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Relation between platelet microaggregates and ankle brachial index in patients with peripheral arterial disease
Thrombosis Research (2006) 117, 263 — 269
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REGULAR ARTICLE
Relation between platelet microaggregates and ankle brachial index in patients with peripheral arterial disease Takashi Kudoha, Tomohiro Sakamotoa,*, Shinzo Miyamotoa, Kunihiko Matsuib, Sunao Kojimaa, Seigo Sugiyamaa, Michihiro Yoshimuraa, Yukio Ozakic, Hisao Ogawaa a
Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan b Department of General Medicine, Kumamoto University Hospital, Kumamoto, Japan c Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Nakakoma, Japan Received 29 November 2004; received in revised form 17 March 2005; accepted 18 March 2005 Available online 17 May 2005
KEYWORDS Peripheral arterial disease; Platelet aggregation; Ankle brachial index
Abstract Introduction: Peripheral arterial disease is one of the systemic atherosclerotic diseases, and patients with the disorder are classified in the high risk group of coronary artery disease. A lower ankle brachial index is a frequent finding in peripheral arterial disease. While platelet microaggregates are a significant predictor of adverse clinical outcome in coronary artery disease, the significance of platelet aggregability in peripheral arterial disease has not been elucidated. Materials and methods: Small platelet aggregates measured using laser-light scattering and ankle brachial index were determined in 42 patients with both coronary artery disease and peripheral arterial disease (peripheral group), 56 patients with only coronary artery disease (coronary group) and 32 patients without both (control group). Results: The level of small platelet aggregates was increased significantly in the peripheral group (4.3 104 [range 2.2 104 to 7.4 104]) compared with both the coronary (1.1 104 [range 0.3 104 to 5.0 104]) and control groups (0.5 104 [range 0.1 104 to 0.9 104]). There was a significant inverse correlation between log small platelet aggregates and ankle brachial index (n = 130, r = 0.422,
Abbreviations: PAD, peripheral arterial disease; CAD, coronary artery disease; ABI, ankle brachial index; SPA, small platelet aggregates; AU, arbitrary units; ADP, adenosine diphosphate. * Corresponding author. Tel.: +81 96 373 5175; fax: +81 96 362 3256. E-mail address: [email protected] (T. Sakamoto). 0049-3848/$ - see front matter D 2005 Published by Elsevier Ltd. doi:10.1016/j.thromres.2005.03.011
264
T. Kudoh et al. p b 0.001). Multivariate logistic regression analysis revealed that a lower ankle brachial index (b 0.90) was an independent determinant of increased levels of small platelet aggregates. Conclusions: Platelet aggregability was increased in patients with peripheral arterial disease with the degree of platelet aggregation being closely associated with ankle brachial index. It is possible that this change in platelet activity may be one mechanism to explain why a lower ankle brachial index is a predictor of poor prognosis in patients with peripheral arterial disease. D 2005 Published by Elsevier Ltd.
Peripheral arterial disease (PAD) is most commonly a manifestation of atherosclerosis in the lower limbs distal to the aortic bifurcation. Previous studies have shown that the presence of PAD is a strong indicator of systemic atherosclerosis [1], and there is evidence that patients with PAD have an increased risk of cardiovascular morbidity and mortality, particularly that of coronary artery disease (CAD), compared with patients without PAD [2—7]. It has been shown that the prevalence of CAD in patients with PAD is at least 40% [8—10]. Platelet aggregation is a useful biological marker for predicting coronary events and mortality in survivors of myocardial infarction [11]. However, platelet aggregation measured by conventional methods such as light transmission has the limitation of relatively low sensitivity for detecting the initial aggregation process, that is characterized by the formation of small platelet aggregates [12,13]. In contrast, a platelet aggregometer using laserlight scattering provide precise quantitative evaluation of both the size and number of platelet aggregates [14]. We reported recently that high levels of platelet microaggregates correlated with adverse clinical outcome in patients with CAD [15]. It is therefore likely that patients with CAD with increased numbers of small platelet aggregates (SPA) may represent a patient population with more advanced systemic atherosclerosis. Several investigators have reported a relationship between blood coagulation and fibrinolytic parameters in patients with PAD [16—20] and there are also a few reports on platelet activation in PAD [21,22]. However, comparison of platelet aggregability in PAD with other atherosclerotic vascular diseases has not been studied extensively. Despite CAD and PAD both being classified clinically as atherosclerotic disorders, CAD patients do not always have PAD. This suggests that patients with CAD complicated with PAD have more advanced atherosclerotic characteristics compared with patients with CAD alone. We hypothesized that platelet hyperaggregability was observed in patients with CAD complicated with
PAD and platelet aggregability increased in parallel with severity of the disease. Therefore, the primarily, we compared platelet aggregability in patients with different severity of vascular disease, categorized as either the presence or absence of CAD and/ or PAD. In addition, the relationship between ankle brachial index (ABI), an index of PAD severity, and platelet aggregability was also examined.
Patients in a clinical study and methods Study population Patients who complained of chest pain on admission to our institution were enrolled prospectively in the study. To determine the required number of patients, we performed power calculation based on our previous data. This study’s target enrollment was approximately 120 patients. After written informed consent had been obtained all the patients (91 males and 39 females; mean age 71 F 1 years, range 53—85) underwent diagnostic cardiac catheterization to confirm the diagnosis of CAD. CAD was defined as the presence of organic stenosis N 70% in at least one major coronary artery. As shown in Fig. 1, the patients were divided into three groups with the first group containing patients with an ABI b 0.90 in at least one leg regardless of the presence of CAD (PAD group), the second group containing patients with CAD whose ABI z 0.90 (CAD group) and the third group containing the remaining patients who did not have either CAD or PAD (control group). Enrollment was continued prospectively until each group was matched for age and gender. At the end of enrollment the groups had the following characteristics: PAD group (n = 42); CAD group (n = 56); and control group (n = 32). The entire PAD group and 51 (91%) of the CAD group were administered oral aspirin (100 mg/day). No patients were taking antiplatelet drugs other than aspirin. The protocol of the study was approved by the ethics committee of our institution.
Platelet aggregation in PAD
265
Measurement of ABI
Patients with chest pain, n=130
Cardiac catheterization and ABI check
Presence of PAD (ABI < 0.90)
YES
PAD group, n=42 (39 with CAD, 3 without CAD)
NO YES Presence of CAD
CAD group, n=56
These measurements were carried out using an autonomic waveform analyzer, from PWV/ABI (Colin, Co., Ltd., Komaki, Japan) that is able to record bilateral ABI, electrocardiography and heart sounds simultaneously [26]. The patient was examined in the supine position, with the electrocardiogram electrodes placed on both wrists, a microphone for detecting heart sounds placed on the left edge of the sternum, and pressure cuffs applied around both the brachia and ankles. In all patients, ABI was measured after at least 5 min rest on a bed.
NO
Statistical analysis Control group, n=32
Figure 1 A flow diagram of the enrollment procedures and selection of the study groups.
Blood sampling Blood samplings for the measurement of platelet aggregation were obtained from the antecubital vein in the morning. With the patients in the recumbent position, a 21-gauge needle was inserted into the vein and the blood was collected into two tubes, the initial tube was used for the clinical chemistry analysis. The second tube containing 3.8% sodium citrate anticoagulant was used for the determination of platelet aggregation. The samples for platelet aggregation measurement were allowed to stand for 15 min at room temperature and were then centrifuged at 150 g for 10 min at room temperature to obtain platelet-rich plasma. The remaining samples were centrifuged at 300 g for 10 min at room temperature to obtain platelet-poor plasma.
Measurement of platelet aggregation Platelet aggregation was measured by a PA-200 aggregometer (Kowa Co. Ltd., Tokyo, Japan), which determines the size and number of platelet aggregates using laser-light scattering. The validity of this method has been confirmed by our previous studies and also by other investigations [14,15,23—25] that assessed platelet aggregation in blood samples in the presence of 1.0 AM adenosine diphosphate (ADP). ADP was added to the platelet-rich plasma 60 s after the start of measurement with platelet aggregation being evaluated as the maximum value of light intensity induced by ADP.
All data are expressed as mean F S.D. The comparison of continuous data among the three groups was performed using a one-way analysis of variance. When a statistically significant difference was detected the data were then analyzed by a post hoc analysis, the Scheffe’s F test. The SPA data did not distribute normally in the present study. Therefore, the Kruskal—Wallis test with Tukey—Kramer test after log transformation was used to evaluate pairwise mean SPA differences among three groups. We used an overall significance level of a = 0.05 for all pairwise comparisons of the mean potencies data. The Spearman’s rank correlation test was used to evaluate the relationship between log SPA and ABI. The cutoff point between high and low levels of platelet aggregation was set at the 90th percentile of the distribution of the SPA (1.96 104 AU) in the control subjects. To evaluate whether ABI levels were an independent risk factor for the difference between patients with either high or low levels of SPA, a multivariate logistic regression analysis was performed using the following factors as categorical covariates: current smoking (individuals who smoke any tobacco product at least once a day, including those who smoke everyday and who smoke any tobacco product, but not everyday: according to World Health Organization’s standard definitions of smoking), hypertension (blood pressure level z 140/90 mm Hg or requiring antihypertensive medication), diabetes mellitus (according to World Health Organization criteria), hypercholesterolemia (serum total cholesterol level N 220 mg/dl or the use of lipid-lowering medications). The frequency data were compared using the v 2 test. A p value b 0.05 was considered as statistically significant. The statistical analyses were carried out using personal computer-based software, StatView (ver.5.0 for Windows).
266
T. Kudoh et al. p<0.0001
Results p<0.0001
The clinical characteristics of the study population are summarized in Table 1. There were no differences between the three groups in the serum levels of total cholesterol, HDL cholesterol, LDL cholesterol, or triglyceride. The frequency of diabetes mellitus and smoking was significantly higher in the PAD group than in the other 2 groups ( p b 0.05). The number of coronary risk factors was comparable in the PAD and CAD groups, but was lower in the control group. The majority of the PAD group (93%) had coronary artery disease. The severity of the PAD was classified by the Fontaine classification (stage I: 12 patients, II: 25 patients, III: 5 patients).
11
Platelet aggregates among the three groups Fig. 2 shows the level of log SPA in the three groups. The level of log SPA was increased significantly in the PAD group (10.67 [range 10.01 to 11.19]) compared with the CAD (9.25 [range 8.07 to 10.80]) and control groups (8.40 [range 7.14 to 9.06]) ( p b 0.0001 for comparison between any two mean value). Log platelet aggregates measured using the optical
Table 1
Log Small Platelet Aggregates
Patients characteristics
12
p<0.0001
10 9 8 7 6 PAD (n=42)
CAD (n=56)
Control (n=32)
Figure 2 Comparison of small platelet aggregates in the three groups. The mean level of log SPA was higher in the PAD group than in the CAD group, that in turn was higher than in the control group.
density method was also increased significantly in the PAD (2.63 [range 2.25 to 3.21]) and CAD groups (2.46 [range 1.96 to 2.84]) compared with the control group (1.95 [1.59 range to 2.36]) ( p b 0.0001). However, the difference between the PAD and CAD groups was not significant.
Clinical characteristics of the study groups
Variables
PAD group (n = 42)
CAD group (n = 56)
Control group (n = 32)
Age (years) Sex males/females Hypertension Diabetes mellitus Obesity (BMI N 25 kg/m2) Smoking Ankle brachial index HbA1c (%) Total-cholesterol (mg/dl) HDL-cholesterol (mg/dl) LDL-cholesterol (mg/dl) Triglyceride (mg/dl) Number of risk factors SPA (AU) (median [IQR])
72 F 7 31/11 35 (83)y 26 (62)**,y 5 (12)y 20 (48)**,y 0.68 F 0.20**,# 6.4 F 1.4*,# 199 F 48 48 F 13 131 F 40 132 F 72 2.6 F 1.0# 4.3 104 (2.2 104—7.4 104)
71 F 7 37/19 44 (79)y 18 (32) 11 (20) 5 (9) 1.09 F 0.09 5.8 F 0.8 196 F 39 49 F 10 132 F 35 135 F 48 2.2 F 1.2 1.1 104 (0.3 104—5.0 104)
70 F 7 23/9 17 (53) 4 (12) 9 (28) 7 (21) 1.12 F 0.07 5.6 F 0.8 177 F 35 52 F 15 112 F 27 103 F 48 1.6 F 1.4 0.5 104 0.1 104—0.9 104)
3 (7) 10 (24)# 13 (31)# 16 (38)#
0 (0) 17 (30)# 23 (41)# 16 (29)#
32 (100) 0 0 0
Coronary angiographic data 0-vessel 1-vessel 2-vessel 3-vessel
p b 0.05 vs. CAD, **p b 0.01 vs. CAD, #p b 0.01 vs. Control, yp b 0.05 vs. Control. Data are expressed as mean value F S.D. or number (%) of subjects. Results of SPA levels are expressed as the median value (25th to 75th percentile range). BMI=body mass index, BP=blood pressure, HbA1c=hemoglobin A1c, HDL=high-density lipoprotein, LDL=low-density lipoprotein, PAD=peripheral arterial disease, CAD=coronary artery disease, SPA=small platelet aggregates, IQR=interquartile range.
Platelet aggregation in PAD
267 atherosclerotic vascular disease as evidence from an epidemiological study showed that a high percentage of patients with PAD have atherosclerosis in systemic vascular beds other than lower extremities [27]. This implies that atherosclerotic state may be more advanced in patients with both PAD and CAD compared with patients with CAD alone who have more localized atherosclerosis. In our study, small platelet aggregability appeared to be higher in patients with combined PAD and CAD, or in those with more advanced atherosclerosis. This finding suggests that SPA may be an index of systemic progression of atherosclerosis. In general, the severity of PAD is evaluated by measuring ABI [28] with this procedure being almost equal to angiography for detecting PAD [29]. In the present study, we demonstrated an inverse correlation between SPA and ABI, a finding that implied SPA may provide a quantitative index for the severity of PAD. In this regard, more increased SPA would be produced in cases of more advanced PAD (Fig. 3). In contrast, we found no significant correlation between large platelet aggregates and ABI. Formation of platelet aggregates induced by physiological concentrations of ADP occurs in two phases, namely primary and secondary aggregation [30]. While there is a close relationship between SPA and primary aggregates [23], large platelet aggregates are generated after the formation of SPA and are therefore equivalent to secondary aggregation. The process of forming large platelet aggregates from SPA may be modified by several factors such as neurohormonal factors, whereas SPA are formed primarily and therefore correlate directly with the severity of atherosclerosis as measured by lower levels of ABI. Taken together, our findings indicate that large platelet aggregates may be a blunt and indirect index of atherosclerosis and we speculate
Table 2 Multivariate logistic regression analysis: variables differentiating between patients with high and low levels of SPA Variables
Odds ratio
95% CI
p value
Ankle brachial index b 0.90 Diabetes mellitus Hypercholesterolemia Hypertension Smoking
5.09 0.64 1.19 0.51 0.72
1.97—13.14 0.26—1.53 0.53—2.66 0.21—1.26 0.28—1.85
b 0.001 0.312 0.672 0.145 0.495
In multivariate logistic regression analysis, lower levels of ABI were the independent variables of higher SPA (90th percentile of the distribution of the platelet aggregate levels in the control subjects).
Correlation of small platelet aggregates and ABI The left-hand panel of Fig. 3 shows the relationship between log SPA and ABI in the combined data of the subjects. Although there was a significant inverse correlation between log SPA and ABI (r = 0.422, p b 0.001), there was no significant correlation between log large platelet aggregates and ABI (Fig. 3, right-hand panel). Multivariate logistic regression analysis showed a significant association between high levels of SPA and lower levels of ABI (b 0.90). Therefore, lower levels of ABI were found to be independent determinants of high levels of SPA (Table 2).
Discussion This study demonstrated that the mean number of SPA was significantly higher in patients with both PAD and CAD than in patients with CAD alone or patients without either of these disorders. PAD is a clinical manifestation of diffuse
Log Large Platelet Aggregates
Log Small Platelet Aggregates
13.0
11.0
9.0
7.0
p< 0.0001 r= -0.42 n= 130
12.0 10.0
8.0 6.0
p= N.S. r= -0.17 n= 130
4.0
5.0 0.0
0.2
0.4
0.6
0.8
ABI
1.0
1.2
1.4
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
ABI
Figure 3 Correlation between SPA and ABI in the combined data of the three study groups. There was a significant inverse correlation between log SPA and ABI (r = 0.422, p b 0.001, left-hand panel), whereas there was no significant correlation between log large platelet aggregates and ABI (right-hand panel).
268 this may be the reason why only SPA correlated with ABI. Previous studies have shown that the severity of PAD is closely associated with the risk of myocardial infarction, ischemic stroke, and death from vascular causes [31—33]. As ABI becomes lower, the risk of cardiovascular events increases [9,31]. In our study we found a close relationship between SPA and ABI, with lower levels of ABI being a significant and independent variable that differentiated between patients with high or low levels of SPA. In a previous study we showed high levels of platelet microaggregates correlated with adverse clinical outcome in patients with CAD [15] and therefore increased formation of SPA may be one of the mechanisms responsible for poor prognosis in patients with more advanced PAD. There is considerable evidence that antiplatelet agents reduce the risk of nonfatal myocardial infarction, stroke, and death from vascular causes [34]. Among the range of antiplatelet agents, the use of aspirin has been reported to modify the prognosis of patients with PAD by decreasing the incidence of associated cardiac and cerebrovascular events [35]. However, in the present study, SPA remained at high levels in spite of treatment with aspirin in the patients with PAD. Other investigators have also reported that aspirin did not inhibit the formation of SPA effectively [24], and therefore it appears that the use of aspirin alone may be insufficient in the management of patients with PAD. More potent antiplatelet agents than aspirin or combination therapy of these agents with aspirin may be necessary in order to achieve a better prognosis in patients with PAD.
Study limitation The present study was a cross-sectional investigation and was limited by a relatively small number of patients. Therefore, there may have been some inherent limitations such as selection bias between cases and controls. Accordingly, a further study in a larger cohort of patients is necessary in order to clarify the usefulness and significance of SPA measurements in patients with atherosclerotic disorders.
Conclusions Small platelet aggregation was increased in patients with PAD compared to patients without the disorder. The finding of a significant inverse correlation between small platelet aggregability and ABI indicated that the measurement of platelet
T. Kudoh et al. aggregability in patients with PAD may be a useful index for assessing the severity of the disease.
Acknowledgments This study was supported in part by a Research Grant for Cardiovascular Disease (14A-1, 14C-4) from the Ministry of Health and Welfare, a grant-inaid for scientific research (B(2)15390248 and C(2)14570680) from the Ministry of Education, Science, and Culture in Japan and a Smoking Research Foundation grant for Biomedical Research, Tokyo, Japan.
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269 [24] Tohgi H, Takahashi H, Watanabe K, Kuki H, Shirasawa Y. Development of large platelet aggregates from small aggregates as determined by laser-light scattering: effects of aggregant concentration and antiplatelet medication. Thromb Haemost 1996;75:838 – 43. [25] Sakamoto T, Ogawa H, Kawano H, Hirai N, Miyamoto S, Takazoe K, et al. Rapid change of platelet aggregability in acute hyperglycemia. Detection by a novel laser-light scattering method. Thromb Haemost 2000;83:475 – 9. [26] Yamashina A, Tomiyama H, Takeda K, Tsuda H, Arai T, Hirose K, et al. Validity, reproducibility, and clinical significance of noninvasive brachial-ankle pulse wave velocity measurement. Hypertens Res 2002;25:359 – 64. [27] Weitz JI, Byrne J, Clagett GP, Farkouh ME, Porter JM, Sackett DL, et al. Diagnosis and treatment of chronic arterial insufficiency of the lower extremities: a critical review. Circulation 1996;94:3026 – 49. [28] Raines JK, Darling RC, Buth J, Brewster DC, Austen WG. Vascular laboratory criteria for the management of peripheral vascular disease of the lower extremities. Surgery 1976;79:21 – 9. [29] Bernstein E, Fronek A. Current status of noninvasive tests in the diagnosis of peripheral arterial disease. Surg Clin North Am 1982;62:473 – 87. [30] Elwood PC, Renaud S, Sharp DS, Beswick AD, O’Brien JR, Yarnell JW. Ischemic heart disease and platelet aggregation. The Caerphilly collaborative heart disease study. Circulation 1991;83:38 – 44. [31] McKenna M, Wolfson S, Kuller L. The ratio of ankle and arm arterial pressure as an independent predictor of mortality. Atherosclerosis 1991;87:119 – 28. [32] Murabito JM, Evans JC, Larson MG, Nieto K, Levy D, Wilson PW. The ankle-brachial index in the elderly and risk of stroke, coronary disease, and death: the Framingham study. Arch Intern Med 2003;163:1939 – 42. [33] Papamichael CM, Lekakis JP, Stamatelopoulos KS, Papaioannou TG, Alevizaki MK, Cimponeriu AT, et al. Ankle-brachial index as a predictor of the extent of coronary atherosclerosis and cardiovascular events in patients with coronary artery disease. Am J Cardiol 2000;86:615 – 8. [34] Collaborative overview of randomised trials of antiplatelet therapy-II: maintenance of vascular graft or arterial patency by antiplatelet therapy. Antiplatelet trialists’ collaboration. BMJ 1994;308:159 – 68. [35] Jackson MR, Clagett GP. Antithrombotic therapy in peripheral arterial occlusive disease. Chest 2001;119(1 Suppl): 283S – 99S.
intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
Relation between platelet microaggregates and ankle brachial index in patients with peripheral arterial disease Takashi Kudoha, Tomohiro Sakamotoa,*, Shinzo Miyamotoa, Kunihiko Matsuib, Sunao Kojimaa, Seigo Sugiyamaa, Michihiro Yoshimuraa, Yukio Ozakic, Hisao Ogawaa a
Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan b Department of General Medicine, Kumamoto University Hospital, Kumamoto, Japan c Department of Clinical and Laboratory Medicine, Yamanashi Medical University, Nakakoma, Japan Received 29 November 2004; received in revised form 17 March 2005; accepted 18 March 2005 Available online 17 May 2005
KEYWORDS Peripheral arterial disease; Platelet aggregation; Ankle brachial index
Abstract Introduction: Peripheral arterial disease is one of the systemic atherosclerotic diseases, and patients with the disorder are classified in the high risk group of coronary artery disease. A lower ankle brachial index is a frequent finding in peripheral arterial disease. While platelet microaggregates are a significant predictor of adverse clinical outcome in coronary artery disease, the significance of platelet aggregability in peripheral arterial disease has not been elucidated. Materials and methods: Small platelet aggregates measured using laser-light scattering and ankle brachial index were determined in 42 patients with both coronary artery disease and peripheral arterial disease (peripheral group), 56 patients with only coronary artery disease (coronary group) and 32 patients without both (control group). Results: The level of small platelet aggregates was increased significantly in the peripheral group (4.3 104 [range 2.2 104 to 7.4 104]) compared with both the coronary (1.1 104 [range 0.3 104 to 5.0 104]) and control groups (0.5 104 [range 0.1 104 to 0.9 104]). There was a significant inverse correlation between log small platelet aggregates and ankle brachial index (n = 130, r = 0.422,
Abbreviations: PAD, peripheral arterial disease; CAD, coronary artery disease; ABI, ankle brachial index; SPA, small platelet aggregates; AU, arbitrary units; ADP, adenosine diphosphate. * Corresponding author. Tel.: +81 96 373 5175; fax: +81 96 362 3256. E-mail address: [email protected] (T. Sakamoto). 0049-3848/$ - see front matter D 2005 Published by Elsevier Ltd. doi:10.1016/j.thromres.2005.03.011
264
T. Kudoh et al. p b 0.001). Multivariate logistic regression analysis revealed that a lower ankle brachial index (b 0.90) was an independent determinant of increased levels of small platelet aggregates. Conclusions: Platelet aggregability was increased in patients with peripheral arterial disease with the degree of platelet aggregation being closely associated with ankle brachial index. It is possible that this change in platelet activity may be one mechanism to explain why a lower ankle brachial index is a predictor of poor prognosis in patients with peripheral arterial disease. D 2005 Published by Elsevier Ltd.
Peripheral arterial disease (PAD) is most commonly a manifestation of atherosclerosis in the lower limbs distal to the aortic bifurcation. Previous studies have shown that the presence of PAD is a strong indicator of systemic atherosclerosis [1], and there is evidence that patients with PAD have an increased risk of cardiovascular morbidity and mortality, particularly that of coronary artery disease (CAD), compared with patients without PAD [2—7]. It has been shown that the prevalence of CAD in patients with PAD is at least 40% [8—10]. Platelet aggregation is a useful biological marker for predicting coronary events and mortality in survivors of myocardial infarction [11]. However, platelet aggregation measured by conventional methods such as light transmission has the limitation of relatively low sensitivity for detecting the initial aggregation process, that is characterized by the formation of small platelet aggregates [12,13]. In contrast, a platelet aggregometer using laserlight scattering provide precise quantitative evaluation of both the size and number of platelet aggregates [14]. We reported recently that high levels of platelet microaggregates correlated with adverse clinical outcome in patients with CAD [15]. It is therefore likely that patients with CAD with increased numbers of small platelet aggregates (SPA) may represent a patient population with more advanced systemic atherosclerosis. Several investigators have reported a relationship between blood coagulation and fibrinolytic parameters in patients with PAD [16—20] and there are also a few reports on platelet activation in PAD [21,22]. However, comparison of platelet aggregability in PAD with other atherosclerotic vascular diseases has not been studied extensively. Despite CAD and PAD both being classified clinically as atherosclerotic disorders, CAD patients do not always have PAD. This suggests that patients with CAD complicated with PAD have more advanced atherosclerotic characteristics compared with patients with CAD alone. We hypothesized that platelet hyperaggregability was observed in patients with CAD complicated with
PAD and platelet aggregability increased in parallel with severity of the disease. Therefore, the primarily, we compared platelet aggregability in patients with different severity of vascular disease, categorized as either the presence or absence of CAD and/ or PAD. In addition, the relationship between ankle brachial index (ABI), an index of PAD severity, and platelet aggregability was also examined.
Patients in a clinical study and methods Study population Patients who complained of chest pain on admission to our institution were enrolled prospectively in the study. To determine the required number of patients, we performed power calculation based on our previous data. This study’s target enrollment was approximately 120 patients. After written informed consent had been obtained all the patients (91 males and 39 females; mean age 71 F 1 years, range 53—85) underwent diagnostic cardiac catheterization to confirm the diagnosis of CAD. CAD was defined as the presence of organic stenosis N 70% in at least one major coronary artery. As shown in Fig. 1, the patients were divided into three groups with the first group containing patients with an ABI b 0.90 in at least one leg regardless of the presence of CAD (PAD group), the second group containing patients with CAD whose ABI z 0.90 (CAD group) and the third group containing the remaining patients who did not have either CAD or PAD (control group). Enrollment was continued prospectively until each group was matched for age and gender. At the end of enrollment the groups had the following characteristics: PAD group (n = 42); CAD group (n = 56); and control group (n = 32). The entire PAD group and 51 (91%) of the CAD group were administered oral aspirin (100 mg/day). No patients were taking antiplatelet drugs other than aspirin. The protocol of the study was approved by the ethics committee of our institution.
Platelet aggregation in PAD
265
Measurement of ABI
Patients with chest pain, n=130
Cardiac catheterization and ABI check
Presence of PAD (ABI < 0.90)
YES
PAD group, n=42 (39 with CAD, 3 without CAD)
NO YES Presence of CAD
CAD group, n=56
These measurements were carried out using an autonomic waveform analyzer, from PWV/ABI (Colin, Co., Ltd., Komaki, Japan) that is able to record bilateral ABI, electrocardiography and heart sounds simultaneously [26]. The patient was examined in the supine position, with the electrocardiogram electrodes placed on both wrists, a microphone for detecting heart sounds placed on the left edge of the sternum, and pressure cuffs applied around both the brachia and ankles. In all patients, ABI was measured after at least 5 min rest on a bed.
NO
Statistical analysis Control group, n=32
Figure 1 A flow diagram of the enrollment procedures and selection of the study groups.
Blood sampling Blood samplings for the measurement of platelet aggregation were obtained from the antecubital vein in the morning. With the patients in the recumbent position, a 21-gauge needle was inserted into the vein and the blood was collected into two tubes, the initial tube was used for the clinical chemistry analysis. The second tube containing 3.8% sodium citrate anticoagulant was used for the determination of platelet aggregation. The samples for platelet aggregation measurement were allowed to stand for 15 min at room temperature and were then centrifuged at 150 g for 10 min at room temperature to obtain platelet-rich plasma. The remaining samples were centrifuged at 300 g for 10 min at room temperature to obtain platelet-poor plasma.
Measurement of platelet aggregation Platelet aggregation was measured by a PA-200 aggregometer (Kowa Co. Ltd., Tokyo, Japan), which determines the size and number of platelet aggregates using laser-light scattering. The validity of this method has been confirmed by our previous studies and also by other investigations [14,15,23—25] that assessed platelet aggregation in blood samples in the presence of 1.0 AM adenosine diphosphate (ADP). ADP was added to the platelet-rich plasma 60 s after the start of measurement with platelet aggregation being evaluated as the maximum value of light intensity induced by ADP.
All data are expressed as mean F S.D. The comparison of continuous data among the three groups was performed using a one-way analysis of variance. When a statistically significant difference was detected the data were then analyzed by a post hoc analysis, the Scheffe’s F test. The SPA data did not distribute normally in the present study. Therefore, the Kruskal—Wallis test with Tukey—Kramer test after log transformation was used to evaluate pairwise mean SPA differences among three groups. We used an overall significance level of a = 0.05 for all pairwise comparisons of the mean potencies data. The Spearman’s rank correlation test was used to evaluate the relationship between log SPA and ABI. The cutoff point between high and low levels of platelet aggregation was set at the 90th percentile of the distribution of the SPA (1.96 104 AU) in the control subjects. To evaluate whether ABI levels were an independent risk factor for the difference between patients with either high or low levels of SPA, a multivariate logistic regression analysis was performed using the following factors as categorical covariates: current smoking (individuals who smoke any tobacco product at least once a day, including those who smoke everyday and who smoke any tobacco product, but not everyday: according to World Health Organization’s standard definitions of smoking), hypertension (blood pressure level z 140/90 mm Hg or requiring antihypertensive medication), diabetes mellitus (according to World Health Organization criteria), hypercholesterolemia (serum total cholesterol level N 220 mg/dl or the use of lipid-lowering medications). The frequency data were compared using the v 2 test. A p value b 0.05 was considered as statistically significant. The statistical analyses were carried out using personal computer-based software, StatView (ver.5.0 for Windows).
266
T. Kudoh et al. p<0.0001
Results p<0.0001
The clinical characteristics of the study population are summarized in Table 1. There were no differences between the three groups in the serum levels of total cholesterol, HDL cholesterol, LDL cholesterol, or triglyceride. The frequency of diabetes mellitus and smoking was significantly higher in the PAD group than in the other 2 groups ( p b 0.05). The number of coronary risk factors was comparable in the PAD and CAD groups, but was lower in the control group. The majority of the PAD group (93%) had coronary artery disease. The severity of the PAD was classified by the Fontaine classification (stage I: 12 patients, II: 25 patients, III: 5 patients).
11
Platelet aggregates among the three groups Fig. 2 shows the level of log SPA in the three groups. The level of log SPA was increased significantly in the PAD group (10.67 [range 10.01 to 11.19]) compared with the CAD (9.25 [range 8.07 to 10.80]) and control groups (8.40 [range 7.14 to 9.06]) ( p b 0.0001 for comparison between any two mean value). Log platelet aggregates measured using the optical
Table 1
Log Small Platelet Aggregates
Patients characteristics
12
p<0.0001
10 9 8 7 6 PAD (n=42)
CAD (n=56)
Control (n=32)
Figure 2 Comparison of small platelet aggregates in the three groups. The mean level of log SPA was higher in the PAD group than in the CAD group, that in turn was higher than in the control group.
density method was also increased significantly in the PAD (2.63 [range 2.25 to 3.21]) and CAD groups (2.46 [range 1.96 to 2.84]) compared with the control group (1.95 [1.59 range to 2.36]) ( p b 0.0001). However, the difference between the PAD and CAD groups was not significant.
Clinical characteristics of the study groups
Variables
PAD group (n = 42)
CAD group (n = 56)
Control group (n = 32)
Age (years) Sex males/females Hypertension Diabetes mellitus Obesity (BMI N 25 kg/m2) Smoking Ankle brachial index HbA1c (%) Total-cholesterol (mg/dl) HDL-cholesterol (mg/dl) LDL-cholesterol (mg/dl) Triglyceride (mg/dl) Number of risk factors SPA (AU) (median [IQR])
72 F 7 31/11 35 (83)y 26 (62)**,y 5 (12)y 20 (48)**,y 0.68 F 0.20**,# 6.4 F 1.4*,# 199 F 48 48 F 13 131 F 40 132 F 72 2.6 F 1.0# 4.3 104 (2.2 104—7.4 104)
71 F 7 37/19 44 (79)y 18 (32) 11 (20) 5 (9) 1.09 F 0.09 5.8 F 0.8 196 F 39 49 F 10 132 F 35 135 F 48 2.2 F 1.2 1.1 104 (0.3 104—5.0 104)
70 F 7 23/9 17 (53) 4 (12) 9 (28) 7 (21) 1.12 F 0.07 5.6 F 0.8 177 F 35 52 F 15 112 F 27 103 F 48 1.6 F 1.4 0.5 104 0.1 104—0.9 104)
3 (7) 10 (24)# 13 (31)# 16 (38)#
0 (0) 17 (30)# 23 (41)# 16 (29)#
32 (100) 0 0 0
Coronary angiographic data 0-vessel 1-vessel 2-vessel 3-vessel
p b 0.05 vs. CAD, **p b 0.01 vs. CAD, #p b 0.01 vs. Control, yp b 0.05 vs. Control. Data are expressed as mean value F S.D. or number (%) of subjects. Results of SPA levels are expressed as the median value (25th to 75th percentile range). BMI=body mass index, BP=blood pressure, HbA1c=hemoglobin A1c, HDL=high-density lipoprotein, LDL=low-density lipoprotein, PAD=peripheral arterial disease, CAD=coronary artery disease, SPA=small platelet aggregates, IQR=interquartile range.
Platelet aggregation in PAD
267 atherosclerotic vascular disease as evidence from an epidemiological study showed that a high percentage of patients with PAD have atherosclerosis in systemic vascular beds other than lower extremities [27]. This implies that atherosclerotic state may be more advanced in patients with both PAD and CAD compared with patients with CAD alone who have more localized atherosclerosis. In our study, small platelet aggregability appeared to be higher in patients with combined PAD and CAD, or in those with more advanced atherosclerosis. This finding suggests that SPA may be an index of systemic progression of atherosclerosis. In general, the severity of PAD is evaluated by measuring ABI [28] with this procedure being almost equal to angiography for detecting PAD [29]. In the present study, we demonstrated an inverse correlation between SPA and ABI, a finding that implied SPA may provide a quantitative index for the severity of PAD. In this regard, more increased SPA would be produced in cases of more advanced PAD (Fig. 3). In contrast, we found no significant correlation between large platelet aggregates and ABI. Formation of platelet aggregates induced by physiological concentrations of ADP occurs in two phases, namely primary and secondary aggregation [30]. While there is a close relationship between SPA and primary aggregates [23], large platelet aggregates are generated after the formation of SPA and are therefore equivalent to secondary aggregation. The process of forming large platelet aggregates from SPA may be modified by several factors such as neurohormonal factors, whereas SPA are formed primarily and therefore correlate directly with the severity of atherosclerosis as measured by lower levels of ABI. Taken together, our findings indicate that large platelet aggregates may be a blunt and indirect index of atherosclerosis and we speculate
Table 2 Multivariate logistic regression analysis: variables differentiating between patients with high and low levels of SPA Variables
Odds ratio
95% CI
p value
Ankle brachial index b 0.90 Diabetes mellitus Hypercholesterolemia Hypertension Smoking
5.09 0.64 1.19 0.51 0.72
1.97—13.14 0.26—1.53 0.53—2.66 0.21—1.26 0.28—1.85
b 0.001 0.312 0.672 0.145 0.495
In multivariate logistic regression analysis, lower levels of ABI were the independent variables of higher SPA (90th percentile of the distribution of the platelet aggregate levels in the control subjects).
Correlation of small platelet aggregates and ABI The left-hand panel of Fig. 3 shows the relationship between log SPA and ABI in the combined data of the subjects. Although there was a significant inverse correlation between log SPA and ABI (r = 0.422, p b 0.001), there was no significant correlation between log large platelet aggregates and ABI (Fig. 3, right-hand panel). Multivariate logistic regression analysis showed a significant association between high levels of SPA and lower levels of ABI (b 0.90). Therefore, lower levels of ABI were found to be independent determinants of high levels of SPA (Table 2).
Discussion This study demonstrated that the mean number of SPA was significantly higher in patients with both PAD and CAD than in patients with CAD alone or patients without either of these disorders. PAD is a clinical manifestation of diffuse
Log Large Platelet Aggregates
Log Small Platelet Aggregates
13.0
11.0
9.0
7.0
p< 0.0001 r= -0.42 n= 130
12.0 10.0
8.0 6.0
p= N.S. r= -0.17 n= 130
4.0
5.0 0.0
0.2
0.4
0.6
0.8
ABI
1.0
1.2
1.4
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
ABI
Figure 3 Correlation between SPA and ABI in the combined data of the three study groups. There was a significant inverse correlation between log SPA and ABI (r = 0.422, p b 0.001, left-hand panel), whereas there was no significant correlation between log large platelet aggregates and ABI (right-hand panel).
268 this may be the reason why only SPA correlated with ABI. Previous studies have shown that the severity of PAD is closely associated with the risk of myocardial infarction, ischemic stroke, and death from vascular causes [31—33]. As ABI becomes lower, the risk of cardiovascular events increases [9,31]. In our study we found a close relationship between SPA and ABI, with lower levels of ABI being a significant and independent variable that differentiated between patients with high or low levels of SPA. In a previous study we showed high levels of platelet microaggregates correlated with adverse clinical outcome in patients with CAD [15] and therefore increased formation of SPA may be one of the mechanisms responsible for poor prognosis in patients with more advanced PAD. There is considerable evidence that antiplatelet agents reduce the risk of nonfatal myocardial infarction, stroke, and death from vascular causes [34]. Among the range of antiplatelet agents, the use of aspirin has been reported to modify the prognosis of patients with PAD by decreasing the incidence of associated cardiac and cerebrovascular events [35]. However, in the present study, SPA remained at high levels in spite of treatment with aspirin in the patients with PAD. Other investigators have also reported that aspirin did not inhibit the formation of SPA effectively [24], and therefore it appears that the use of aspirin alone may be insufficient in the management of patients with PAD. More potent antiplatelet agents than aspirin or combination therapy of these agents with aspirin may be necessary in order to achieve a better prognosis in patients with PAD.
Study limitation The present study was a cross-sectional investigation and was limited by a relatively small number of patients. Therefore, there may have been some inherent limitations such as selection bias between cases and controls. Accordingly, a further study in a larger cohort of patients is necessary in order to clarify the usefulness and significance of SPA measurements in patients with atherosclerotic disorders.
Conclusions Small platelet aggregation was increased in patients with PAD compared to patients without the disorder. The finding of a significant inverse correlation between small platelet aggregability and ABI indicated that the measurement of platelet
T. Kudoh et al. aggregability in patients with PAD may be a useful index for assessing the severity of the disease.
Acknowledgments This study was supported in part by a Research Grant for Cardiovascular Disease (14A-1, 14C-4) from the Ministry of Health and Welfare, a grant-inaid for scientific research (B(2)15390248 and C(2)14570680) from the Ministry of Education, Science, and Culture in Japan and a Smoking Research Foundation grant for Biomedical Research, Tokyo, Japan.
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