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Renal function and aspirin resistance in patients with coronary artery disease
Thrombosis Research 130 (2012) e103–e106
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Regular Article
Renal function and aspirin resistance in patients with coronary artery disease A.D. Blann ⁎, 1, N. Kuzniatsova 1, S. Velu, G.Y.H. Lip University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Birmingham, B18 7QH, UK
a r t i c l e
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Article history: Received 30 January 2012 Received in revised form 19 June 2012 Accepted 27 June 2012 Available online 17 July 2012 Keywords: Aspirin resistance Chronic renal failure Coronary artery disease Platelet aggregation
a b s t r a c t Aspirin resistance and chronic renal failure are both potentially important clinical issues in coronary artery disease. To test the hypothesis of a relationship between the two, we recruited 169 stable outpatients with proven coronary artery disease (myocardial infarction, coronary artery bypass grafting, intra-coronary stents) taking 75 mg aspirin daily. Blood was taken for light transmission aggregometry to agonists arachidonic acid (0.5 mg/mL) and adenosine diphosphate (10 μmol/L), for platelet marker soluble P selectin (enzyme linked immunosorbent assay), resting and stimulated expression of CD62P (flow cytometry) and for renal function (estimated glomerular filtration rate). The estimated glomerular filtration rate was lower when aspirin resistance was defined by response to arachidonic acid after 3, 5 and 7 minutes (approximately 30% of patients) (pb 0.021), and when defined by response to adenosine diphosphate after 3 minutes (approximately 17% of patients)(p =0.015) compared to those who were sensitive to aspirin. Mean [standard deviation] soluble P selectin levels were 57 [23] ng/mL in 49 patients with aspirin resistance, and 50 [15] ng/mL in the 119 aspirin sensitive patients (p=0.02). Estimated glomerular filtration rate correlated inversely with platelet CD62P expression at rest (r=−0.22, p =0.004), and when stimulated by arachidonic acid (r=−0.21, p=0.007) and by adenosine diphosphate (r= −0.17, p=0.023). Aspirin resistance was more than twice as prevalent in those with the greatest renal disease (50% of patients) compared to those with the best renal function (21.4%). Our data point to a weak relationship between worsening glomerular filtration rate and aspirin resistance. Nevertheless, we suspect that failure of patients to be fully responsive to aspirin may be important in the pathophysiology of thrombosis in renal dysfunction. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
Introduction Although anti-platelet therapy is the mainstay of cardiovascular disease, that fact that thrombosis still occurs despite the use of 75 mg/d aspirin implies that in some subjects (perhaps 10%) this agent is sub-effective [1,2]. This has lead to the concept of a ‘suboptimal response to aspirin’, also described as ‘high on-treatment platelet reactivity’ or ‘aspirin resistance’ (AR) [3,4]. This suboptimal response in patients undergoing percutaneous coronary intervention has been associated with increased risk of thrombosis and increased major adverse clinical events in long term follow-up [5–9]. In patients with stable coronary artery disease, the use of a lower dose of aspirin increases the likelihood of persistent platelet activation [10], and in-stent thrombosis is associated with AR [11,12]. However, research into AR is confounded by variation in the dose of aspirin, and the lack of a clear consensus of a definition, due partly to numerous laboratory methods [4–14]. Cardiovascular disease is an important and possibly fatal consequence of chronic kidney disease (CKD), and accounts for 40% of all CKD deaths [15]. Even in the absence of cardiovascular disease, CKD ⁎ Corresponding author. Tel./fax: +44 121 507 5076. E-mail address: [email protected] (A.D. Blann). 1 Equal contribution, as joint first authors.
brings an increased risk of thrombosis, the dominant pathophysiology of which is likely to be abnormalities of platelet function [16–18]. Indeed, use of aspirin is recommended in selected groups of patients at high risk of cardiovascular disease, such as in diabetic nephropathy, arterio-venous fistulae and dialysis [19–21]. Furthermore, both obesity and diabetes are likely risk factors for AR [22,23]. The present study was designed to test the hypothesis that in patients with existing coronary artery disease, and therefore on 75 mg aspirin daily, there is a relationship between CKD and AR. We tested our hypothesis in stable outpatients attending a cardiology clinic. The estimated glomerular filtration rate (eGFR) was taken to be a surrogate of renal function, and we used several different definitions of platelet activity/function and AR. To provide a perspective for the values of the research indices, we recruited control subjects not taking aspirin. Subjects and Methods We recruited 169 patients with proven coronary artery disease, all of whom were prescribed 75 mg aspirin daily. There was no standard time of day for this therapy. We also recruited 35 controls from Hospital staff who were free of CAD. Clinical and demographic data is shown in Table 1. No control was taking aspirin or anti-hypertension medication, and no patient was within 3 months of their index event. Exclusion criteria for all subjects were current use of oral or parenteral
0049-3848/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.06.026
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Table 1 Demographic, clinical (including pharmaceutical) and laboratory details of subjects. Demographic details
Patients
Controls
Age (years) Sex (men / women) Body mass index (kg/m2)
64 [10] 138 / 31 27.6 [4.0]
62 [9] 24 / 11 26.7 [5.2]
Clinical and laboratory details
n
(%)
n
Myocardial infarction CABG Percutaneous coronary intervention Diabetes History of stroke Smokers Peripheral artery disease Statins ACE inhibitor/ARB Beta blocker Calcium channel blocker Diuretic Nitrate Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (beats/minute) Total cholesterol (mmol/L) HDL cholesterol (mmol/L) eGFR (mls/min/1.73 m2) Thromboxane B2 (ng/mL) Soluble P selectin (ng/mL) CD62P expression (%)
78 45 105
(46.1) (26.6) (62.1)
0 0 0
37 13 28 15 155 134 119 50 46 34 136 [20] 76 [11] 66 [12] 4.1 [1.0] 1.3 [0.3] 71.5 [13.5] 3.8 [3.0-4.8] 52 [17] 20.3 [6.9]
(21.9) (7.7) (16.6) (8.9) (91.7) (79.3) (70.4) (29.6) (27.2) (20.1)
0 0 3 0 2 0 0 0 0 0 139 [16] 81 [8] 69 [11] 5.5 [1.1] 1.6 [0.3] 91.7 [1.9] 5.5 [4.3-6.5] 45 [8] 17.3 [4.9]
%
(8.6) (5.7)
Data mean [standard deviation]. CABG=coronary artery bypass graft, ACE=angiotensin converting enzyme, ARB=angiotensin receptor blocker, HDL=high density lipoprotein, eGFR=estimated glomerular filtration rate.
anticoagulation or other anti-platelet drugs (clopidogrel, prasugrel), bleeding abnormalities, and/or significant hepatic, neoplastic, renal (requiring dialysis), connective tissue disease or inflammatory disease. A sample of serum was taken for creatinine to allow the eGFR to be calculated. Based on this, subjects were placed in a CKD stage; Stage 1 eGFR >90 ml/mim/1.732, Stage 2 eGFR 60–89, Stage 3a eGFR 49–59, Stage 3b eGFR 30–48, Stage 4 eGFR 15–29, Stage 5 eGFR b 15 ml/min/ 1.732 [24]. The project had the approval of the Local Research Ethics Committee and written informed consent was obtained from each subject. Venous blood was taken into 3.2% sodium citrate for light transmission aggregometry (LTA), flow cytometry and soluble P selectin (ELISA, R&D System, Abingdon, UK). Thromboxane B2 was assessed in serum, collected after the blood was clotted at 30 minutes at 37 °C (ELISA, Diagnostica Stago, Reading, UK). The latter, levels of which are reduced by aspirin, was measured to help confirm compliance. LTA was performed on 270 μL aliquots of platelet rich plasma (1000 rpm, 10 minutes) with a 4-channel PAP aggregometer (Alpha Labs, Basingstoke, UK) according to standard protocols using 30 μL agonists arachidonic acid (AA, Sigma Aldrich, UK, 0.5 mg/mL) and adenosine diphosphate (ADP, Sigma Aldrich, UK, 10 μmol/L). Percentage of light transmission was collected at 3, 5 and 7 minutes after the addition of the agonist. A normal response to an agonist is an increase the light transmittance to, for example, 50% of baseline, indicates aggregation. In the presence of aspirin, this response is blunted so that less aggregation takes place, for example, to 15%. It follows that a high aggregation response (e.g. 50%) to an agonist in the presence of aspirin implies platelet function is unimpaired, and this may be due to AR. Using commonly published criteria, we classified patients as being AR if their response to AA was >20%, and if their response to ADP was >70% [11,22,23,25]. Intra-assay coefficients of variation were 3.5% for the response to ADP, and 1.8% for the response to AA. In whole blood flow cytometry (FACScalibor, Becton Dickinson, Oxford, UK), we took increased expression of CD62P as a surrogate
of platelet activation [26–29]. Briefly, the platelet cloud was identified in forward and side scatter, gated, and platelet identity confirmed by CD62P-APC and CD42a-PerCP (defining platelet gpIX)(both mAbs Becton Dickinson, Oxford, UK) in accordance with international guidelines [30]. Specificity was set by isotype controls. Reaction tubes were incubated with ADP at 2 × 10 −4 M for two minutes, AA at 125 μg/mL for five minutes, or saline before addition of antibodies to detect the resting and activated expression of CD62P as mean fluorescence intensity. The intra-assay coefficients of variation (CV) for CD62P expression by resting and ADP-stimulated platelets was 3.7% and 0.9% respectively (n= 5 determinations). The intra-assay CVs for CD62P expression by AA-activated platelets were 3.7% for the percent of CD62P positive cells and 6.6% for their mean fluorescence intensity (n=4 determinations). The eGFR (units: ml/min/1.73 m 2) was calculated from an equation inputting serum creatinine (routine Hospital service), age, sex, and African ancestry (24). We based our sample size calculation on the likelihood of an AR rate of 25% [5,11,12,14] so that n = 160 patients would provide about n = 40 being AR and n = 120 aspirin sensitive. This sample size provides the power at 1-beta = 0.8 and alpha b0.05 for a correlation coefficient of 0.2 [31]. We recruited consecutive patients slightly in excess of this number to obtain better confidence. Thirty five control subjects free of CAD were recruited to provide a local perspective of expected data, a simple comparison, and demonstration of the expected difference in the patients due to aspirin – no formal comparison with patient data was intended. Data distributed normally are presented as mean and standard deviation, were analysed by student's t test, and were correlated according to Pearson's method. Data distributed non-normally are presented as median and inter-quartile range and analysed by the Mann-Whitney U test. Categorical data were analysed by chi-squared testing. With n >> 100 we have power for a multivariate regression analysis to determine what we feel may be seven major influences on the GFR. Pb 0.05 was considered statistically significant.
Results Differences in platelet responses to the agonists between patients and controls are shown in Table 2. Markedly lower % light transmittance to all agonist conditions in patients with CAD are as expected and indicate the influence of aspirin. This is supported by significantly lower thromboxane B2 in the patients (pb 001). Taking the 95th percentile of the control group as defining a normal thromboxane level, 35 patients (20.7%) had a thromboxane level within the ‘normal’ range, implying aspirin resistance. Soluble P selectin was higher in the patients (p= 0.017), and patient's platelets were more likely to be expressing CD62P than were platelets from the controls (p= 0.019). Patients had lower total cholesterol, HDL-cholesterol, diastolic blood pressure and eGFR than the controls (all p b 0.01), the former doubtless due to cardiovascular pharmacotherapy. There was no difference in the proportion of smokers between the groups.
Table 2 Responses of platelets from control subjects and patients with CAD to agonists arachidonic acid and adenosine diphosphate at different time points. Agonist and time when aggregation defined
Control subjects not on aspirin
All patients with CAD on aspirin
P value
AA : 3 minutes AA : 5 minutes AA : 7 minutes ADP : 3 minutes ADP : 5 minutes ADP : 7 minutes
69.0 73.9 75.5 68.0 72.0 73.3
18.8 18.9 18.4 58.3 57.5 54.5
b0.001 b0.001 b0.001 b0.001 b0.001 b0.001
[14.5] [14.8] [15.4] [10.3] [11.0] [11.3]
[13.5] [13.1] [13.4] [13.5] [15.1] [15.9]
Data is mean [SD] % light transmittance. Analyses by t test. AA – arachidonic acid, ADP adenosine diphosphate. The time refers to the number of minutes exposure to each agonist at which point platelet response was defined.
A.D. Blann et al. / Thrombosis Research 130 (2012) e103–e106
Differences in eGFR according to platelet responses to AA and ADP in patients resistant or sensitive to aspirin are shown in Table 3. GFR was lower regardless of the definition of AR. This difference became statistically significant when AR was defined by response to AA after 3, 5 and 7 minutes, and when defined by response to ADP after 3 minutes. The AA 3 minute response was the most sensitive at defining AR. Soluble P selectin was mean 57 [standard deviation 23] ng/mL in 49 patients deemed to have AR, and was 50 [15] ng/mL in 119 deemed to be aspirin sensitive (p = 0.02). Similarly, thromboxane B2 was median 3.6 ng/mL [IQR 2.8-4.2] in AR and was 3.9 [3.0-4.9] ng/mL in those sensitive to aspirin (p = 0.11). BMI in the AR patients was 28 [4.3] kg/m2 and was 27.4 [3.8] in those sensitive to aspirin (p= 0.33). BMI failed to correlate with unstimulated, AA stimulated, or ADP stimulated mean fluorescence expression of CD62P (r=−0.066, p = 0.40, r = 0.037, p = 0.64 and r = −0.045, p = 0.56 respectively). GFR and BMI failed to correlate (r= −0.124, p = 0.11). The proportion of patients resistant to aspirin was linked strongly to their CKD class (I - 21.4%, Class II - 28.1%, Class IIIa – 42.9%, Class IIIb – 50%, p = 0.012)(24). The frequency of AR more than doubled between CKD I and CKD IIIb. The correlations between eGFR and the aggregation responses were as follows: AA 3 mins, r = − 0.19, p = 0.011; AA 5 mins, r = − 0.20, p = 0.008; AA 7 mins r = − 0.20, p = 0.009; ADP 3 mins r = 0.055, p = 0.483; ADP 5 mins r = − 0.02 p = 0.76; ADP 7 min r = − 0.06, p = 0.47. eGFR correlated inversely with platelet expression of CD62P when unstimulated (r = − 0.22, p = 0.004), when stimulated by AA (r = − 0.21, p = 0.006), or by ADP (r = − 0.18, p = 0.021). Although AA increased the expression of CD62P by a median of 26%, and ADP increased expression by a median of 166%, the magnitude of these increases were unrelated to the eGFR (r = − 0.116, p = 0.14 and r = − 0.044, p = 0.57 respectively). In a multivariate regression analysis to determine factors independently related to the eGFR, we entered diabetes, BMI, the platelet count, serum thromboxane B2, soluble P selectin, and the definition of AR by AA and ADP responses at 3 minutes. Only the latter two indices were independently related to GFR (both p b 0.001), r2 = 15.8%. Discussion The successful treatment of coronary artery disease and cerebrovascular disease is frustrated by aspirin resistance (AR) [3–14]. Cardiovascular disease and thrombosis are common and often fatal complications of CKD, possibly due to excessive platelet activity [15–21]. Using the surrogate of eGFR, we report an association between AR and CKD. Patients who were AR (defined by both AA and ADP responses) had lower eGFR and had higher soluble P selectin than patients who were sensitive to aspirin. Some suggest that AR may reflect lack of compliance, which in one study accounted for nearly half the AR [32]. This seems unlikely in our case as there is no difference in thromboxane B2 in those who are AR compared to those with a normal response to aspirin, implying, as a Table 3 The relationship between glomerular filtration rate and resistance or sensitivity to aspirin defined by different agonists in the patients with coronary artery disease. Agonist and time when aggregation defined
Aspirin Resistant
Aspirin Sensitive
P value
AA : 3 minutes AA : 5 minutes AA : 7 minutes ADP : 3 minutes ADP : 5 minutes ADP : 7 minutes
68.7 69.2 69.0 72.0 72.3 70.2
75.1 74.9 74.9 79.5 76.8 76.2
0.009 0.021 0.018 0.015 0.106 0.25
[15.7] [n = 49] [15.8] [n = 51] [15.9] [n = 48] [14.8] [n = 26] [14.7] [n = 33] [13.9] [n = 25]
[13.7] [n = 119] [13.8] [n = 116] [13.8] [n = 119] [12.0] [n = 141] [13.8] [n = 134] [13.9] [n = 142]
Data is mean [SD] glomerular filtration rate: units ml/min/1.73 m2. Analyses by t test. [n] refers to number of subjects in each group. AA – arachidonic acid, ADP=adenosine diphosphate. The time refers to the number of minutes exposure to each agonist at which point platelet response was defined.
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group, equal suppression of the cyclo-oxygenase pathway [33,34]. In addition, we believe that the patients are highly motivated to take their aspirin because of their heart disease. There was a weak inverse correlation between eGFR and the resting and agonist-stimulated expression of CD62P. As the platelet expression of CD62P (P selectin), and increased soluble P selectin both mark platelet activation [18,25–28,35,36], thus patients with the worse GFR have the greatest degree of platelet activation. However, the relationship between the expression of CD62P and eGFR, although mathematically significant, is weak, with a correlation coefficient of around 0.2. Notably, despite being on aspirin, patients still have increased platelet activity (soluble P selectin, surface expression of CD62P), compared to the healthy controls, reflecting continuing alpha granule degranulation [34]. Indeed, this alone may partially explain the continuing thrombosis risk despite the use of this drug and supports the hypothesis that aspirin does not inhibit the degranulation of alpha granules or the appearance of P-selectin at the surface of the platelet [36–39]. It follows that for more comprehensive platelet suppression, concurrent use of additional agents (such as clopidogrel) may be valuable. Indeed, 300 mg aspirin/day can overcome 75 mg/day AR [40], but the long term haemorrhagic consequences of this are unknown. Tanrikulu et al. [21] has recently reported an increased frequency of AR in stages 3/4 CKD (in 25% of patients) and in patients on dialysis (46%) compared to eurenal controls (17%)(p b 0.001). although the cardiovascular status of these subjects is unclear, our data broadly supports this finding. Although our data point to more active platelets and AR in those CAD patients with the worse renal function (as does that of Tanikulu et al in a different population), these relationships are not necessary causal. However, it seems more plausible that worsening renal disease is more likely to precipitate platelet activation/ dysfunction than vica versa, perhaps by the combined effects of increased urea and/or creatinine. A further difficulty in comparing our data with that of Tanikulu [21] is the relatively mild disease in our patients: the lowest eGFR was 36 ml/min/1.73 m 2, and no patient had CKD stage 4 disease. In our patients, the presence of LTA-defined AR is unrelated to obesity, unlike data focusing on diabetes where platelet activity was defined by expression of CD62P [23]. Using a different technology (Verify-Now Aspirin) in 468 stable CAD patients, Lee et al found AR in 27%, in whom renal insufficiency was a univariate predictor (p = 0.009) of AR, although this dropped out in a multivariate analysis [10]. They also reported that AR was dependent on the dose of aspirin. Gum et al, using the PFA-100 and LTA, reported that AR to a high dose of 325 mg aspirin was not influenced by renal disease [41]. At this higher dose, less then 10% of patients were AR, unsurprisingly implying a dose-response effect. As our study is limited by its cross-sectional nature, further speculations are restricted. A further limitation is that we cannot tell if AR is related to ‘standard’ aspirin, or aspirin that comes in an enteric coating. But perhaps the greatest limitation is the very definition of AR, which can be by Verify-Now [5,12,21,25,26], light transmission or whole blood aggregometry [7,22,25,26,40], and the PTA-100 [14,22,25]), and also by the dose of aspirin (such as 75, 80, 100, 150 or 325 mg/day [11,13,22,26]). Even the time point to define AR in aggregometry varies – we used 3, 5 and 7 minutes, Madsen et al used 6 minutes [13]. All of the issues lead to difficulty in cross-study comparison [4,26]. Aspirin exerts its effects via cyclo-oxygenase metabolism, a pathway and also known to be active in the endothelium [42]. It is therefore conceivable that insufficient functioning aspirin contributes to dysfunction (possibly, glomerular endothelial dysfunction) of the renal vasculature, and thus to a fall in GFR. An alternative mechanism may be an increase in platelet turnover [43], likely to be present in the patients and more pronounced in those with renal disease. Nevertheless, more robust data are likely to come from both short term studies,
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such as the effects of treatments on GFR, including dialysis, and long term studies, where the relationship between GFR and AR can be tested in their effect on clinical deterioration to the requirement for dialysis and/or mortality. Although the relationship is weak (correlation coefficient 0.17 – 0.22) and prevents in depth speculation about causality, we suggest that renal function may be a factor to be considered in future studies on AR in cardiovascular disease. Acknowledgements Dr Kuzniatsova was supported by a Fellowship from the European Cardiology Society. We thank Balu Balakrishnan for general scientific support with ELISAs and platelet aggregometry, and Eduard Shantsila for flow cytometry support. References [1] Antithrombotic Trialists' Collaboration. Collaborative meta-analysis of randomised trails of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002;324:71–86. [2] Keller TT, Squizzato A, Middeldorp S. Clopidogrel plus aspirin versus aspirin alone for preventing cardiovascular disease. Cochrane Database Syst Rev 2007;18(CD005158). [3] Gasparyan AY, Watson T, Lip GY. The role of aspirin in cardiovascular prevention: implications of aspirin resistance. J Am Coll Cardiol 2008;51:1829–43. [4] Sharma RK, Reddy HK, Singh VN, Sharma R, Voelker DJ, Bhatt G. Aspirin and clopidogrel hyporesponsiveness and nonresponsiveness in patients with coronary artery stenting. Vasc Health Risk Manag 2009;5:965–72. [5] Chen WH, Cheng X, Lee PY, Ng W, Kwok JY, Tse HF, et al. Aspirin resistance and adverse clinical events in patients with coronary artery disease. Am J Med 2007;120:631–5. [6] Chen WH, Lee PY, Ng W, Tse HF, Lau CP. Aspirin resistance is associated with a high incidence of myonecrosis after non-urgent percutaneous coronary intervention despite clopidogrel pretreatment. J Am Coll Cardiol 2004;43:1122–6. [7] Beigel R, Hod H, Fefer P, Asher E, Novikov I, Shenkman B, et al. Relation of aspirin failure to clinical outcome and to platelet response to aspirin in patients with acute myocardial infarction. Am J Cardiol 2011;107:339–42. [8] Storey R. Variability of response to antiplatelet therapy. Eur Heart J Suppl 2008;10: A21–7. [9] Chen WH, Lee PY, Ng W, Kwok JY, Cheng X, Lee SW, et al. Relation of aspirin resistance to coronary flow reserve in patients undergoing elective percutaneous coronary intervention. Am J Cardiol 2005;96:760–3. [10] Lee P, Chen W, Ng W, Cheng X, Kwok JY, Tse HF, et al. Low-dose aspirin increases aspirin resistance in patients with coronary artery disease. Am J Med 2005;118: 723–7. [11] Wenaweser P, Dörffler-Melly J, Imboden K, Windecker S, Togni M, Meier B, et al. Stent thrombosis is associated with an impaired response to antiplatelet therapy. J Am Coll Cardiol 2005;45:1748–52. [12] Rajendran S, Parikh D, Shugman I, French JK, Juergens CP. High on treatment platelet reactivity and stent thrombosis. Heart Lung Circ 2011;20:525–31. [13] Madsen EH, Saw J, Kristensen SR, Schmidt EB, Pittendreigh C, Maurer-Spurej E. Long-term aspirin and clopidogrel response evaluated by light transmission aggregometry, VerifyNow, and thrombelastography in patients undergoing percutaneous coronary intervention. Clin Chem 2010;56:839–47. [14] Dawson J, Quinn T, Lees KR, Walters MR. Microembolic Signals and Aspirin Resistance in Patients with Carotid Stenosis. Cardiovasc Ther 2012;30:234–9. [15] Parmar MS. Chronic renal disease. Br Med J 2002;325:85–90. [16] Wattanakit K, Cushman M. Chronic kidney disease and venous thromboembolism: epidemiology and mechanisms. Curr Opin Pulm Med 2009;15:408–12. [17] Casserly LF, Dember LM. Thrombosis in end-stage renal disease. Semin Dial 2003;16:245–56. [18] Landray MJ, Wheeler DC, Lip GY, Newman DJ, Blann AD, McGlynn FJ, et al. Inflammation, endothelial dysfunction, and platelet activation in patients with chronic kidney disease: the chronic renal impairment in Birmingham (CRIB) study. Am J Kidney Dis 2004;43:244–53. [19] James MT, Hemmelgarn BR, Tonelli M. Early recognition and prevention of chronic kidney disease. Lancet 2010;375:1296–309. [20] Osborn G, Escofet X, Da Silva A. Medical adjuvant treatment to increase patency of arteriovenous fistulae and grafts. Cochrane Database Syst Rev 2008;4(CD002786).
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Renal function and aspirin resistance in patients with coronary artery disease A.D. Blann ⁎, 1, N. Kuzniatsova 1, S. Velu, G.Y.H. Lip University of Birmingham Centre for Cardiovascular Sciences, City Hospital, Birmingham, B18 7QH, UK
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Article history: Received 30 January 2012 Received in revised form 19 June 2012 Accepted 27 June 2012 Available online 17 July 2012 Keywords: Aspirin resistance Chronic renal failure Coronary artery disease Platelet aggregation
a b s t r a c t Aspirin resistance and chronic renal failure are both potentially important clinical issues in coronary artery disease. To test the hypothesis of a relationship between the two, we recruited 169 stable outpatients with proven coronary artery disease (myocardial infarction, coronary artery bypass grafting, intra-coronary stents) taking 75 mg aspirin daily. Blood was taken for light transmission aggregometry to agonists arachidonic acid (0.5 mg/mL) and adenosine diphosphate (10 μmol/L), for platelet marker soluble P selectin (enzyme linked immunosorbent assay), resting and stimulated expression of CD62P (flow cytometry) and for renal function (estimated glomerular filtration rate). The estimated glomerular filtration rate was lower when aspirin resistance was defined by response to arachidonic acid after 3, 5 and 7 minutes (approximately 30% of patients) (pb 0.021), and when defined by response to adenosine diphosphate after 3 minutes (approximately 17% of patients)(p =0.015) compared to those who were sensitive to aspirin. Mean [standard deviation] soluble P selectin levels were 57 [23] ng/mL in 49 patients with aspirin resistance, and 50 [15] ng/mL in the 119 aspirin sensitive patients (p=0.02). Estimated glomerular filtration rate correlated inversely with platelet CD62P expression at rest (r=−0.22, p =0.004), and when stimulated by arachidonic acid (r=−0.21, p=0.007) and by adenosine diphosphate (r= −0.17, p=0.023). Aspirin resistance was more than twice as prevalent in those with the greatest renal disease (50% of patients) compared to those with the best renal function (21.4%). Our data point to a weak relationship between worsening glomerular filtration rate and aspirin resistance. Nevertheless, we suspect that failure of patients to be fully responsive to aspirin may be important in the pathophysiology of thrombosis in renal dysfunction. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved.
Introduction Although anti-platelet therapy is the mainstay of cardiovascular disease, that fact that thrombosis still occurs despite the use of 75 mg/d aspirin implies that in some subjects (perhaps 10%) this agent is sub-effective [1,2]. This has lead to the concept of a ‘suboptimal response to aspirin’, also described as ‘high on-treatment platelet reactivity’ or ‘aspirin resistance’ (AR) [3,4]. This suboptimal response in patients undergoing percutaneous coronary intervention has been associated with increased risk of thrombosis and increased major adverse clinical events in long term follow-up [5–9]. In patients with stable coronary artery disease, the use of a lower dose of aspirin increases the likelihood of persistent platelet activation [10], and in-stent thrombosis is associated with AR [11,12]. However, research into AR is confounded by variation in the dose of aspirin, and the lack of a clear consensus of a definition, due partly to numerous laboratory methods [4–14]. Cardiovascular disease is an important and possibly fatal consequence of chronic kidney disease (CKD), and accounts for 40% of all CKD deaths [15]. Even in the absence of cardiovascular disease, CKD ⁎ Corresponding author. Tel./fax: +44 121 507 5076. E-mail address: [email protected] (A.D. Blann). 1 Equal contribution, as joint first authors.
brings an increased risk of thrombosis, the dominant pathophysiology of which is likely to be abnormalities of platelet function [16–18]. Indeed, use of aspirin is recommended in selected groups of patients at high risk of cardiovascular disease, such as in diabetic nephropathy, arterio-venous fistulae and dialysis [19–21]. Furthermore, both obesity and diabetes are likely risk factors for AR [22,23]. The present study was designed to test the hypothesis that in patients with existing coronary artery disease, and therefore on 75 mg aspirin daily, there is a relationship between CKD and AR. We tested our hypothesis in stable outpatients attending a cardiology clinic. The estimated glomerular filtration rate (eGFR) was taken to be a surrogate of renal function, and we used several different definitions of platelet activity/function and AR. To provide a perspective for the values of the research indices, we recruited control subjects not taking aspirin. Subjects and Methods We recruited 169 patients with proven coronary artery disease, all of whom were prescribed 75 mg aspirin daily. There was no standard time of day for this therapy. We also recruited 35 controls from Hospital staff who were free of CAD. Clinical and demographic data is shown in Table 1. No control was taking aspirin or anti-hypertension medication, and no patient was within 3 months of their index event. Exclusion criteria for all subjects were current use of oral or parenteral
0049-3848/$ – see front matter. Crown Copyright © 2012 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2012.06.026
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Table 1 Demographic, clinical (including pharmaceutical) and laboratory details of subjects. Demographic details
Patients
Controls
Age (years) Sex (men / women) Body mass index (kg/m2)
64 [10] 138 / 31 27.6 [4.0]
62 [9] 24 / 11 26.7 [5.2]
Clinical and laboratory details
n
(%)
n
Myocardial infarction CABG Percutaneous coronary intervention Diabetes History of stroke Smokers Peripheral artery disease Statins ACE inhibitor/ARB Beta blocker Calcium channel blocker Diuretic Nitrate Systolic blood pressure (mm Hg) Diastolic blood pressure (mm Hg) Heart rate (beats/minute) Total cholesterol (mmol/L) HDL cholesterol (mmol/L) eGFR (mls/min/1.73 m2) Thromboxane B2 (ng/mL) Soluble P selectin (ng/mL) CD62P expression (%)
78 45 105
(46.1) (26.6) (62.1)
0 0 0
37 13 28 15 155 134 119 50 46 34 136 [20] 76 [11] 66 [12] 4.1 [1.0] 1.3 [0.3] 71.5 [13.5] 3.8 [3.0-4.8] 52 [17] 20.3 [6.9]
(21.9) (7.7) (16.6) (8.9) (91.7) (79.3) (70.4) (29.6) (27.2) (20.1)
0 0 3 0 2 0 0 0 0 0 139 [16] 81 [8] 69 [11] 5.5 [1.1] 1.6 [0.3] 91.7 [1.9] 5.5 [4.3-6.5] 45 [8] 17.3 [4.9]
%
(8.6) (5.7)
Data mean [standard deviation]. CABG=coronary artery bypass graft, ACE=angiotensin converting enzyme, ARB=angiotensin receptor blocker, HDL=high density lipoprotein, eGFR=estimated glomerular filtration rate.
anticoagulation or other anti-platelet drugs (clopidogrel, prasugrel), bleeding abnormalities, and/or significant hepatic, neoplastic, renal (requiring dialysis), connective tissue disease or inflammatory disease. A sample of serum was taken for creatinine to allow the eGFR to be calculated. Based on this, subjects were placed in a CKD stage; Stage 1 eGFR >90 ml/mim/1.732, Stage 2 eGFR 60–89, Stage 3a eGFR 49–59, Stage 3b eGFR 30–48, Stage 4 eGFR 15–29, Stage 5 eGFR b 15 ml/min/ 1.732 [24]. The project had the approval of the Local Research Ethics Committee and written informed consent was obtained from each subject. Venous blood was taken into 3.2% sodium citrate for light transmission aggregometry (LTA), flow cytometry and soluble P selectin (ELISA, R&D System, Abingdon, UK). Thromboxane B2 was assessed in serum, collected after the blood was clotted at 30 minutes at 37 °C (ELISA, Diagnostica Stago, Reading, UK). The latter, levels of which are reduced by aspirin, was measured to help confirm compliance. LTA was performed on 270 μL aliquots of platelet rich plasma (1000 rpm, 10 minutes) with a 4-channel PAP aggregometer (Alpha Labs, Basingstoke, UK) according to standard protocols using 30 μL agonists arachidonic acid (AA, Sigma Aldrich, UK, 0.5 mg/mL) and adenosine diphosphate (ADP, Sigma Aldrich, UK, 10 μmol/L). Percentage of light transmission was collected at 3, 5 and 7 minutes after the addition of the agonist. A normal response to an agonist is an increase the light transmittance to, for example, 50% of baseline, indicates aggregation. In the presence of aspirin, this response is blunted so that less aggregation takes place, for example, to 15%. It follows that a high aggregation response (e.g. 50%) to an agonist in the presence of aspirin implies platelet function is unimpaired, and this may be due to AR. Using commonly published criteria, we classified patients as being AR if their response to AA was >20%, and if their response to ADP was >70% [11,22,23,25]. Intra-assay coefficients of variation were 3.5% for the response to ADP, and 1.8% for the response to AA. In whole blood flow cytometry (FACScalibor, Becton Dickinson, Oxford, UK), we took increased expression of CD62P as a surrogate
of platelet activation [26–29]. Briefly, the platelet cloud was identified in forward and side scatter, gated, and platelet identity confirmed by CD62P-APC and CD42a-PerCP (defining platelet gpIX)(both mAbs Becton Dickinson, Oxford, UK) in accordance with international guidelines [30]. Specificity was set by isotype controls. Reaction tubes were incubated with ADP at 2 × 10 −4 M for two minutes, AA at 125 μg/mL for five minutes, or saline before addition of antibodies to detect the resting and activated expression of CD62P as mean fluorescence intensity. The intra-assay coefficients of variation (CV) for CD62P expression by resting and ADP-stimulated platelets was 3.7% and 0.9% respectively (n= 5 determinations). The intra-assay CVs for CD62P expression by AA-activated platelets were 3.7% for the percent of CD62P positive cells and 6.6% for their mean fluorescence intensity (n=4 determinations). The eGFR (units: ml/min/1.73 m 2) was calculated from an equation inputting serum creatinine (routine Hospital service), age, sex, and African ancestry (24). We based our sample size calculation on the likelihood of an AR rate of 25% [5,11,12,14] so that n = 160 patients would provide about n = 40 being AR and n = 120 aspirin sensitive. This sample size provides the power at 1-beta = 0.8 and alpha b0.05 for a correlation coefficient of 0.2 [31]. We recruited consecutive patients slightly in excess of this number to obtain better confidence. Thirty five control subjects free of CAD were recruited to provide a local perspective of expected data, a simple comparison, and demonstration of the expected difference in the patients due to aspirin – no formal comparison with patient data was intended. Data distributed normally are presented as mean and standard deviation, were analysed by student's t test, and were correlated according to Pearson's method. Data distributed non-normally are presented as median and inter-quartile range and analysed by the Mann-Whitney U test. Categorical data were analysed by chi-squared testing. With n >> 100 we have power for a multivariate regression analysis to determine what we feel may be seven major influences on the GFR. Pb 0.05 was considered statistically significant.
Results Differences in platelet responses to the agonists between patients and controls are shown in Table 2. Markedly lower % light transmittance to all agonist conditions in patients with CAD are as expected and indicate the influence of aspirin. This is supported by significantly lower thromboxane B2 in the patients (pb 001). Taking the 95th percentile of the control group as defining a normal thromboxane level, 35 patients (20.7%) had a thromboxane level within the ‘normal’ range, implying aspirin resistance. Soluble P selectin was higher in the patients (p= 0.017), and patient's platelets were more likely to be expressing CD62P than were platelets from the controls (p= 0.019). Patients had lower total cholesterol, HDL-cholesterol, diastolic blood pressure and eGFR than the controls (all p b 0.01), the former doubtless due to cardiovascular pharmacotherapy. There was no difference in the proportion of smokers between the groups.
Table 2 Responses of platelets from control subjects and patients with CAD to agonists arachidonic acid and adenosine diphosphate at different time points. Agonist and time when aggregation defined
Control subjects not on aspirin
All patients with CAD on aspirin
P value
AA : 3 minutes AA : 5 minutes AA : 7 minutes ADP : 3 minutes ADP : 5 minutes ADP : 7 minutes
69.0 73.9 75.5 68.0 72.0 73.3
18.8 18.9 18.4 58.3 57.5 54.5
b0.001 b0.001 b0.001 b0.001 b0.001 b0.001
[14.5] [14.8] [15.4] [10.3] [11.0] [11.3]
[13.5] [13.1] [13.4] [13.5] [15.1] [15.9]
Data is mean [SD] % light transmittance. Analyses by t test. AA – arachidonic acid, ADP adenosine diphosphate. The time refers to the number of minutes exposure to each agonist at which point platelet response was defined.
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Differences in eGFR according to platelet responses to AA and ADP in patients resistant or sensitive to aspirin are shown in Table 3. GFR was lower regardless of the definition of AR. This difference became statistically significant when AR was defined by response to AA after 3, 5 and 7 minutes, and when defined by response to ADP after 3 minutes. The AA 3 minute response was the most sensitive at defining AR. Soluble P selectin was mean 57 [standard deviation 23] ng/mL in 49 patients deemed to have AR, and was 50 [15] ng/mL in 119 deemed to be aspirin sensitive (p = 0.02). Similarly, thromboxane B2 was median 3.6 ng/mL [IQR 2.8-4.2] in AR and was 3.9 [3.0-4.9] ng/mL in those sensitive to aspirin (p = 0.11). BMI in the AR patients was 28 [4.3] kg/m2 and was 27.4 [3.8] in those sensitive to aspirin (p= 0.33). BMI failed to correlate with unstimulated, AA stimulated, or ADP stimulated mean fluorescence expression of CD62P (r=−0.066, p = 0.40, r = 0.037, p = 0.64 and r = −0.045, p = 0.56 respectively). GFR and BMI failed to correlate (r= −0.124, p = 0.11). The proportion of patients resistant to aspirin was linked strongly to their CKD class (I - 21.4%, Class II - 28.1%, Class IIIa – 42.9%, Class IIIb – 50%, p = 0.012)(24). The frequency of AR more than doubled between CKD I and CKD IIIb. The correlations between eGFR and the aggregation responses were as follows: AA 3 mins, r = − 0.19, p = 0.011; AA 5 mins, r = − 0.20, p = 0.008; AA 7 mins r = − 0.20, p = 0.009; ADP 3 mins r = 0.055, p = 0.483; ADP 5 mins r = − 0.02 p = 0.76; ADP 7 min r = − 0.06, p = 0.47. eGFR correlated inversely with platelet expression of CD62P when unstimulated (r = − 0.22, p = 0.004), when stimulated by AA (r = − 0.21, p = 0.006), or by ADP (r = − 0.18, p = 0.021). Although AA increased the expression of CD62P by a median of 26%, and ADP increased expression by a median of 166%, the magnitude of these increases were unrelated to the eGFR (r = − 0.116, p = 0.14 and r = − 0.044, p = 0.57 respectively). In a multivariate regression analysis to determine factors independently related to the eGFR, we entered diabetes, BMI, the platelet count, serum thromboxane B2, soluble P selectin, and the definition of AR by AA and ADP responses at 3 minutes. Only the latter two indices were independently related to GFR (both p b 0.001), r2 = 15.8%. Discussion The successful treatment of coronary artery disease and cerebrovascular disease is frustrated by aspirin resistance (AR) [3–14]. Cardiovascular disease and thrombosis are common and often fatal complications of CKD, possibly due to excessive platelet activity [15–21]. Using the surrogate of eGFR, we report an association between AR and CKD. Patients who were AR (defined by both AA and ADP responses) had lower eGFR and had higher soluble P selectin than patients who were sensitive to aspirin. Some suggest that AR may reflect lack of compliance, which in one study accounted for nearly half the AR [32]. This seems unlikely in our case as there is no difference in thromboxane B2 in those who are AR compared to those with a normal response to aspirin, implying, as a Table 3 The relationship between glomerular filtration rate and resistance or sensitivity to aspirin defined by different agonists in the patients with coronary artery disease. Agonist and time when aggregation defined
Aspirin Resistant
Aspirin Sensitive
P value
AA : 3 minutes AA : 5 minutes AA : 7 minutes ADP : 3 minutes ADP : 5 minutes ADP : 7 minutes
68.7 69.2 69.0 72.0 72.3 70.2
75.1 74.9 74.9 79.5 76.8 76.2
0.009 0.021 0.018 0.015 0.106 0.25
[15.7] [n = 49] [15.8] [n = 51] [15.9] [n = 48] [14.8] [n = 26] [14.7] [n = 33] [13.9] [n = 25]
[13.7] [n = 119] [13.8] [n = 116] [13.8] [n = 119] [12.0] [n = 141] [13.8] [n = 134] [13.9] [n = 142]
Data is mean [SD] glomerular filtration rate: units ml/min/1.73 m2. Analyses by t test. [n] refers to number of subjects in each group. AA – arachidonic acid, ADP=adenosine diphosphate. The time refers to the number of minutes exposure to each agonist at which point platelet response was defined.
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group, equal suppression of the cyclo-oxygenase pathway [33,34]. In addition, we believe that the patients are highly motivated to take their aspirin because of their heart disease. There was a weak inverse correlation between eGFR and the resting and agonist-stimulated expression of CD62P. As the platelet expression of CD62P (P selectin), and increased soluble P selectin both mark platelet activation [18,25–28,35,36], thus patients with the worse GFR have the greatest degree of platelet activation. However, the relationship between the expression of CD62P and eGFR, although mathematically significant, is weak, with a correlation coefficient of around 0.2. Notably, despite being on aspirin, patients still have increased platelet activity (soluble P selectin, surface expression of CD62P), compared to the healthy controls, reflecting continuing alpha granule degranulation [34]. Indeed, this alone may partially explain the continuing thrombosis risk despite the use of this drug and supports the hypothesis that aspirin does not inhibit the degranulation of alpha granules or the appearance of P-selectin at the surface of the platelet [36–39]. It follows that for more comprehensive platelet suppression, concurrent use of additional agents (such as clopidogrel) may be valuable. Indeed, 300 mg aspirin/day can overcome 75 mg/day AR [40], but the long term haemorrhagic consequences of this are unknown. Tanrikulu et al. [21] has recently reported an increased frequency of AR in stages 3/4 CKD (in 25% of patients) and in patients on dialysis (46%) compared to eurenal controls (17%)(p b 0.001). although the cardiovascular status of these subjects is unclear, our data broadly supports this finding. Although our data point to more active platelets and AR in those CAD patients with the worse renal function (as does that of Tanikulu et al in a different population), these relationships are not necessary causal. However, it seems more plausible that worsening renal disease is more likely to precipitate platelet activation/ dysfunction than vica versa, perhaps by the combined effects of increased urea and/or creatinine. A further difficulty in comparing our data with that of Tanikulu [21] is the relatively mild disease in our patients: the lowest eGFR was 36 ml/min/1.73 m 2, and no patient had CKD stage 4 disease. In our patients, the presence of LTA-defined AR is unrelated to obesity, unlike data focusing on diabetes where platelet activity was defined by expression of CD62P [23]. Using a different technology (Verify-Now Aspirin) in 468 stable CAD patients, Lee et al found AR in 27%, in whom renal insufficiency was a univariate predictor (p = 0.009) of AR, although this dropped out in a multivariate analysis [10]. They also reported that AR was dependent on the dose of aspirin. Gum et al, using the PFA-100 and LTA, reported that AR to a high dose of 325 mg aspirin was not influenced by renal disease [41]. At this higher dose, less then 10% of patients were AR, unsurprisingly implying a dose-response effect. As our study is limited by its cross-sectional nature, further speculations are restricted. A further limitation is that we cannot tell if AR is related to ‘standard’ aspirin, or aspirin that comes in an enteric coating. But perhaps the greatest limitation is the very definition of AR, which can be by Verify-Now [5,12,21,25,26], light transmission or whole blood aggregometry [7,22,25,26,40], and the PTA-100 [14,22,25]), and also by the dose of aspirin (such as 75, 80, 100, 150 or 325 mg/day [11,13,22,26]). Even the time point to define AR in aggregometry varies – we used 3, 5 and 7 minutes, Madsen et al used 6 minutes [13]. All of the issues lead to difficulty in cross-study comparison [4,26]. Aspirin exerts its effects via cyclo-oxygenase metabolism, a pathway and also known to be active in the endothelium [42]. It is therefore conceivable that insufficient functioning aspirin contributes to dysfunction (possibly, glomerular endothelial dysfunction) of the renal vasculature, and thus to a fall in GFR. An alternative mechanism may be an increase in platelet turnover [43], likely to be present in the patients and more pronounced in those with renal disease. Nevertheless, more robust data are likely to come from both short term studies,
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such as the effects of treatments on GFR, including dialysis, and long term studies, where the relationship between GFR and AR can be tested in their effect on clinical deterioration to the requirement for dialysis and/or mortality. Although the relationship is weak (correlation coefficient 0.17 – 0.22) and prevents in depth speculation about causality, we suggest that renal function may be a factor to be considered in future studies on AR in cardiovascular disease. Acknowledgements Dr Kuzniatsova was supported by a Fellowship from the European Cardiology Society. We thank Balu Balakrishnan for general scientific support with ELISAs and platelet aggregometry, and Eduard Shantsila for flow cytometry support. References [1] Antithrombotic Trialists' Collaboration. Collaborative meta-analysis of randomised trails of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002;324:71–86. [2] Keller TT, Squizzato A, Middeldorp S. Clopidogrel plus aspirin versus aspirin alone for preventing cardiovascular disease. Cochrane Database Syst Rev 2007;18(CD005158). [3] Gasparyan AY, Watson T, Lip GY. The role of aspirin in cardiovascular prevention: implications of aspirin resistance. J Am Coll Cardiol 2008;51:1829–43. [4] Sharma RK, Reddy HK, Singh VN, Sharma R, Voelker DJ, Bhatt G. Aspirin and clopidogrel hyporesponsiveness and nonresponsiveness in patients with coronary artery stenting. Vasc Health Risk Manag 2009;5:965–72. [5] Chen WH, Cheng X, Lee PY, Ng W, Kwok JY, Tse HF, et al. Aspirin resistance and adverse clinical events in patients with coronary artery disease. Am J Med 2007;120:631–5. [6] Chen WH, Lee PY, Ng W, Tse HF, Lau CP. Aspirin resistance is associated with a high incidence of myonecrosis after non-urgent percutaneous coronary intervention despite clopidogrel pretreatment. J Am Coll Cardiol 2004;43:1122–6. [7] Beigel R, Hod H, Fefer P, Asher E, Novikov I, Shenkman B, et al. Relation of aspirin failure to clinical outcome and to platelet response to aspirin in patients with acute myocardial infarction. Am J Cardiol 2011;107:339–42. [8] Storey R. Variability of response to antiplatelet therapy. Eur Heart J Suppl 2008;10: A21–7. [9] Chen WH, Lee PY, Ng W, Kwok JY, Cheng X, Lee SW, et al. Relation of aspirin resistance to coronary flow reserve in patients undergoing elective percutaneous coronary intervention. Am J Cardiol 2005;96:760–3. [10] Lee P, Chen W, Ng W, Cheng X, Kwok JY, Tse HF, et al. Low-dose aspirin increases aspirin resistance in patients with coronary artery disease. Am J Med 2005;118: 723–7. [11] Wenaweser P, Dörffler-Melly J, Imboden K, Windecker S, Togni M, Meier B, et al. Stent thrombosis is associated with an impaired response to antiplatelet therapy. J Am Coll Cardiol 2005;45:1748–52. [12] Rajendran S, Parikh D, Shugman I, French JK, Juergens CP. High on treatment platelet reactivity and stent thrombosis. Heart Lung Circ 2011;20:525–31. [13] Madsen EH, Saw J, Kristensen SR, Schmidt EB, Pittendreigh C, Maurer-Spurej E. Long-term aspirin and clopidogrel response evaluated by light transmission aggregometry, VerifyNow, and thrombelastography in patients undergoing percutaneous coronary intervention. Clin Chem 2010;56:839–47. [14] Dawson J, Quinn T, Lees KR, Walters MR. Microembolic Signals and Aspirin Resistance in Patients with Carotid Stenosis. Cardiovasc Ther 2012;30:234–9. [15] Parmar MS. Chronic renal disease. Br Med J 2002;325:85–90. [16] Wattanakit K, Cushman M. Chronic kidney disease and venous thromboembolism: epidemiology and mechanisms. Curr Opin Pulm Med 2009;15:408–12. [17] Casserly LF, Dember LM. Thrombosis in end-stage renal disease. Semin Dial 2003;16:245–56. [18] Landray MJ, Wheeler DC, Lip GY, Newman DJ, Blann AD, McGlynn FJ, et al. Inflammation, endothelial dysfunction, and platelet activation in patients with chronic kidney disease: the chronic renal impairment in Birmingham (CRIB) study. Am J Kidney Dis 2004;43:244–53. [19] James MT, Hemmelgarn BR, Tonelli M. Early recognition and prevention of chronic kidney disease. Lancet 2010;375:1296–309. [20] Osborn G, Escofet X, Da Silva A. Medical adjuvant treatment to increase patency of arteriovenous fistulae and grafts. Cochrane Database Syst Rev 2008;4(CD002786).
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