, 105 patients were under dual anti-platelet treatment, and no patient with low clopidogrel response status was observed according to LTAADP assay (Fig. 2).
Fig. 2. Scatter plot of LTAAA and LTAADP assays in patients under dual anti-platelet treatment. (“△” represents patients from LTAAA assay and “▽” represents patients from LTAADP assay). Horizontal dotted lines indicate test-specific cut-off values for HAPR on left, low clopidogrel response on right. The arrow indicates the zone within which patients are considered HAPR or low clopidogrel response.
The measurement of platelet aggregation by LTAAA and urinary 11dhTxB2 segregated patients into two distinct groups according to their HAPR status (Fig. 3). In Table 1, 104 patients were classified as HAPR based on urinary 11dhTxB2 levels (prevalence of 32.2%), while only forty patients were classified as HAPR according to LTAAA assay (prevalence of 12.4%). In terms of general medications, no obvious differences were observed between HAPR and no-HAPR groups, regardless of the assay methods. The Spearman correlation coefficients and kappa statistics were calculated to assess the correlation and agreement between LTAAA and urinary 11dhTxB2 assays. Although measurement of urinary 11dhTxB2 has been considered a specific indicator of detecting platelet inhibition by aspirin, the results demonstrated a poor correlation with the gold standard LTA assay (r = 0.063, P = 0.2612). Likewise, the agreement between the two assays in relation to the classification of HAPR status was weak (kappa = 0.053, P = 0.259). With a mean follow-up time of 12 months, cardiovascular events occurred in 39 patients (unstable angina pectoris in 7, non-fatal myocardial infarction in 1, recurrent ischemic stroke in 2, PCI in 5, and progression of coronary artery confirmed by coronary angiography in 31). The outcomes occurred more frequently in HAPR patients who were diagnosed by LTA assay as compared with no-HAPR patients (22.5% versus 10.6%, P = 0.039, OR = 2.45, 95% CI = 1.06–5.63). However, the HAPR
Table 1 Baseline characteristic of patients with and without HAPR status in the study. Variables
Mean age, y Male, n (%) BMI, kg/m2 Risk factors/past medical history Hypertension g, n (%) Diabetes, n (%) Current smoking, n (%) Hyperlipidemia, n (%) Prior MI, n (%) Prior PCI, n (%) Prior CVA, n (%) Essential medicines Clopidogrel, n (%) ACEI/ARB, n (%) β-Blocker, n (%) Calcium channel blocker, n (%) Nitrates, n (%) Statin, n (%) Outcomes n = 39 Cardiovascular events and death, n (%)
Urine 11dhTXb2
LTA analysis
HAPR (n = 104)
No-AR (n = 219)
P value
HAPR (n = 40)
No-AR (n = 283)
P value
76.0 ± 10.7 78 (75.0) 25.21 ± 3.4
75.5 ± 10.9 161 (73.5) 23.88 ± 3.5
0.008 0.892 0.383
76.1 ± 11.0 29 (72.5) 25.9 ± 3.5
75.3 ± 9.9 210 (74.2) 23.7 ± 3.6
0. 003 0.848 0.204
84 (80.8) 60 (57.7) 32 (30.8) 89 (85.6) 25 (24.0) 42 (40.4) 9 (8.7)
153 (70.8) 101 (46.1) 48 (21.9) 173 (79.0) 48 (21.9) 74 (33.8) 23 (10.5)
0.059 0.057 0.098 0.174 0.672 0.265 0.693
33 (82.5) 26 (65.0) 15 (37.5) 36 (90.0) 12 (30.0) 19 (47.5) 3 (7.5)
206 (72.8) 135 (47.7) 65 (23.0) 226 (79.9) 61 (21.6) 97 (34.3) 29 (10.2)
0.248 0.044 0.052 0.193 0.231 0.115 0.780
37 (35.6) 53 (51.0) 69 (66.3) 46 (44.2) 38 (36.5) 85 (81.7)
68 (31.1) 89 (40.6) 126 (57.0) 93 (42.5) 70 (32.0) 161 (73.5)
0.447 0.093 0.116 0.810 0.450 0.125
15 (37.5) 20 (50.0) 29 (72.5) 18 (45.0) 15 (37.5) 31 (62.0)
90 (31.8) 122 (43.1) 166 (58.7) 121 (42.8) 93 (32.9) 215 (76.0)
0.475 0.497 0.120 0.865 0.593 0.053
16 (15.4)
23 (10.5%)
0.207
9 (22.5)
30 (10.6)
0.039
HAPR, high-on aspirin platelet response; BMI, body mass index; MI, myocardial infarction; PCI, percutaneous coronary intervention; CVA: cardiovascular accident; ACEI/ARB, angiotensin antagonist inhibitor/ angiotensin receptor blocker.
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T. Liu et al. / Gene 571 (2015) 23–27
Fig. 3. Scatter plot of the results of LTAAA and urine 11dTxB2 assays in the patients. X axis presets patients with HAPR from LTAAA platelet aggregation assay, Y axis indices patients with HAPR by urinary 11-dehydrothromboxane B2 (11dTxB2) measurement. Horizontal and vertical dotted lines indicate test-specific cut-off values for HAPR by LTAAA and 11dTxB2 assay respectively. The arrow indicates the zone within which patients are considered HAPR. Abbreviation: HAPR, High on aspirin platelet response; LTA, Light Transmission Aggregometry.
status, as determined by urinary 11dTXB2 measurement, did not show significant correlation with the outcomes (Table 1). 4. Discussion To the best of our knowledge, our study is the first to compare simultaneously two different assays in the elderly Chinese population, in order to evaluate the prevalence of HAPR. Using LTAAA assay, we observed that the elderly CAD patients (mean age 75.8) had a high prevalence of HAPR (12.4%), while Lordkipanize et al. study showed that the prevalence of high platelet reactivity was 4% by LTAAA assay in the population with the mean age of 66.5 years[19]. Further, Nauder et al. found a 0.4% prevalence of high platelet reactivity in their study population with an average age of 45 years (Faraday et al., 2006). Moreover, Hovens et al. in a recent systematic review showed a pooled unadjusted prevalence of 6% using LTAAA assay (Hovens et al., 2007). Several factors might be responsible for the heterogeneity among these studies (Bennett et al., 2008; Topcuoglu et al., 2011). On one hand, factors such as aspirin dosage and different racial backgrounds would affect the estimated prevalence of HAPR. On the other hand, because age was considered as an important risk factor in HAPR, the relatively advanced age in our enrolled population might explain the high prevalence. Among the assays available to estimate the antiplatelet effect of aspirin, LTAAA assay is considered as a gold standard because of its relatively high specificity for platelets, and AA is used as the agonist to exploit the specific pathways affected by aspirin (Tantry et al., 2005). However, its requirement for highly skilled measurement and poor standardization of precise method restricts the wide applications of this technology. In our study, the LTAAA assays were all carried out by professional technicians at the First Hospital of Peking University, thus ensuring the accuracy of the measurements, which is another advantage of this study. It is well established that TxB2 is the major metabolite of platelet TxA2 in plasma. The presence of its metabolite, 11dhTxB2, is believed to be predominantly attributable to platelet activation, and 11dhTxB2 levels decrease after aspirin treatment (Catella et al., 1986). Some studies suggest that methods directly measuring urinary 11dhTxB2 might be better in assessing platelet reactivity and predicting the risk of cardiovascular events (Eikelboom et al., 2002). Using the 11dhTxB2 method, Dharmasaroja et al. enrolled 101 CAD patients and found prevalence of HAPR of 40% (Dharmasaroja and Sae-Lim, 2014). Similarly, in Lordkipanize et al. study, a 22.9% prevalence of HAPR was observed (Lordkipanidze et al., 2007). Thus, the 32.2% prevalence of HAPR found
in the current study is generally in agreement with earlier studies by others. However, the weak agreement among different platelet function tests and poor correlation with clinical outcomes raise questions about which method is reliable in assessing aspirin responses (Faraday et al., 2006; Dharmasaroja and Sae-Lim, 2014; Lordkipanidze et al., 2007). Lordkipanize et al. compared six methods to assess platelet function and found poor correlations and low agreements among the various assays (Lordkipanidze et al., 2007). Pornpatr et al. found a poor correlation and a low agreement when comparing VerifyNow with urinary 11dhTxB2 assays (Dharmasaroja and Sae-Lim, 2014). Likewise, Fallahi et al. observed a 6.8% rate of HAPR by VerifyNow assay and a poor correlation with urinary 11dhTxB2 (Fallahi et al., 2013). Similarly, we found a poor correlation between urinary 11dhTxB2 measurement and LTAAA assay. Our study found that HAPR patients, diagnosed by both urinary 11dhTxB2 measurement and LTAAA assay, were prone to be older, with higher BMI, and have more co-morbidities, such as hypertension, diabetes, current smoking and hyperlipidemia. In previous studies, females demonstrated an increased baseline platelet reactivity and a greater residual platelet activity after taking aspirin when compared with men, while in our study these differences were not observed. Furthermore, it was widely acknowledged in previous studies that patients with poor aspirin response might suffer from higher risk of cardiovascular events than those with normal aspirin response (Krasopoulos et al., 2008). In our study, patients with HAPR tend to experience a higher risk of cardiovascular events, and HAPR status based on LTAAA was significantly related to clinical outcomes. Nevertheless, in Pornpatr's study, patients with poor aspirin response diagnosed by urinary 11dhTxB2 measurement display a more close correlation with clinical outcomes than LTAAA assay. The variance in the results may be due to the relatively small scale of their study, and the definition of poor aspirin response. Although the measurement of urinary 11dhTxB2 is platelet specific, it was observed that at times of stress such as inflammation, non-platelet derived TXA2 might be produced, while the LTAAA assay reflects the current activity of the platelets toward a defined stimulus. Based on the previous studies, our research confirmed poor correlation and low agreement between the two tests, and revealed that LTAAA assay might be more reliable in predicting risks of cardiovascular events. This study has several strengths and limitations. Firstly, this is the first study to compare two methods on platelet function in the Chinese elderly population. Meanwhile, in patients under dual antiplatelet medications, ADP-induced platelet aggregation was used to exclude patients with low clopidogrel response. Secondly, the LTAAA assays in our study were all carried out by the same professional technicians, which ensured the accuracy and reproducibility of the measurement. With regard to limitations, other detection methods such as PFA-100, VerifyNow assay, or Thrombelastogram were not included in this study. In addition, variations in lifestyle factors, such as diet, smoking history, and physical activity, can affect the outcomes in any study. Therefore, more patients should be included in further studies to confirm our conclusions, and further research is still needed to identify the best method to evaluate aspirin response. In conclusion, we performed a prospective study with follow-up results of cardiovascular events and mortality. Our data showed a poor correlation and a low agreement between the two tests in evaluating aspirin response. A significant correlation between LTAAA assay and clinical outcomes was observed. Therefore, the findings of our study can be added to the growing evidences that consider LTAAA assay as a more stable and accurate method to assess platelet function on antiplatelet treatment.
Competing financial interests There are no conflicts of interest.
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Acknowledgments The work was supported by grants from the International Science & Technology Cooperation Projects of China (2013DFA30860), the National Science & Technology Pillar Program of China (2012BAI 37B00), and Beijing Huikangyisheng Sci-Tech Co., Ltd. (00020140616003). References Angiolillo, D.J., Fernandez-Ortiz, A., Bernardo, E., Ramirez, C., Sabate, M., et al., 2005. Platelet function profiles in patients with type 2 diabetes and coronary artery disease on combined aspirin and clopidogrel treatment. Diabetes 54, 2430–2435. Angiolillo, D.J., Bernardo, E., Ramirez, C., Costa, M.A., Sabate, M., et al., 2006. Insulin therapy is associated with platelet dysfunction in patients with type 2 diabetes mellitus on dual oral antiplatelet treatment. J. Am. Coll. Cardiol. 48, 298–304. Angiolillo, D.J., Bernardo, E., Sabate, M., Jimenez-Quevedo, P., Costa, M.A., et al., 2007. Impact of platelet reactivity on cardiovascular outcomes in patients with type 2 diabetes mellitus and coronary artery disease. J. Am. Coll. Cardiol. 50, 1541–1547. Bennett, D., Yan, B., Macgregor, L., Eccleston, D., Davis, S.M., 2008. A pilot study of resistance to aspirin in stroke patients. J. Clin. Neurosci. 15, 1204–1209. Catella, F., Healy, D., Lawson, J.A., FitzGerald, G.A., 1986. 11-Dehydrothromboxane B2: a quantitative index of thromboxane A2 formation in the human circulation. Proc. Natl. Acad. Sci. U. S. A. 83, 5861–5865. Cattaneo, M., 2011. The clinical relevance of response variability to antiplatelet therapy. Hematol. Am. Soc. Hematol. Educ. Prog. 2011, 70–75. Dharmasaroja, P.A., Sae-Lim, S., 2014. Comparison of aspirin response measured by urinary 11-dehydrothromboxane B2 and VerifyNow aspirin assay in patients with ischemic stroke. J. Stroke Cerebrovasc. Dis. 23, 953–957. Eikelboom, J.W., Hirsh, J., Weitz, J.I., Johnston, M., Yi, Q., et al., 2002. Aspirin-resistant thromboxane biosynthesis and the risk of myocardial infarction, stroke, or cardiovascular death in patients at high risk for cardiovascular events. Circulation 105, 1650–1655. Eikelboom, J.W., Hankey, G.J., Thom, J., Bhatt, D.L., Steg, P.G., et al., 2008. Incomplete inhibition of thromboxane biosynthesis by acetylsalicylic acid: determinants and effect on cardiovascular risk. Circulation 118, 1705–1712. Fallahi, P., Katz, R., Toma, I., Li, R., Reiner, J., et al., 2013. Aspirin insensitive thrombophilia: transcript profiling of blood identifies platelet abnormalities and HLA restriction. Gene 520, 131–138.
27
Faraday, N., Becker, D.M., Yanek, L.R., Herrera-Galeano, J.E., Segal, J.B., et al., 2006. Relation between atherosclerosis risk factors and aspirin resistance in a primary prevention population. Am. J. Cardiol. 98, 774–779. Gum, P.A., Kottke-Marchant, K., Welsh, P.A., White, J., Topol, E.J., 2003. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J. Am. Coll. Cardiol. 41, 961–965. Gurbel, P.A., Bliden, K.P., DiChiara, J., Newcomer, J., Weng, W., et al., 2007. Evaluation of dose-related effects of aspirin on platelet function: results from the Aspirin-Induced Platelet Effect (ASPECT) study. Circulation 115, 3156–3164. Harrison, P., 2000. Progress in the assessment of platelet function. Br. J. Haematol. 111, 733–744. Harrison, P., Frelinger, A.L., Furman, M.I., Michelson, A.D., 2007. Measuring antiplatelet drug effects in the laboratory. Thromb. Res. 120, 323–336. Hovens, M.M., Snoep, J.D., Eikenboom, J.C., van der Bom, J.G., Mertens, B.J., et al., 2007. Prevalence of persistent platelet reactivity despite use of aspirin: a systematic review. Am. Heart J. 153, 175–181. Jack, D.B., 1997. One hundred years of aspirin. Lancet 350, 437–439. Krasopoulos, G., Brister, S.J., Beattie, W.S., Buchanan, M.R., 2008. Aspirin “resistance” and risk of cardiovascular morbidity: systematic review and meta-analysis. BMJ 336, 195–198. Lordkipanidze, M., Pharand, C., Schampaert, E., Turgeon, J., Palisaitis, D.A., et al., 2007. 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. 28, 1702–1708. Michelson, A.D., 2004. Platelet function testing in cardiovascular diseases. Circulation 110, e489–e493. Nicholson, N.S., Panzer-Knodle, S.G., Haas, N.F., Taite, B.B., Szalony, J.A., et al., 1998. Assessment of platelet function assays. Am. Heart J. 135, S170–S178. Patrono, C., Coller, B., FitzGerald, G.A., Hirsh, J., Roth, G., 2004. Platelet-active drugs: the relationships among dose, effectiveness, and side effects: the Seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 126, 234S–264S. Patrono, C., Garcia Rodriguez, L.A., Landolfi, R., aigent, C., 2005. Low-dose aspirin for the prevention of atherothrombosis. N. Engl. J. Med. 353, 2373–2383. Tantry, U.S., Bliden, K.P., Gurbel, P.A., 2005. Resistance to antiplatelet drugs: current status and future research. Expert. Opin. Pharmacother. 6, 2027–2045. Topcuoglu, M.A., Arsava, E.M., Ay, H., 2011. Antiplatelet resistance in stroke. Expert. Rev. Neurother. 11, 251–263.