Resistance to antiplatelet therapy, especially aspirin or clopidogrel, triggers other therapies for patients with coronary heart disease (CHD). Enhanced external counterpulsation (EECP) is a noninvasive, pneumatic technique that provides beneficial effects for patients with CHD. However, the physiological effects of EECP have not been fully studied, and the role of EECP on platelet function remains poorly understood.
Methods
A total of 168 patients with CHD were finally selected from the Second Xiangya Hospital and randomly assigned to either a control group or EECP group. The control group accepted only standard medical treatment, while the EECP group accepted standard medical treatment and EECP treatment. Blood samples were collected from patients at baseline and after EECP, and platelet aggregation was assessed. Changes in platelet aggregation were compared before and after treatment.
Results
There was no difference in the basal levels of arachidonic acid (AA) induced platelet maximum aggregation ratio (MAR) between the two groups. The AA-induced platelet MAR was significantly decreased after EECP therapy. The logistic analysis showed that low HDL-C was not favorable for the decrease in platelet aggregation.
Conclusion
EECP therapy is favorable for lowering platelet aggregation in patients with CHD, especially the AA-induced platelet aggregation ratio.
Hinweise
The original version of this article was revised: Originally, the article was published electronically on the publisher’s internet portal (currently SpringerLink) on 21 January 2021 with open access.
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Abkürzungen
CHD
Coronary heart disease
LDL-C
Low-density lipoprotein cholesterol
HDL-C
High-density lipoprotein cholesterol
MAR
Maximum aggregation ratio
BMI
Body mass index
ACS
Acute coronary syndromes
Introduction
Coronary heart disease (CHD) is a major cause of death worldwide. Our knowledge of platelets, especially activated platelets, which play a vital role in the pathophysiology of CHD, has increased substantially over the past 50 years. This increased understanding has identified various treatment strategies for CHD, especially acute coronary syndromes (ACS), by targeting key mediators of platelet activation and aggregation processes.
Aspirin is the first choice for antiplatelet therapy, and is also fundamental for the secondary prevention of CHD. Aspirin has been shown to reduce 34% of non-fatal myocardial infarction (MI) [1] and decrease the risk of new atherothrombotic events by 25–30% [2‐4]. However, due to the risk of side effects such as bleeding and gastrointestinal associated with aspirin, there is a need to identify new antiplatelet drugs. Clopidogrel has been used as another important antiplatelet therapy due to its strong antiplatelet effect and significantly lower gastrointestinal side effects compared with aspirin [5]. The Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA) study showed that the combination of aspirin and clopidogrel reduced cardiovascular risk by 6.9% [6]. A meta-analysis of 16 randomized controlled studies, including 17,000 high-risk patients with coronary heart disease, showed that aspirin combined with clopidogrel reduced major cardiovascular events by 20% [7]. Therefore, the current combination of aspirin and clopidogrel represents the basic treatment for patients with ACS [8]; however, with the expansion of clinical research and the extended follow-up time, not all patients have effective antiplatelet therapy. Previous reports have shown that even in patients taking aspirin, the cardiovascular risk remains as high as 24% [9]. There are also statistics suggesting that ischemic events still occur in approximately 15% of high-risk patients, despite the use of dual antiplatelet drugs [10]. Therefore, patients exhibit low reactivity or even resistance to antiplatelet therapy consisting of aspirin and/or clopidogrel. Aspirin or clopidogrel resistance indicates that in patients undergoing aspirin and/or clopidogrel treatment, the platelet function measured by the laboratory has not reached the target value after treatment [11, 12]. Thus, testing a patient’s platelet function can help understand the patients’ responsiveness to aspirin and/or clopidogrel treatment. Typically, only the ability of platelets to form thrombi is tested, (i.e., the function of platelet aggregation), since the primary clinical purpose of using antiplatelet drugs is to prevent thrombosis. By detecting the function of platelet aggregation, patients with ineffective, insufficient, or excessive antiplatelet drugs can be identified in time, and the efficacy of antiplatelet drugs can be monitored and objectively evaluated to guide the necessary adjustment of clinical medications, which can help ensure that the treatment of CHD achieves favorable results while avoiding or reducing potential side effects.
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Enhanced external counterpulsation (EECP) is a noninvasive physical therapy device. Its main function is to increase venous blood return, enhance the preload and output of the heart, and reduce systemic resistance [13]. During EECP, the patient’s calf, thigh, and buttocks are tied with airbags, and the diastole and systole of the heart are sensed by the electrocardiogram. The inflatable airbag is inflated from the calf, thigh, to the buttocks in the early stage of cardiac cycle. The air is then immediately deflated before the next contraction of the heart, thereby reducing the work of the heart by decreasing the resistance of the surrounding blood vessels [14]. EECP is widely used for the treatment and prevention of cardiovascular diseases, especially CHD. There is substantial evidence-based research for the use of EECP in the treatment of CHD. The first randomized, placebo-controlled, double-blind trial to study EECP for stable angina was the multi-center study of enhanced external counterpulsation (MUST-EECP) study, which began in 1995 [15]. The study showed that the symptom of chest pain episodes and use of nitrate medications were significantly reduced in patients following EECP treatment, and there was also an increase in exercise tolerance. More importantly, the benefit was found to last for more than one year in 70% of patients. As early as 2002, the American College of Cardiology/American Heart Association (ACC/AHA) incorporated EECP into the clinical treatment guidelines for CHD. In 2006, the European Society of Cardiology (ESC) and the Chinese Medical Association also included EECP in the clinical treatment guidelines for CHD.
At present, research into the mechanism by which EECP benefiting patients has primarily focused on its effect on hemodynamics, which has been proven to affect platelet activity. However, it remains unclear whether EECP has an effect on platelet aggregation in the circulation. Our goal is to investigate the effects of EECP on platelet aggregation in patients with CHD by measuring the platelet aggregation function before and after EECP treatment, and help explore the underlying mechanism of EECP to benefit cardiovascular disease.
Methods
Subjects
All participants who were diagnosed with clinical documented CHD through clinical presentation, ischemic electrocardiographic abnormalities, and coronary angiography findings that showed ≥50% stenosis in at least one main coronary artery with no recent angina attacks, admitted to our hospital between 2018 and 2019 were enrolled. Patients were required to have been in a clinical condition for at least 6 months. All patients provided informed consent. The exclusion criteria for this study were as follows: (a) blood pressure > 180/100 mmHg; (b) uncontrolled arrhythmia (e.g., atrial fibrillation, atrial flutter, and frequent ventricular premature beats); (c) severe valvular lesions; (d) hemorrhagic disease, or bleeding tendency, abnormal platelet count (< 100 × 109/L or > 300 × 109/L), or INR > 2.0; (e) various congenital heart diseases or heart valve diseases; (f) patients with severe heart failure, especially those with grade III and IV and left ventricular ejection fraction <30%; (g) peripheral vascular diseases (e.g., arterial occlusive disease of the extremities, thrombophlebitis, and venous thrombosis); (h) significant pulmonary hypertension; (i) arterial dissection or aneurysm; (j) infection; (k) pregnancy. The study was approved by the Medical Ethics Committee of the Second Xiangya Hospital of Central South University.
Study Design
A total of 168 subjects were enrolled. All participants were randomly placed into two groups: (1) the control group (88 patients); and (2) EEPC treatment group (80 patients). All patients received essential medication for CHD, which included aspirin (81 mg per day) and/or clopidogrel (75 mg per day), β-receptor blockers, angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB), and statins. Patients in the control group only received essential medication each day. No digoxin or diuretics was given to patients. However, in addition to receiving the same basic medical treatment as the control group, the patients in the EECP group received a typical course of EECP treatment, which consists of 35 sessions, provided 1 h daily, 5 days per week for 7 weeks. In diastole, the cuffs were inflated to 260 mmHg and the timing of the inflation was individually adjusted according to the electrocardiogram to provide the highest diastolic/systolic blood pressure ratio during treatment. The P-ECP/TI EECP equipment from PSK (Chongqing, China) was used. All of the EECP treatment sessions were carried out in the hospital under professional guidance.
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Clinical and Biochemical Measurements
Patient’s clinical characteristics were recorded, including gender, age, body mass index (BMI), as well as history of hypertension, diabetes, and smoking. The anthropometric measurements (weight, height, and BMI) were assessed after overnight fasting for at least 10 h. Antecubital vein blood samples were collected for monitoring in all subjects at baseline and at the end of seven weeks in the early morning during a fasting state. The blood platelet, plasma high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglyceride concentrations were measured using a Hitachi 7170A analyzer (Tokyo, Japan) by a specialist at our hospital who was unaware of the study allocation. Plasma vWF was determined by ELISA kit (Shanghai, China) according to the manufacturer’s protocol. Serum sample aliquots were stored in a freezer at −80 °C.
Measurement of Platelet Aggregation Function
A PL-11 platelet function analyzer (SINNOWA Medical Science & Technology Co., Nanjing, China) was used for platelet aggregation testing as previously described [16]. Platelet aggregation was induced by arachidonic acid (AA) and adenosine diphosphate (ADP). PL-11 counts single platelets and platelet aggregation is measured as the loss of single platelets. It contains an automated impedance-based hematology analyzer and agonist kits. The whole procedure was automatically done after transferring 500 µl citrated blood sample into a polycarbonate tube and inserting it into the detecting position. Blood sample in the polycarbonate tube was mixed gently during the whole testing process. Platelet count was detected in duplicate at the start and the mean value of platelet count was set as the baseline. There was a short interval between each test point for system cleaning. When 25 µl AA (0.31 mM, final concentration) or ADP (4uM, final concentration) was automatically trickled into blood sample after second detecting time. The single platelet counting dropped when aggregates formed became too large to be counted as single platelets. PL-11 counted platelet several times till it detected the lowest level. The system calculated the maximal platelet aggregation ratio according to the following formula:
Blood samples were stored at room temperature before being tested. The entire procedure was required to be performed within 1 h after sampling.
Statistical Analysis
Data were analyzed using Statistical Package for Social Sciences version 21.0. Plots were created using GraphPad Prism version 6.0 (GraphPad Software, Inc., La Jolla, California). Comparisons between categorical data were performed with x2 tests. We analyzed whether the data collected confirmed to a normal distribution. The mean ± standard deviation (SD) was calculated for data with a normal distribution, and the median was calculated for data with a non-normal distribution and were compared by non-parametric tests. The baseline levels of the two groups were compared using an independent t-test, while comparisons of the continuous variables before and after EECP treatment were assessed using a paired t-test. Logistic regression was used to analyze the gender, age, triglyceride, HDL-C, and LDL-C that affected the changes of AA-induced platelet aggregation of EECP. The changes of AA-induced MAR after EECP treatment is defined as dependent variable (“1” for decrease, “0” for the opposite), and gender (“1” for male, “0” for female), age, triglyceride, HDL-C, and LDL-C are defined as independent variables. Statistical significance was assumed at P < 0.05.
Results
Participant Characteristics at Baseline
All 168 participants completed the study; no subject died or dropped out due to severe side effects during the entire study. The baseline characteristics of the patients are shown in Table 1. There was no significant difference regarding gender, age, BMI, or history of hypertension, diabetes, or smoking between the two groups. Similarly, the levels of LDL-C, HDL-C, triglyceride, total cholesterol (TC), and platelets were also comparable between the groups at baseline. For individual, cardiac management medication remains consistent throughout the study.
Table 1
Baseline characteristics of study participants
Baseline characteristics
Control group (n = 88)
EECP group (n = 80)
P value
Gender (male/female)
65/23
60/20
0.87
Age (years)
60.7 ± 10.9
61.0 ± 10.3
0.89
BMI (kg/m2)
25.0 ± 2.5
24.1 ± 2.6
0.29
Hypertention (n, %)
61 (69.3)
52 (65)
0.55
Diabetes mellitus (n, %)
24 (27.3)
23 (28.7)
0.83
Current smoker (n, %)
43 (48.9)
44 (55)
0.43
LDL-C (mmol/L)
2.52 ± 0.91
2.28 ± 0.89
0.09
HDL-C (mmol/L)
1.07 ± 0.25
1.10 ± 025
0.40
Triglyceride (mmol/L)
1.75 ± 0.88
1.71 ± 0.91
0.77
TC (mmol/L)
4.03 ± 1.06
3.80 ± 1.02
0.16
Aspirin used (n, %)
88 (100)
80(100)
1
Clopidogrel used (n, %)
61 (69.3)
51 (63.8)
0.08
Statin used (n, %)
88 (100)
80 (100)
1
β-blocker used (n, %)
67 (76.1)
63 (78.7)
0.69
ACEI/ARB used (n, %)
51 (58)
58 (72.5)
0.07
BMI, body mass index; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; TC, total cholesterol; ACEI, angiotensin-converting enzyme inhibitors; ARB, angiotensin receptor blockers
Efficacy Changes after EECP Treatment
A total of 12 out of 88 patients in the control group and 22 out of 80 patients in the EECP treatment group had an improvement of one CCS angina class (P = 0.02) (Table 2). The NT-proBNP in EECP group was significantly lower after EECP performance. However, there was no changes of the EF before and after treatment. There were 35 out of 88 patients in the control group and 20 out of 80 patients in the EECP treatment group having angina symptom recurrent (P = 0.04) during 5.2 months follow-up (3–12 months). However, no one suffered from myocardial infarction, death, or readmission to hospital due to cardiovascular disease.
Table 2
Characteristics before and after treatment
Characteristic
Control group
EECP group
Baseline
7 weeks
Baseline
7 weeks
AA-induced MAR (%)
All patients
31.6 ± 15.1
28.8 ± 13.0
34.7 ± 13.9
26.2 ± 9.7*
Patients with aspirin only
33.8 ± 14.3
31.2 ± 15.3
34.8 ± 11.5
24.9 ± 7.2*
Patients with both aspirin and clopidogrel
30.6 ± 12.9
27.8 ± 9.7
34.7 ± 15.2
26.9 ± 10.9*
ADP-induced MAR (%)
64.3 ± 11.5
62.3 ± 11.4
61.2 ± 13.6
60.1 ± 12.3
EF (%)
54.7 ± 8.4
54.8 ± 8.1
55.2 ± 9.1
55.3 ± 9.0
NT-proBNP (pg/ml)
303.0 ± 83.1
307.6 ± 87.0
292.1 ± 79.8
214.3 ± 91.5*
BP (mmHg)
112.6 ± 11.3
113.1 ± 10.9
113.4 ± 12.1
112.5 ± 12.0
CCS angina class (n)
Ι
0
3
0
10
ΙΙ
63
69
60
62
ΙΙΙ
25
16
20
8
IV
0
0
0
0
Platelet (×109/L)
205.5 ± 55.2
206.9 ± 53.4
207.6 ± 54.9
208.7 ± 58.4
vWF (%)
151 ± 55
146 ± 57
159 ± 49
137 ± 40*
*means P < 0.05
Maximal Platelet Aggregation Ratio before and after EECP
There was no significant difference between the AA- (31.6 ± 15.1 vs. 34.7 ± 13.9) and ADP- (64.3 ± 11.5 vs. 61.2 ± 13.6) induced MAR at baseline between the two groups. After EECP treatment, the AA-induced MAR in the EECP group was significantly decreased (34.7 ± 13.9 vs. 26.2 ± 9.7), regardless of whether clopidogrel was used. There was no change of ADP-induced MAR (Table 2).
Factors Affecting the Change in Platelet Aggregation Function by EECP
Our data shows that not all patients in the EECP group had a decreased platelet MAR after performing EECP (Fig. 1). Therefore, we hypothesize that there may be other factors that impact the efficiency of EECP on platelet aggregation. A logistic regression analysis was used to determine these factors. The results show that low HDL-C is not conducive to the reduction of platelet aggregation (P = 0.039), while gender, age, TG, and LDL-C had no significant effect on the change of the platelet MAR (Table 3).
Fig. 1
The AA-induced platelet maximum aggregation ratio change in EECP group before and after EECP treatment
Table 3
The result of logistic regression analysis
Characteristic
Label
Estimatestd
Error
Exp
P
Age
X1
0.67
0.63
1.95
0.29
Gender
X2
−0.32
0.026
0.97
0.22
LDL-C
X3
−0.13
0.34
0.88
0.70
HDL-C
X4
2.29
1.11
9.96
0.039
TG
X5
−0.366
0.28
0.69
0.19
×
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Discussion
Our results demonstrate that in the EECP group, the AA-induced platelet aggregation ratio was significantly decreased after a combination of basic medication and EECP treatment, which suggests that EECP therapy could lower the platelet aggregation ratio.
There’s no change of platelet count before and after EECP treatment, which indicates that EECP lowering AA-induced platelet aggregation is not through affecting the number of platelet. Although the mechanism of how EECP lowers platelet aggregation ratio in patients with CHD is unknown, there are several hypotheses regarding how EECP benefits the cardiovascular system. One theory is that it may be related to the effect of EECP on endothelial function. Endothelial dysfunction will expose the underlying endothelial tissue to the blood, causing increased platelet activation. Studies have shown that EECP can improve endothelial function in patients with symptomatic coronary artery disease [17]. Our result showed that vWF levels were lower after EECP treatment (159 ± 49 vs. 137 ± 40), which can also indicate that EECP may decrease platelet activity by improving endothelial function.
The effect on inflammation may be another mechanism by which EECP reduces the platelet aggregation ratio. During an inflammatory response, there is an increased permeability of the blood vessels and a variety of activated substances are emitted from the surface of the blood vessel lumen. These activated substances can bind to receptors on the surface of platelets, causing platelet deformation and activation. Studies have shown that EECP therapy can increase blood flow shear stress, which reduces proinflammatory biomarkers (e.g., monocyte chemoattractant protein) and reduces signal transduction, as well as the expression of adhesion molecules in circulation [18].
It has been well-established that high blood flow shear stress can cause enhanced platelet activation [19, 20], which is contradictory to the findings of our study; however, perhaps this is dependent on how the EECP device works. The EECP balloon is inflated during diastole, which results in increased circulating shear stress. However, because it deflates immediately before the heart contracts, the platelets are not exposed to conditions of such high shear stress for long periods of time. In addition, Rubenstein et al. [21] found that short-term exposure to high shear stress did not cause an increase in platelet activity or thrombogenic capacity. This indicates that high shear stress alone may not activate platelets, but the length of time that platelets are exposed to a high shear stress environment can affect platelet activation. Therefore, the time during which EECP increases the shear stress is too short to induce platelet activation, but it is sufficient to improve endothelial function and lower inflammatory factors.
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Although the overall results show that EECP reduces platelet MAR in patients with CHD, not all patients undergoing EECP exhibit a decreased AA-induced platelet MAR. Therefore, it is speculated that there may be other factors that affect the reduction in the platelet aggregation ratio. The logistic analysis shows that a low HDL-C is an unfavorable factor for reducing AA-induced platelet MAR. In addition, Jastrzebska et al. showed that dyslipidemia could affect the therapeutic efficacy of aspirin [22]. By analyzing the blood lipids in the aspirin-resistant and the aspirin-sensitive patients, the authors found that the plasma HDL-C in patients in the aspirin-resistance group was significantly lower than that in the aspirin-sensitive group. Similarly, other researchers have also found that patients with higher HDL-C levels respond better to aspirin. These findings are consistent with our results, which indicates that there is a better efficiency of lowering the platelet aggregation ratio in response to EECP therapy in patients with higher HDL-C levels. HDL-C may also represent a novel therapeutic target for regulating the platelet aggregation ratio.
The results showed that EECP treatment improved CCS angina classes, and decreased angina attack and NT-proBNP. However, the EF did not change. It may be due to the relatively short EECP treatment duration (7 weeks). In addition, the EF in most patients was normal, which made it more difficult to get a huge improvement in a short time.
We acknowledge that there are several limitations associated with this study. First, since the sample size is small, the data would be more convincing if the sample size was enlarged. Second, it would be better if more data such as FcγRIIa, PLCγ2 phosphorylation, which play a role in platelet activation [23], can be measured. Last, it would be better to present the difference in platelet aggregation according the treatment duration such as one week, two, …seven weeks. It would be helpful to appreciate the improvement of EECP treatment.
Conclusion
In conclusion, the results of this study suggest that EECP therapy is favorable for lowering platelet activation in patients with CHD, especially the AA-induced platelet aggregation ratio. EECP may be considered as a means of decreasing platelet aggregation in patients suffering recurrent angina attack while they are receiving anti-angina medication treatment. We also found that low HDL-C levels are not conducive to reducing the AA-induced platelet aggregation mediated by EECP. These findings suggest that HDL-C may represent a novel therapeutic target for regulating the platelet aggregation ratio.
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Acknowledgements
We thank all patients who participated in this study, Xiaoxiu Wang for platelet aggregation ratio measurement, and the Department of Clinical Laboratory, Second Xiangya Hospital, for their assistance.
Availability of Data and Material
Some restrictions will apply.
Compliance with Ethical Standards
Declarations
Not applicable.
Conflict of Interest
The authors declare no conflict of interest.
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