Adenosine Diphosphate–Induced Platelet Aggregation Might Contribute to Poor Outcomes in Atrial Fibrillation–Related Ischemic Stroke Jae-Hyung Choi, MD, Jae-Kwan Cha, MD, PhD and Jae-Taeck Huh, MD, PhD
Systemic atherosclerosis is involved in ischemic damages and cardioembolism after atrial fibrillation (AF)–related ischemic stroke (IS). Platelet activation is a critical factor in systemic atherosclerosis; however, there is little information regarding the role of platelet activation on the outcome of AF-related IS. We investigated the relationship between adenosine diphosphate (ADP)–induced platelet aggregation and the long-term outcomes of AF-related IS. We studied 249 patients who were exclusively treated with anticoagulation therapy after they had experienced AF-related IS. We evaluated their platelet function 5 days after admission to the hospital by using an optic platelet aggregometer test. We also assessed the prognoses of patients 90 days after the AF-related IS. Our results showed that ADP-induced platelet aggregation was positively correlated with CHA2DS2-VASc scores (r 5 .285, P , .01). Totally, 107 (43.0%) patients had a poor outcome at 90 days after IS. Univariate analysis showed that the following factors significantly contribute to a poor outcome: older age (odds ratio [OR] 5 1.07, confidence interval [CI] 1.04-1.10, P , .01), a history of stroke (OR 5 3.24, CI 1.61-6.53, P ,.01), high scores on the National Institutes of Health Stroke Scale (NIHSS; OR 5 1.25, CI 1.18-1.32, P , .01), increased white blood cell counts (OR 5 1.12, CI 1.02-1.24, P , .01), high CHA2DS2-VASc scores ($5, OR 5 7.31, CI 3.36-15.93, P 5 .025), and the highest tertile of ADP-induced platelet aggregation ($72%, OR 5 3.17, CI 1.67-5.99, P , .01). Of these factors, high NIHSS scores (OR 5 1.27, CI 1.20-1.36, P , .01), high CHA2DS2-VASc scores (OR 5 4.69, CI 1.21-18.14, P 5 .03), and the highest tertile of ADP-induced platelet aggregation (OR 5 2.49, CI 1.17-5.27, P 5 .02) were independently associated with a poor outcome at 90 days after IS. Therefore, our results suggest that platelet activation might affect the outcome of AF-related IS. Key Words: Platelet aggregation—atrial fibrillation—ischemic stroke—adenosine diphosphate. Ó 2014 by National Stroke Association
Introduction Atrial fibrillation (AF) is the most common cause of cardiac embolism, accounting for approximately 77% of the high-risk cardiac sources of embolism in ischemic From the Stroke Center, Dong-A University Hospital, Busan, Korea. Received July 30, 2013; revision received September 29, 2013; accepted October 9, 2013. Address correspondence to Jae-Kwan Cha, MD, PhD, Department of Neurology, College of Medicine, Dong-A University, 1,3Ga, Dongdaeshin-Dong, Seo-Gu, Busan 602-715, South Korea. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.10.011
stroke (IS).1 The incidence of AF-related IS has increased considerably in recent years because of increases in the elderly population.2 The risk of recurrence and mortality is higher after AF-related IS than other types of IS. CHADS2 is a scoring system that is used to select patients eligible for anticoagulation therapy, and it has a significant predictive value for mortality in patients with AF-related IS.3,4 Recently, the CHA2DS2-VASc score was introduced in clinical practice. This new scoring system added the following predictors to the CHADS2 score: (1) vascular disease (myocardial infarction, complex aortic plaque, and peripheral artery disease), (2) age between 65 and 74 years, and (3) female sex. The prediction of thromboembolism with the
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CHA2DS2-VASc scoring system was modestly better than that with the CHADS2 score.5 Several studies have, thus far, reported that an increase in CHADS2/CHA2DS2-VASc scores was related to high mortality and poor outcomes in patients who experienced AF-related IS.6-8 The exact mechanism responsible for the higher mortality rates associated with AF during stroke has not yet been elucidated. However, it can be presumed that these scoring systems indicate the likelihood of an AF-related IS and the burden of systemic atherosclerosis in AF.9 Therefore, the presence of systemic atherosclerosis might increase the mortality rate after AFrelated IS. Thus far, there has been little evidence to clarify the role of systemic atherosclerosis in AF-related IS. Atherosclerosis is a systemic process, mediated by several factors, including endothelial cells, inflammatory cells, tissue factors, and platelets. Platelet activation is involved in all the key steps that comprise the progression of atherosclerosis.10 Many studies have shown that platelet activation is a critical factor that determines the outcome of atherothrombosis, including myocardial infarction and stroke.11,12 Therefore, we hypothesized that increased platelet activation could be related to the atheroma burden and potentiated ischemic damage in AF-related IS. To test this hypothesis, we investigated whether CHA2DS2-VASc scores correlate with the extent of ADP-induced platelet aggregation, and we then assessed whether ADP-induced platelet aggregation influenced the outcome in AF-related IS.
For brain imaging studies, we examined the brain computed tomography and/or magnetic resonance imaging and magnetic resonance angiogram of all patients. The modified Rankin Scale was calculated at baseline and at 90 days after IS. A poor outcome was defined as a modified Rankin Scale score greater than 2 at 90 days after IS. This study was approved by the local ethics committee.
Optical Platelet Aggregometer We evaluated the extent of platelet aggregation in our patients on day 5 after their admission to the stroke center. For this, 30 mL of whole blood was anticoagulated with 3.2% of sodium citrate. Platelet-rich plasma (PRP) was prepared by centrifugation at 160g for 10 minutes at room temperature. The platelet count of the PRP was adjusted to 200,000/mm3 using platelet poor plasma obtained by centrifugation at 4000g for 5 minutes. For this, we used an optical aggregometer (Chrono Log, 560VS). Platelets were stimulated with arachidonic acid (.5 mg/mL) or adenosine diphosphate (ADP, 10 mM). We measured the extent of platelet aggregation for a period of 6 minutes after stimulation with either one of the agonists, AA or ADP. The results are expressed as the percent aggregation (percent aggregation is the estimated percentage difference in the light that is transmitted by PPP and PRP). This method has been verified previously.13,14
Statistical Analysis
Subjects and Methods The present study retrospectively recruited 3758 patients who were registered on the stroke registry of Dong-A University, Busan, Korea, between March 2000 and September 2012 after an acute ischemic stroke (AIS; within 72 hours of their ischemic events). We included the following patients in our study population: (1) those who had an AF-related stroke mechanism, (2) those who were not given any antiplatelet agents during the admission period, (3) those who could be followed-up 90 days after AIS, (4) and those in whom platelet function was evaluated according to the predefined method. Stroke neurologists assessed baseline National Institutes of Health Stroke Scale (NIHSS) for all the patients included in our study. On the day of admission, the medical history of each patient was recorded, and complete physical and neurologic examinations were performed. All patients were subject to a standard investigative protocol, including routine blood tests and transcranial Doppler ultrasonography, electrocardiography, and transthoracic echocardiography in selected cases. We calculated the pre-admission CHA2DS2-VASc score for each patient according to previously published data,6-8 and patients were categorized in 3 groups based on their CHA2DS2-VASc score: 0-2, 3-4, and 5 or more.
Categorical variables were summarized according to counts and relative frequencies and numeric variables according to mean 6 standard deviation or median (Q1Q3). Patients were divided into 3 subgroups according to CHA2DS2-VASc scores (0-2, 3-4, $5). We used Pearson correlation coefficients to test for correlations between CHA2DS2-VASc scores and the extent of ADP-induced platelet aggregation. Clinical and laboratory findings were compared between the 3 groups. The significance of intergroup differences was assessed by chi-square analysis for categorical variables and analysis of variance or Kruskal–Wallis test for continuous variables. Post hoc analyses of differences between the 3 groups were performed with Scheffe and Bonferroni tests. Comparisons between good and poor outcomes at 90 days after IS were made using t tests for continuous variables and chi-square or Wilcoxon rank sum tests for categorical variables. The odds ratio (OR) for comparison of the 2 groups was summarized, together with the 95% confidence interval and P value, using logistic regression. The data for ADP-induced platelet aggregation were stratified based on tertiles. In addition, a multivariate model was created using a backward elimination method. The probability was set at .10 for removal. ORs were also adjusted for factors affecting the response variables. P values less than .05
ADP-INDUCED PLATELET AGGREGATION AND THE OUTCOMES OF AF-RELATED IS
were considered statistically significant. All statistical analyses were carried out using SAS, version 9.1.3, statistical software.
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Total 3758 pts with AIS
Results 846 pts with AF-related
During the observation period, 3758 patients were admitted to our stroke center. Of the 3758 patients, 249 were recruited for the present study following the selection steps as described in Figure 1. The mean age of our group of patients was 69.5 6 10.9, the median NIHSS was 8.0 (3.0-15.0), and the median CHA2DS2-VASc score was 3.0 (2.0-4.0). The median value of ADP-induced platelet aggregation was 48% (mean 6 SD, 63.0% 6 18.6%). Table 1 shows the differences in the clinical and laboratory findings of the 3 subgroups categorized according to CHA2DS2-VASc scores (0-2, 3-4, $5). The prevalence of concomitant coronary arterial diseases increased incrementally as the CHA2DS2VASc scores increased (Table 1, P , .01). Patients who had a CHA2DS2-VASc score of 5 or more had significantly higher values of ADP-induced platelet aggregation compared with those in the other 2 groups (Table 1). In addition, there was a positive correlation between CHA2DS2-VASc scores and values of ADP-induced platelet aggregation (r 5 .285, P , .01, Fig 2). At 90 days post-IS, 107 (43.0%) patients had a poor outcome. The group of patients with a poor outcome had a significantly higher median CHA2DS2-VASc score than patients with a good outcome. There were significant differences in the mean values for ADP-induced platelet aggregation (P , .01) in the 3 groups categorized according to CHA2DS2-VASc score (0-2, 3-4, $5). Patients were also stratified into 3 subgroups according to tertiles of ADP values (#56%, 57%-71%, #72%), and patients who were stratified to the highest tertile of ADP-induced platelet aggregation had a significantly higher incidence of poor outcome end points than those stratified to the lowest tertile (P , .01; Table 2). Univariate analysis showed that the following factors significantly contributed to the occurrence of a poor outcome: older age (OR 5 1.07, confidence interval [CI] 1.04-1.10, P , .01), history of stroke (OR 5 3.24, CI 1.616.53, P , .01), higher NIHSS results (OR 5 1.25, CI 1.181.32, P , .01), increased white blood cell (WBC) counts (OR 5 1.12, CI 1.02-1.24, P , .01), high CHA2DS2-VASc score ($5, OR 5 7.31, CI 3.36-15.93, P ,.01), and the highest tertile of ADP-induced platelet aggregation ($72%, OR 5 3.17, CI 1.67-5.99, P , .01, Table 2). We performed multiple logistic regression analysis, using a backward elimination method in which the probability for removal was set at .10. This allowed us to evaluate the independent significance of ADP-induced platelet aggregation on primary events after AF-related IS. Our results showed that the severity of neurologic deficit, as expressed by NIHSS (OR 5 1.27, CI 1.20-1.36, P , .01), a higher CHA2DS2-VASc score (OR 5 4.69, CI 1.21 to 18.14,
Excluded 380 pts without platelet function test 466 pts with AF-related IS 3 pts with malignancy 3 pts with follow-up loss 10 pts with other causes
450 pts with AF-related IS Excluded 201 pts using antiplatelet agents Final inclusion, 249 pts Figure 1. Flow chart of the selection process of study subjects. Abbreviations: AF, atrial fibrillation; AIS, acute ischemic stroke, stroke within 72 hours; Pts, patients.
P 5 .03), and the highest tertile of ADP-induced platelet aggregation (OR 5 2.49, CI 1.17 to 5.27, P 5.02), was independently associated with a poor outcome 90 days after AF-related IS (Table 3).
Discussion A previous study reported that cerebral atherosclerosis is more common in patients with high CHA2DS2-VASc scores than in those with low CHA2DS2-VASc scores. Further, patients with high CHA2DS2-VASc scores were shown to have a higher risk of atherothrombotic and cardioembolic stroke than those with low CHA2DS2-VASc scores.9 Our findings corroborate these results, and we found that the prevalence of coronary artery disease increased as the CHA2DS2-VASc scores increased, suggesting that this score indicates the presence of systemic atherosclerosis. In addition, our study was unique in that it assessed the relationship between the extent of ADP-induced platelet aggregation and the CHA2DS2-VASc score. It was not known whether CHA2DS2-VASc scores, which indicate the status of atheroma burden in AF-related IS, might be associated with enhanced platelet activation. In the present study, ADP-induced platelet aggregation significantly correlated with CHA2DS2-VASc scores in AFrelated IS. Many previous studies have shown that ADP-induced platelet aggregation is a key component in the progression of atherothrombosis.14-16 Therefore, it
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Table 1. Clinical and laboratory findings in the 3 groups categorized according to CHA2DS2-VASc scores CHA2DS2-VASc
Male (%) Age (y, mean 6 SD) HT (%) CAD (%) Previous stroke (%) CHF (%) NIHSS (median, IQR) WBC (3103/dL) CRP (mg/dL) Statin (%) ADP (mean 6 SD) ADP (median IQR) Low #56 (%) Middle 57-71 (%) High $72 (%)
0-2 (n 5 85)
3-4 (n 5 113)
$5 (n 5 51)
P value
71 (83.5) 60.9 6 11.0* 31 (36.5) 8 (9.4) 1 (1.2) 5 (5.9) 7.0 [2.0-14.0] 7.9 6 2.5 .6 6 2.1 52 (61.2) 56.1 6 17.0* 59.0 [44.0-67.0]* 38 (44.7) 32 (37.6) 15 (17.6)
63 (55.8) 72.8 6 7.6* 79 (69.9) 18 (15.9) 16 (14.2) 14 (12.4) 8.0 [4.0-15.0] 8.2 6 2.8 .6 6 1.1 67 (59.3) 64.7 6 19.4* 68.0 [52.0-76.0]* 35 (31.0) 37 (32.7) 41 (36.3)
16 (31.4) 76.7 6 7.0* 46 (90.2) 22 (43.1) 25 (49.0) 13 (25.5) 10.0 [3.0-16.0] 8.3 6 2.4 1.0 6 1.7 31 (60.8) 70.6 6 15.5* 73.0 [62.0-82.0]* 9 (17.6) 14 (27.5) 28 (54.9)
,.01 ,.01 ,.01 ,.01 ,.01 ,.01 .27 .65 .38 .96 ,.01 ,.01 ,.01
Abbreviations: ADP, adenosine diphosphate; CAD, coronary artery diseases; CHF, congestive heart failure; CRP, C-reactive protein; HT, hypertension; NIHSS, National Institutes of Health Stroke Scale; WBC, white blood cell. P value was calculated by analysis of variance, c2 test, and Kruskal-Wallis test. *Post hoc by Scheffe and Bonferroni correction.
is plausible that ADP-induced platelet aggregation could reveal the status of atheroma burden in AF-related IS, especially because high CHA2DS2-VASc scores and ADP-induced platelet aggregation are independently associated with a poor outcome after AF-related IS. It has been suggested that the platelet activation reciprocally stimulates systemic atherosclerosis. Several previous studies have also shown that ADP-induced platelet aggregation is involved in ischemic damage after atherothrombosis,11,12 and we previously reported that unregulated ADP-induced platelet aggregation increased the rate of mortality in patients with AIS who were treated with aspirin only.17 Considering these findings, the enhanced ADP-induced platelet aggregation found in patients with high CHA2DS2-VASc scores could 120 r = 0.285 p< 0.001
100
ADP
80 60 40 20 0 0
1
2
3
4
5
6
7
Figure 2. Association between CHA2DS2-VASc scores and ADP-induced platelet aggregation values. ADP-induced platelet aggregation increased as CHA2DS2-VASc scores increased. There was a positive correlation between the CHA2DS2-VAS score and the value of ADP-induced platelet aggregation (r 5 .326, P , .01). Pearson correlation coefficients for correlations between CHA2DS2-VASc scores and the extent of ADP-induced platelet aggregation. Abbreviation: ADP, adenosine diphosphate.
potentiate the development of thrombus and then aggravate ischemic neuronal damage. Moreover, unregulated ADP-induced platelet aggregation might increase the risk of recurrent ischemic attack during the chronic phase of AF-related IS. Nevertheless, we were not able to clearly demonstrate this in the present study because our focus was on assessing functional outcome at 90 days post-IS, rather than study the detail of events, such as early neurologic deterioration and recurrent IS, after an AF-related IS. Our study is the first to show a relationship between the extent of platelet activation and CHA2DS2-VASc scores, which, in turn, indicates the degree of atheroma burden in AF-related IS. In addition, our findings suggest that platelet activation might have an important role in the progression of ischemic damage and in the coagulation mechanism in AF-related IS. Although we were able to demonstrate that there is enhanced platelet activation in patients with high CHA2DS2-VASc scores who experience AF-related IS, we were not able to show the need for combination treatment with an antiplatelet agent and warfarin in AFrelated IS. Antiplatelet agents can effectively reduce platelet activation, but previous studies have shown that antiplatelet agents in combination with warfarin increased hemorrhagic complications in IS.18,19 There have, thus far, not been any guidelines regarding the use of antiplatelet– warfarin combination therapy in AF-related IS, except in special cases.20 However, in the future, we will consider more specific therapeutic strategy including using antiplatelet agents on the top of warfarin in AF-related stroke showing higher platelet activation.
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Table 2. Comparison of clinical and laboratory findings between good and poor outcomes in atrial fibrillation–related ischemic stroke 90-d outcome
Sex Female (%) Male (%) Age (y) HT (%) CAD (%) Previous stroke (%) CHF (%) NIHSS WBC (3103/dL) Glucose (mg/dL) Cholesterol (mg/dL) CRP (mg/dL) Statin (%) CHA2DS2-VASc 0-2 (%) 3-4 (%) $5 (%) ADP (mean 6 SD) ADP (median IQR) Low #56 (%) Middle 57-71 (%) High $72 (%)
Good (n 5 142)
Poor (n 5 107)
Total
52 (36.6) 90 (63.4) 66.7 6 10.8 83 (58.5) 23 (16.2) 14 (9.9) 14 (9.9) 4.0 [2.0-9.0] 7.8 6 2.34 138.1 6 44.9 178.6 6 36.6 .5 6 1.0 90 (63.4) 3.0 [2.0-3.0] 64 (45.1) 63 (44.4) 15 (10.6) 59.4 6 19.6 62.0 [48.0-71.0] 56 (39.4) 52 (36.6) 34 (23.9)
47 (43.9) 60 (56.1) 73.3 6 9.8 73 (68.2) 25 (23.4) 28 (26.2) 18 (16.8) 15.0 [10.0-18.0] 8.6 6 2.9 143.0 6 51.7 179.0 6 37.1 .9 6 2.2 60 (56.1) 4.0 [3.0-5.0] 21 (19.6) 50 (46.7) 36 (33.6) 67.8 6 16.1 70.0 [59.0-78.0] 26 (24.3) 31 (29.0) 50 (46.7)
99 (39.8) 150 (60.2) 69.5 6 10.9 156 (62.7) 48 (19.3) 42 (16.9) 32 (12.9) 8.0 [3.0-15.0] 8.1 6 2.6 140.2 6 47.9 178.8 6 36.8 .7 6 1.7 150 (60.2) 3.0 [2.0-4.0] 85 (34.1) 113 (45.4) 51 (20.5) 63.0 6 18.6 65.0 [53.0-76.0] 82 (32.9) 83 (33.3) 84 (33.7)
P value
.24 ,.01 .12 .16 ,.01 .10 ,.01 .02 .42 .94 .07 .24 ,.01 ,.01 ,.01 ,.01 ,.01
Abbreviations: ADP, adenosine diphosphate; CAD, coronary artery diseases; CHF, congestive heart failure; CRP, C-reactive protein; HT, hypertension; NIHSS, National Institutes of Health Stroke Scale; WBC, white blood cell. Good: mRS score #2 at 90 d after ischemic event; poor: mRS $3 at 90 d after ischemic event. P values were calculated by c2 test, independent sample t test, Wilcoxon rank sum test between good and poor group.
Table 3. Univariate and multivariate logistic analyses of poor outcome occurrence Univariate
Multivariate
Variable
Reference level
OR (95% CI)
P value
Sex Age HT CAD Previous stroke CHF NIHSS WBC Glucose Cholesterol CRP Statin CHA2DS2-VASC score
M vs. F
.74 [.44-1.23] 1.07 [1.04-1.10] 1.53 [.90-2.58] 1.58 [.84-2.97] 3.24 [1.61-6.53] 1.85 [.87-3.91] 1.25 [1.18-1.32] 1.12 [1.02-1.24] 1.00 [1.00-1.01] 1.00 [.99-1.01] 1.22 [.98-1.52] .74 [.44-1.23] 2.42 [1.31-4.48] 7.31 [3.36-15.93] 1.28 [.68-2.44] 3.17 [1.67-5.99]
.24 ,.01 .12 .16 ,.01 .11 ,.01 .02 .42 .94 .08 .24 ,.01 ,.01 .45 ,.01
ADP
Y vs. N Y vs. N Y vs. N Y vs. N
Y vs. N 3-4 vs. 0-2 $5 vs. 0-2 Middle vs. low High vs. low
OR (95% CI)
P value
1.04 [1.00-1.09]
.07
1.60 [.59-4.34]
.35
1.27 [1.20-1.36] 1.14 [.99-1.31]
,.01 .07
1.11 [.91-1.35]
.31
1.54 [.58-4.08] 4.69 [1.21-18.14]
.39 .03
2.49 [1.17-5.27]
.02
Abbreviations: ADP, adenosine diphosphate; CAD, coronary artery diseases; CHF, congestive heart failure; CI, confidence interval; CRP, Creactive protein; F, female; HT, hypertension; M, male; N, no; NIHSS, National Institutes of Health Stroke Scale; OR, odds ratio; WBC, white blood cell; Y, yes.
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Our study had some limitations: first, this was not a particularly well-designed prospective study, and it was, therefore, difficult to ensure that all biases, which could have affected our data, were excluded. Second, our sample size was so small that the validity of our conclusions could not be confirmed. Finally, we were not able to obtain information regarding the use of antiplatelet agents before admission. However, we excluded any potential effect of antiplatelet use before admission by evaluating ADP-induced platelet aggregation 5 days after admission to hospital. Nevertheless, we cannot confirm that our data revealed the true status of platelet activation without any contamination of antiplatelet agents taken before admission. In conclusion, our results show the importance of platelet activation on the long-term outcomes after AFrelated IS in patients with high CHA2DS2-VASc scores. Acknowledgment: This study was supported by DongA University Research Fund.
References 1. Han SW, Nam HS, Kim SH, et al. Frequency and significance of cardiac sources of embolism in the TOAST classification. Cerebrovasc Dis 2007;24:463-468. 2. Jung KH, Lee SH, Kim BJ, et al. Secular trends in ischemic stroke characteristics in a rapidly developed country: results from the Korean Stroke Registry Study (secular trends in Korean stroke). Cir Cardiovasc Qual Outcomes 2012;5:327-334. 3. Puwanant S, Varr BC, Shrestha K, et al. Role of the CHADS2 score in the evaluation of thromboembolic risk in patients with atrial fibrillation undergoing transesophageal echocardiography before pulmonary vein isolation. J Am Coll Cardiol 2009;54:2032-2039. 4. Rader VJ, Khumri TM, Idupulapati M, et al. Clinical predictors of left atrial thrombus and spontaneous echocardiographic contrast in patients with atrial fibrillation. J Am Soc Echocardiogr 2007;20:1181-1185. 5. Lip GY, Nieuwlaat R, Pisters R, et al. Refining clinical risk stratification for predicting stroke and thromboembolism in atrial fibrillation using a novel risk factor-based approach: the Euro Heart Survey on atrial fibrillation. Chest 2010;137:263-272. 6. Henriksson KM, Farahmand B, Johansson S, et al. Survival after stroke—the impact of CHADS2 score and atrial fibrillation. Int J Cardiol 2010;141:18-23.
J.-H. CHOI ET AL. 7. Sato S, Yazawa Y, Itabashi R, et al. Pre-admission CHADS2 score is related to severity and outcome of stroke. J Neurol Sci 2011;307:149-152. 8. Hong HJ, Kim YD, Cha M-J, et al. Early neurological outcomes according to CHADS2 score in stroke patients with non-valvular atrial fibrillation. Eur J Neurol 2012; 19:284-290. 9. Kim YD, Cha MJ, Kim J, et al. Increases in cerebral atherosclerosis according to CHADS2 scores in stroke patients with nonvalvular atrial fibrillation. Stroke 2011;42: 930-934. 10. Ross R. Mechanisms of disease: atherosclerosis is an inflammatory disease. New Engl J Med 1999;340:115-126. 11. Christie DJ, Kottke-Marchant K, Gorman RT. Hypersensitivity of platelets to adenosine diphosphate in patients with stable cardiovascular disease predicts major adverse events despite antiplatelet therapy. Platelets 2008; 19:104-110. 12. Moghadam S, Htun P, Tomandl B, et al. Hyperresponsiveness of platelets in ischemic stroke. Thromb Haemost 2007;97:974-978. 13. Gum PA, Kottke-Marchant K, Welsh PA, et al. A prospective, blinded determination of the natural history of aspirin resistance among stable patients with cardiovascular disease. J Am Coll Cardiol 2003;41:961-965. 14. Jeon HW, Cha JK. Factors related to progression of middle cerebral artery stenosis determined using transcranial Doppler ultrasonography. J Thromb Thrombolysis 2008; 25:265-269. 15. Geisler T, Langer H, Wydymus M, et al. Low response to clopidogrel is associated with cardiovascular outcome after coronary stent implantation. Eur Heart J 2006; 27:2420-2425. 16. Gurbel PA, Bliden KP, DiChiara J, et al. Evaluation of dose-related effects of aspirin on platelet function: results from the Aspirin-Induced Platelet Effect (ASPECT) study. Circulation 2006;115:3156-3164. 17. Cha J-K, Jeon HW, Kang MJ. ADP-induced platelet aggregation in acute ischemic stroke patients on aspirin therapy. Eur J Neurol 2008;15:1304-1308. 18. Hart RG, Diener HC, Yang S, et al. Intracranial hemorrhage in atrial fibrillation patients during anticoagulation with warfarin or dabigatran: the RE-LY trial. Stroke 2012; 43:1511-1577. 19. Warkentin AE, Donadini MP, Spencer FA, et al. Bleeding risk in randomized controlled trials comparing warfarin and aspirin: a systematic review and meta-analysis. J Thromb Haemost 2012;10:512-520. 20. Furie KL, Kasner SE, Adams RJ, et al. Guidelines for the prevention of stroke in patients with stroke or transient ischemic attack; a guideline for healthcare professionals from the American Heart Association/American Stroke Association. Stroke 2011;42:227-276.
Systemic atherosclerosis is involved in ischemic damages and cardioembolism after atrial fibrillation (AF)–related ischemic stroke (IS). Platelet activation is a critical factor in systemic atherosclerosis; however, there is little information regarding the role of platelet activation on the outcome of AF-related IS. We investigated the relationship between adenosine diphosphate (ADP)–induced platelet aggregation and the long-term outcomes of AF-related IS. We studied 249 patients who were exclusively treated with anticoagulation therapy after they had experienced AF-related IS. We evaluated their platelet function 5 days after admission to the hospital by using an optic platelet aggregometer test. We also assessed the prognoses of patients 90 days after the AF-related IS. Our results showed that ADP-induced platelet aggregation was positively correlated with CHA2DS2-VASc scores (r 5 .285, P , .01). Totally, 107 (43.0%) patients had a poor outcome at 90 days after IS. Univariate analysis showed that the following factors significantly contribute to a poor outcome: older age (odds ratio [OR] 5 1.07, confidence interval [CI] 1.04-1.10, P , .01), a history of stroke (OR 5 3.24, CI 1.61-6.53, P ,.01), high scores on the National Institutes of Health Stroke Scale (NIHSS; OR 5 1.25, CI 1.18-1.32, P , .01), increased white blood cell counts (OR 5 1.12, CI 1.02-1.24, P , .01), high CHA2DS2-VASc scores ($5, OR 5 7.31, CI 3.36-15.93, P 5 .025), and the highest tertile of ADP-induced platelet aggregation ($72%, OR 5 3.17, CI 1.67-5.99, P , .01). Of these factors, high NIHSS scores (OR 5 1.27, CI 1.20-1.36, P , .01), high CHA2DS2-VASc scores (OR 5 4.69, CI 1.21-18.14, P 5 .03), and the highest tertile of ADP-induced platelet aggregation (OR 5 2.49, CI 1.17-5.27, P 5 .02) were independently associated with a poor outcome at 90 days after IS. Therefore, our results suggest that platelet activation might affect the outcome of AF-related IS. Key Words: Platelet aggregation—atrial fibrillation—ischemic stroke—adenosine diphosphate. Ó 2014 by National Stroke Association
Introduction Atrial fibrillation (AF) is the most common cause of cardiac embolism, accounting for approximately 77% of the high-risk cardiac sources of embolism in ischemic From the Stroke Center, Dong-A University Hospital, Busan, Korea. Received July 30, 2013; revision received September 29, 2013; accepted October 9, 2013. Address correspondence to Jae-Kwan Cha, MD, PhD, Department of Neurology, College of Medicine, Dong-A University, 1,3Ga, Dongdaeshin-Dong, Seo-Gu, Busan 602-715, South Korea. E-mail: [email protected]. 1052-3057/$ - see front matter Ó 2014 by National Stroke Association http://dx.doi.org/10.1016/j.jstrokecerebrovasdis.2013.10.011
stroke (IS).1 The incidence of AF-related IS has increased considerably in recent years because of increases in the elderly population.2 The risk of recurrence and mortality is higher after AF-related IS than other types of IS. CHADS2 is a scoring system that is used to select patients eligible for anticoagulation therapy, and it has a significant predictive value for mortality in patients with AF-related IS.3,4 Recently, the CHA2DS2-VASc score was introduced in clinical practice. This new scoring system added the following predictors to the CHADS2 score: (1) vascular disease (myocardial infarction, complex aortic plaque, and peripheral artery disease), (2) age between 65 and 74 years, and (3) female sex. The prediction of thromboembolism with the
Journal of Stroke and Cerebrovascular Diseases, Vol. 23, No. 3 (March), 2014: pp e215-e220
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CHA2DS2-VASc scoring system was modestly better than that with the CHADS2 score.5 Several studies have, thus far, reported that an increase in CHADS2/CHA2DS2-VASc scores was related to high mortality and poor outcomes in patients who experienced AF-related IS.6-8 The exact mechanism responsible for the higher mortality rates associated with AF during stroke has not yet been elucidated. However, it can be presumed that these scoring systems indicate the likelihood of an AF-related IS and the burden of systemic atherosclerosis in AF.9 Therefore, the presence of systemic atherosclerosis might increase the mortality rate after AFrelated IS. Thus far, there has been little evidence to clarify the role of systemic atherosclerosis in AF-related IS. Atherosclerosis is a systemic process, mediated by several factors, including endothelial cells, inflammatory cells, tissue factors, and platelets. Platelet activation is involved in all the key steps that comprise the progression of atherosclerosis.10 Many studies have shown that platelet activation is a critical factor that determines the outcome of atherothrombosis, including myocardial infarction and stroke.11,12 Therefore, we hypothesized that increased platelet activation could be related to the atheroma burden and potentiated ischemic damage in AF-related IS. To test this hypothesis, we investigated whether CHA2DS2-VASc scores correlate with the extent of ADP-induced platelet aggregation, and we then assessed whether ADP-induced platelet aggregation influenced the outcome in AF-related IS.
For brain imaging studies, we examined the brain computed tomography and/or magnetic resonance imaging and magnetic resonance angiogram of all patients. The modified Rankin Scale was calculated at baseline and at 90 days after IS. A poor outcome was defined as a modified Rankin Scale score greater than 2 at 90 days after IS. This study was approved by the local ethics committee.
Optical Platelet Aggregometer We evaluated the extent of platelet aggregation in our patients on day 5 after their admission to the stroke center. For this, 30 mL of whole blood was anticoagulated with 3.2% of sodium citrate. Platelet-rich plasma (PRP) was prepared by centrifugation at 160g for 10 minutes at room temperature. The platelet count of the PRP was adjusted to 200,000/mm3 using platelet poor plasma obtained by centrifugation at 4000g for 5 minutes. For this, we used an optical aggregometer (Chrono Log, 560VS). Platelets were stimulated with arachidonic acid (.5 mg/mL) or adenosine diphosphate (ADP, 10 mM). We measured the extent of platelet aggregation for a period of 6 minutes after stimulation with either one of the agonists, AA or ADP. The results are expressed as the percent aggregation (percent aggregation is the estimated percentage difference in the light that is transmitted by PPP and PRP). This method has been verified previously.13,14
Statistical Analysis
Subjects and Methods The present study retrospectively recruited 3758 patients who were registered on the stroke registry of Dong-A University, Busan, Korea, between March 2000 and September 2012 after an acute ischemic stroke (AIS; within 72 hours of their ischemic events). We included the following patients in our study population: (1) those who had an AF-related stroke mechanism, (2) those who were not given any antiplatelet agents during the admission period, (3) those who could be followed-up 90 days after AIS, (4) and those in whom platelet function was evaluated according to the predefined method. Stroke neurologists assessed baseline National Institutes of Health Stroke Scale (NIHSS) for all the patients included in our study. On the day of admission, the medical history of each patient was recorded, and complete physical and neurologic examinations were performed. All patients were subject to a standard investigative protocol, including routine blood tests and transcranial Doppler ultrasonography, electrocardiography, and transthoracic echocardiography in selected cases. We calculated the pre-admission CHA2DS2-VASc score for each patient according to previously published data,6-8 and patients were categorized in 3 groups based on their CHA2DS2-VASc score: 0-2, 3-4, and 5 or more.
Categorical variables were summarized according to counts and relative frequencies and numeric variables according to mean 6 standard deviation or median (Q1Q3). Patients were divided into 3 subgroups according to CHA2DS2-VASc scores (0-2, 3-4, $5). We used Pearson correlation coefficients to test for correlations between CHA2DS2-VASc scores and the extent of ADP-induced platelet aggregation. Clinical and laboratory findings were compared between the 3 groups. The significance of intergroup differences was assessed by chi-square analysis for categorical variables and analysis of variance or Kruskal–Wallis test for continuous variables. Post hoc analyses of differences between the 3 groups were performed with Scheffe and Bonferroni tests. Comparisons between good and poor outcomes at 90 days after IS were made using t tests for continuous variables and chi-square or Wilcoxon rank sum tests for categorical variables. The odds ratio (OR) for comparison of the 2 groups was summarized, together with the 95% confidence interval and P value, using logistic regression. The data for ADP-induced platelet aggregation were stratified based on tertiles. In addition, a multivariate model was created using a backward elimination method. The probability was set at .10 for removal. ORs were also adjusted for factors affecting the response variables. P values less than .05
ADP-INDUCED PLATELET AGGREGATION AND THE OUTCOMES OF AF-RELATED IS
were considered statistically significant. All statistical analyses were carried out using SAS, version 9.1.3, statistical software.
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Total 3758 pts with AIS
Results 846 pts with AF-related
During the observation period, 3758 patients were admitted to our stroke center. Of the 3758 patients, 249 were recruited for the present study following the selection steps as described in Figure 1. The mean age of our group of patients was 69.5 6 10.9, the median NIHSS was 8.0 (3.0-15.0), and the median CHA2DS2-VASc score was 3.0 (2.0-4.0). The median value of ADP-induced platelet aggregation was 48% (mean 6 SD, 63.0% 6 18.6%). Table 1 shows the differences in the clinical and laboratory findings of the 3 subgroups categorized according to CHA2DS2-VASc scores (0-2, 3-4, $5). The prevalence of concomitant coronary arterial diseases increased incrementally as the CHA2DS2VASc scores increased (Table 1, P , .01). Patients who had a CHA2DS2-VASc score of 5 or more had significantly higher values of ADP-induced platelet aggregation compared with those in the other 2 groups (Table 1). In addition, there was a positive correlation between CHA2DS2-VASc scores and values of ADP-induced platelet aggregation (r 5 .285, P , .01, Fig 2). At 90 days post-IS, 107 (43.0%) patients had a poor outcome. The group of patients with a poor outcome had a significantly higher median CHA2DS2-VASc score than patients with a good outcome. There were significant differences in the mean values for ADP-induced platelet aggregation (P , .01) in the 3 groups categorized according to CHA2DS2-VASc score (0-2, 3-4, $5). Patients were also stratified into 3 subgroups according to tertiles of ADP values (#56%, 57%-71%, #72%), and patients who were stratified to the highest tertile of ADP-induced platelet aggregation had a significantly higher incidence of poor outcome end points than those stratified to the lowest tertile (P , .01; Table 2). Univariate analysis showed that the following factors significantly contributed to the occurrence of a poor outcome: older age (OR 5 1.07, confidence interval [CI] 1.04-1.10, P , .01), history of stroke (OR 5 3.24, CI 1.616.53, P , .01), higher NIHSS results (OR 5 1.25, CI 1.181.32, P , .01), increased white blood cell (WBC) counts (OR 5 1.12, CI 1.02-1.24, P , .01), high CHA2DS2-VASc score ($5, OR 5 7.31, CI 3.36-15.93, P ,.01), and the highest tertile of ADP-induced platelet aggregation ($72%, OR 5 3.17, CI 1.67-5.99, P , .01, Table 2). We performed multiple logistic regression analysis, using a backward elimination method in which the probability for removal was set at .10. This allowed us to evaluate the independent significance of ADP-induced platelet aggregation on primary events after AF-related IS. Our results showed that the severity of neurologic deficit, as expressed by NIHSS (OR 5 1.27, CI 1.20-1.36, P , .01), a higher CHA2DS2-VASc score (OR 5 4.69, CI 1.21 to 18.14,
Excluded 380 pts without platelet function test 466 pts with AF-related IS 3 pts with malignancy 3 pts with follow-up loss 10 pts with other causes
450 pts with AF-related IS Excluded 201 pts using antiplatelet agents Final inclusion, 249 pts Figure 1. Flow chart of the selection process of study subjects. Abbreviations: AF, atrial fibrillation; AIS, acute ischemic stroke, stroke within 72 hours; Pts, patients.
P 5 .03), and the highest tertile of ADP-induced platelet aggregation (OR 5 2.49, CI 1.17 to 5.27, P 5.02), was independently associated with a poor outcome 90 days after AF-related IS (Table 3).
Discussion A previous study reported that cerebral atherosclerosis is more common in patients with high CHA2DS2-VASc scores than in those with low CHA2DS2-VASc scores. Further, patients with high CHA2DS2-VASc scores were shown to have a higher risk of atherothrombotic and cardioembolic stroke than those with low CHA2DS2-VASc scores.9 Our findings corroborate these results, and we found that the prevalence of coronary artery disease increased as the CHA2DS2-VASc scores increased, suggesting that this score indicates the presence of systemic atherosclerosis. In addition, our study was unique in that it assessed the relationship between the extent of ADP-induced platelet aggregation and the CHA2DS2-VASc score. It was not known whether CHA2DS2-VASc scores, which indicate the status of atheroma burden in AF-related IS, might be associated with enhanced platelet activation. In the present study, ADP-induced platelet aggregation significantly correlated with CHA2DS2-VASc scores in AFrelated IS. Many previous studies have shown that ADP-induced platelet aggregation is a key component in the progression of atherothrombosis.14-16 Therefore, it
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Table 1. Clinical and laboratory findings in the 3 groups categorized according to CHA2DS2-VASc scores CHA2DS2-VASc
Male (%) Age (y, mean 6 SD) HT (%) CAD (%) Previous stroke (%) CHF (%) NIHSS (median, IQR) WBC (3103/dL) CRP (mg/dL) Statin (%) ADP (mean 6 SD) ADP (median IQR) Low #56 (%) Middle 57-71 (%) High $72 (%)
0-2 (n 5 85)
3-4 (n 5 113)
$5 (n 5 51)
P value
71 (83.5) 60.9 6 11.0* 31 (36.5) 8 (9.4) 1 (1.2) 5 (5.9) 7.0 [2.0-14.0] 7.9 6 2.5 .6 6 2.1 52 (61.2) 56.1 6 17.0* 59.0 [44.0-67.0]* 38 (44.7) 32 (37.6) 15 (17.6)
63 (55.8) 72.8 6 7.6* 79 (69.9) 18 (15.9) 16 (14.2) 14 (12.4) 8.0 [4.0-15.0] 8.2 6 2.8 .6 6 1.1 67 (59.3) 64.7 6 19.4* 68.0 [52.0-76.0]* 35 (31.0) 37 (32.7) 41 (36.3)
16 (31.4) 76.7 6 7.0* 46 (90.2) 22 (43.1) 25 (49.0) 13 (25.5) 10.0 [3.0-16.0] 8.3 6 2.4 1.0 6 1.7 31 (60.8) 70.6 6 15.5* 73.0 [62.0-82.0]* 9 (17.6) 14 (27.5) 28 (54.9)
,.01 ,.01 ,.01 ,.01 ,.01 ,.01 .27 .65 .38 .96 ,.01 ,.01 ,.01
Abbreviations: ADP, adenosine diphosphate; CAD, coronary artery diseases; CHF, congestive heart failure; CRP, C-reactive protein; HT, hypertension; NIHSS, National Institutes of Health Stroke Scale; WBC, white blood cell. P value was calculated by analysis of variance, c2 test, and Kruskal-Wallis test. *Post hoc by Scheffe and Bonferroni correction.
is plausible that ADP-induced platelet aggregation could reveal the status of atheroma burden in AF-related IS, especially because high CHA2DS2-VASc scores and ADP-induced platelet aggregation are independently associated with a poor outcome after AF-related IS. It has been suggested that the platelet activation reciprocally stimulates systemic atherosclerosis. Several previous studies have also shown that ADP-induced platelet aggregation is involved in ischemic damage after atherothrombosis,11,12 and we previously reported that unregulated ADP-induced platelet aggregation increased the rate of mortality in patients with AIS who were treated with aspirin only.17 Considering these findings, the enhanced ADP-induced platelet aggregation found in patients with high CHA2DS2-VASc scores could 120 r = 0.285 p< 0.001
100
ADP
80 60 40 20 0 0
1
2
3
4
5
6
7
Figure 2. Association between CHA2DS2-VASc scores and ADP-induced platelet aggregation values. ADP-induced platelet aggregation increased as CHA2DS2-VASc scores increased. There was a positive correlation between the CHA2DS2-VAS score and the value of ADP-induced platelet aggregation (r 5 .326, P , .01). Pearson correlation coefficients for correlations between CHA2DS2-VASc scores and the extent of ADP-induced platelet aggregation. Abbreviation: ADP, adenosine diphosphate.
potentiate the development of thrombus and then aggravate ischemic neuronal damage. Moreover, unregulated ADP-induced platelet aggregation might increase the risk of recurrent ischemic attack during the chronic phase of AF-related IS. Nevertheless, we were not able to clearly demonstrate this in the present study because our focus was on assessing functional outcome at 90 days post-IS, rather than study the detail of events, such as early neurologic deterioration and recurrent IS, after an AF-related IS. Our study is the first to show a relationship between the extent of platelet activation and CHA2DS2-VASc scores, which, in turn, indicates the degree of atheroma burden in AF-related IS. In addition, our findings suggest that platelet activation might have an important role in the progression of ischemic damage and in the coagulation mechanism in AF-related IS. Although we were able to demonstrate that there is enhanced platelet activation in patients with high CHA2DS2-VASc scores who experience AF-related IS, we were not able to show the need for combination treatment with an antiplatelet agent and warfarin in AFrelated IS. Antiplatelet agents can effectively reduce platelet activation, but previous studies have shown that antiplatelet agents in combination with warfarin increased hemorrhagic complications in IS.18,19 There have, thus far, not been any guidelines regarding the use of antiplatelet– warfarin combination therapy in AF-related IS, except in special cases.20 However, in the future, we will consider more specific therapeutic strategy including using antiplatelet agents on the top of warfarin in AF-related stroke showing higher platelet activation.
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Table 2. Comparison of clinical and laboratory findings between good and poor outcomes in atrial fibrillation–related ischemic stroke 90-d outcome
Sex Female (%) Male (%) Age (y) HT (%) CAD (%) Previous stroke (%) CHF (%) NIHSS WBC (3103/dL) Glucose (mg/dL) Cholesterol (mg/dL) CRP (mg/dL) Statin (%) CHA2DS2-VASc 0-2 (%) 3-4 (%) $5 (%) ADP (mean 6 SD) ADP (median IQR) Low #56 (%) Middle 57-71 (%) High $72 (%)
Good (n 5 142)
Poor (n 5 107)
Total
52 (36.6) 90 (63.4) 66.7 6 10.8 83 (58.5) 23 (16.2) 14 (9.9) 14 (9.9) 4.0 [2.0-9.0] 7.8 6 2.34 138.1 6 44.9 178.6 6 36.6 .5 6 1.0 90 (63.4) 3.0 [2.0-3.0] 64 (45.1) 63 (44.4) 15 (10.6) 59.4 6 19.6 62.0 [48.0-71.0] 56 (39.4) 52 (36.6) 34 (23.9)
47 (43.9) 60 (56.1) 73.3 6 9.8 73 (68.2) 25 (23.4) 28 (26.2) 18 (16.8) 15.0 [10.0-18.0] 8.6 6 2.9 143.0 6 51.7 179.0 6 37.1 .9 6 2.2 60 (56.1) 4.0 [3.0-5.0] 21 (19.6) 50 (46.7) 36 (33.6) 67.8 6 16.1 70.0 [59.0-78.0] 26 (24.3) 31 (29.0) 50 (46.7)
99 (39.8) 150 (60.2) 69.5 6 10.9 156 (62.7) 48 (19.3) 42 (16.9) 32 (12.9) 8.0 [3.0-15.0] 8.1 6 2.6 140.2 6 47.9 178.8 6 36.8 .7 6 1.7 150 (60.2) 3.0 [2.0-4.0] 85 (34.1) 113 (45.4) 51 (20.5) 63.0 6 18.6 65.0 [53.0-76.0] 82 (32.9) 83 (33.3) 84 (33.7)
P value
.24 ,.01 .12 .16 ,.01 .10 ,.01 .02 .42 .94 .07 .24 ,.01 ,.01 ,.01 ,.01 ,.01
Abbreviations: ADP, adenosine diphosphate; CAD, coronary artery diseases; CHF, congestive heart failure; CRP, C-reactive protein; HT, hypertension; NIHSS, National Institutes of Health Stroke Scale; WBC, white blood cell. Good: mRS score #2 at 90 d after ischemic event; poor: mRS $3 at 90 d after ischemic event. P values were calculated by c2 test, independent sample t test, Wilcoxon rank sum test between good and poor group.
Table 3. Univariate and multivariate logistic analyses of poor outcome occurrence Univariate
Multivariate
Variable
Reference level
OR (95% CI)
P value
Sex Age HT CAD Previous stroke CHF NIHSS WBC Glucose Cholesterol CRP Statin CHA2DS2-VASC score
M vs. F
.74 [.44-1.23] 1.07 [1.04-1.10] 1.53 [.90-2.58] 1.58 [.84-2.97] 3.24 [1.61-6.53] 1.85 [.87-3.91] 1.25 [1.18-1.32] 1.12 [1.02-1.24] 1.00 [1.00-1.01] 1.00 [.99-1.01] 1.22 [.98-1.52] .74 [.44-1.23] 2.42 [1.31-4.48] 7.31 [3.36-15.93] 1.28 [.68-2.44] 3.17 [1.67-5.99]
.24 ,.01 .12 .16 ,.01 .11 ,.01 .02 .42 .94 .08 .24 ,.01 ,.01 .45 ,.01
ADP
Y vs. N Y vs. N Y vs. N Y vs. N
Y vs. N 3-4 vs. 0-2 $5 vs. 0-2 Middle vs. low High vs. low
OR (95% CI)
P value
1.04 [1.00-1.09]
.07
1.60 [.59-4.34]
.35
1.27 [1.20-1.36] 1.14 [.99-1.31]
,.01 .07
1.11 [.91-1.35]
.31
1.54 [.58-4.08] 4.69 [1.21-18.14]
.39 .03
2.49 [1.17-5.27]
.02
Abbreviations: ADP, adenosine diphosphate; CAD, coronary artery diseases; CHF, congestive heart failure; CI, confidence interval; CRP, Creactive protein; F, female; HT, hypertension; M, male; N, no; NIHSS, National Institutes of Health Stroke Scale; OR, odds ratio; WBC, white blood cell; Y, yes.
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Our study had some limitations: first, this was not a particularly well-designed prospective study, and it was, therefore, difficult to ensure that all biases, which could have affected our data, were excluded. Second, our sample size was so small that the validity of our conclusions could not be confirmed. Finally, we were not able to obtain information regarding the use of antiplatelet agents before admission. However, we excluded any potential effect of antiplatelet use before admission by evaluating ADP-induced platelet aggregation 5 days after admission to hospital. Nevertheless, we cannot confirm that our data revealed the true status of platelet activation without any contamination of antiplatelet agents taken before admission. In conclusion, our results show the importance of platelet activation on the long-term outcomes after AFrelated IS in patients with high CHA2DS2-VASc scores. Acknowledgment: This study was supported by DongA University Research Fund.
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