International Science and Technology Publishing

Effects of puerarin on platelet activation markers in patients with acute cerebral infarction
Xinyan Meng^1,2, Yingfeng Mu² , Guofang Chen¹, Deqin Geng²
¹Department of Neurology, Xuzhou Center Hospital,Xuzhou 221009,China
²Department of Neurology, Affiliated Hospital of Xuzhou Medical University,Xuzhou 221009,China
Corresponding author: Deqin Geng, E-mail: gengdeqin@hotmail.com

In the treatment of acute cerebral infarction, many hospitals are using traditional Chinese medicine preparations, but there are different opinions on the treatment effects of these compounds. In this study, platelet activating-related markers in patients with acute cerebral infarction were detected by flow cytometry, showing that, compared with enteric-coated aspirin, puerarin combined with enteric-coated aspirin had better antiplatelet effects. Puerarin is a flavonoid glycoside extracted from the roots of leguminous plants Pueraria lobata or P. edulis. Animal tests have shown that puerarin can inhibit thrombin-induced serotonin release in platelets, which may be associated with inhibition of platelet activation and platelet-mononuclear aggregates. The level of hs-CRP in all patients was mildly decreased after treatment, but the difference was not statistically significant, suggesting that while inflammatory processes may be involved in the pathogenesis of cerebral infarction, neither enteric-coated aspirin nor puerarin could significantly alter the inflammatory process.

1. Materials and methods
1.1 Clinical Data
A total of 60 patients were enrolled in the study, including 31 males and 29 females, all at least 55 years old with an average age of 66 ± 4.2 years. Subjects were first-onset cerebral infarction patients admitted in our department from January 2014 to June 2015 and diagnosed with cerebral infarction according to the diagnostic criteria of the fourth national Cerebrovascular Disease Conference in 1995^〔¹^〕, with confirmation by CT or MRI. All patients were admitted within 24 h after symptom onset. The exclusion criteria included admission 24 h after onset, previous history of stroke or cardiovascular disease, administration of antiplatelet and anticoagulant medicine such as aspirin, clopidogrel or warfarin, and severe liver function abnormalities. The patients were randomly divided into treatment and control groups according to their hospitalization number (30 patients per group). There were no significant differences in sex or age between the two groups.

1.2 Research methods
Venous blood samples were collected from patients after admission and were subjected to blood biochemistry tests, routine blood tests and blood coagulation function tests, as well as examination for high sensitivity C-reactive protein (hs-CRP) and platelet activation markers. The blood was collected without tourniquet, with the first 2 ml of blood discarded. The blood samples were tested immediately after collection, with tests completed within one hour. The patients in the control group were treated with enteric-coated aspirin (Bayer production, Bayaspirin, 0.1 , t.i.d.), edaravone (IVGTT, 30 mg, b.i.d.) and butylphthalide (200 mg, t.i.d.) as the basic treatment, while the treatment group also received puerarin (Jiangsu Chia Tai Tianqing Pharmaceutical Co., Ltd, 0.5 g IVGTT, q.d.) in addition to the above medications. Patients with hypertension or diabetes were treated accordingly. Platelet activation markers, blood biochemistry, routine blood tests, blood coagulation function and hs-CRP were re-examined ten days later, with the results compared for differences.

1.3 Statistical analysis
Data analysis was carried out using the SPSS 16 statistical software package. Comparisons between treatment and control groups were performed using t-tests. Within-group comparisons before and after treatment were performed using paired t-tests. P<0.05 was considered statistically significant.

2. Results
2.1 Before treatment, there was no significant difference between the treatment and control group in the number of white blood cells, percentage of monocytes, platelet count, average platelet volume, platelet distribution width, coagulation function, platelet activation markers, blood glucose, total cholesterol, low density lipoprotein and hs-CRP. The results are listed in Table 1 and Table 2.

Table 1. Pre-treatment comparisons of hs-CRP, CD61, PAC-1, PMA, CD62p and CD42b between treatment and control groups.

group CD42b CD61 PAC-1 CD62p PMA hs-CRP

treatment group (n=30) 85.55±10.62 76.18±9.34 51.56±6.27 34.29±8.21 26.37±6.62 5.45±1.52
control group (n=30) 88.76±11.48 76.53±9.18 47.87±6.61 31.52±7.32 25.36±6.29 5.45±1.36

Table 2. Pre-treatment comparisons of total cholesterol, low density lipoprotein and platelets between treatment and control groups.

group blood glucose total cholesterol low density lipoprotein white blood cells platelet count

treatment group (n=30) 7.26±0.47 4.78±0.35 4.21±0.23 6.23±0.46 179.75±6.58
control group (n=30) 7.68±0.54 4.88±0.33 4.56±0.21 6.34±0.42 189.27±6.74

2.2 In both groups, there was no significant difference in the number of white blood cells, percentage of monocytes, platelet count, average platelet volume, platelet distribution width, blood biochemical parameters and coagulation function before and after treatment. After treatment, CD42b and CD61 in the control group were significantly decreased compared to pre-treatment values, while hs-CSP was nonsignificantly decreased; simultaneously, procaspase activating compound-1 (PAC-1), platelet-monocyte aggregates (PMA) and CD62p were significantly increased in the control group (Table 3).

Table 3. Changes in CD42b, CD61, PAC-1, CD62p, PMA and hs-CRP in the control group before and after treatment.

Time point CD42b CD61 PAC-1 CD62p PMA hs-CRP

pre-treatment (n=30) 88.69±12.21^* 75.32±8.84^* 47.46±5.921* 31.58±7.52^* 25.76±5.35^* 5.45±1.36
post-treatment (n=30) 62.74±8.54 55.55±6.26 58.77±6.78 49.62±6.12 43.67±7.95 5.38±1.40

note:*P<0.01

2.3 After treatment, CD42b, CD61, PAC-1, CD62p and PMA were all significantly decreased in the treatment group, while hs-CRP was nonsignificantly decreased (Table 4). CD42b and CD61 in both the control and treatment groups were significantly decreased compared to pre-treatment values, but there was no statistically significant difference between the two groups after treatment (Table 5).

Table 4. Changes in hs-CRP, PAC-1, CD61, PMA, CD62p and CD42b in the treatment group before and after treatment.

Time point CD42b CD61 PAC-1 CD62p PMA hs-CRP

pre-treatment (n=30) 85.55±10.62^* 76.18±9.34^* 51.56±6.27^* 34.29±8.21^* 26.37±6.62^* 5.45±1.52
post-treatment (n=30) 67.82±8.44 56.63±7.21 36.63±5.12 26.75±6.39 15.38±6.26 5.33±1.34

Table 5. Changes in CD61 and CD42b after treatment in the control and treatment groups.

Time point CD42b CD61

treatment group (n=30) 67.82±8.44 56.63±7.21
control group (n=30) 62.74±8.54 55.55±6.26

note:*P<0.01

3. Discussion
Cerebral infarction is a disease involving many genetic factors. The pathogenesis of the disease is very complicated, and activation of platelets plays a key role^[2].^[2] Platelet activation includes the attachment, accumulation and release of platelets. After platelet activation, granule and plasma membrane glycoproteins of platelets are significantly changed and can be detected as platelet markers. Glycoproteins that can be detected by flow cytometry include 1) PAC-1 (early stage platelet activation marker); 2) GPIb/IX, detected with antibody against CD42b, which is related to platelet adhesion; 3) platelet α-granule membrane protein (GMP-140), detected with antibody against CD62p, the end-stage marker of platelet activation; and 4) GPIIb/IIIa, detectable using antibodies against CD41/CD61, which are mainly associated with platelet aggregation. In addition, PMA was also measured.

Studies have shown that serum levels of PMA, CD61, CD42b, CD62p, PAC-1 and hs-CRP in acute cerebral infarction patients were significantly higher than in healthy control subjects, and that hs-CRP and PMA levels in cerebral infarction patients were positively correlated with each other, suggesting that the activation of platelets and the formation of platelet-monocyte aggregates may play an important role in the pathogenesis of acute cerebral infarction^〔³^〕. Our experimental results showed that after taking enteric-coated aspirin, CD42b and CD61 levels in cerebral infarction patients were significantly decreased, suggesting that the adhesion and aggregation of platelets were inhibited; in contrast, PMA, CD62P and PAC-1 levels did not fall but rather increased, indicating that enteric-coated aspirin had no inhibitory effects on platelet-mononuclear aggregate formation and platelet activation. These results are consistent with the results of animal experiments by Sharpe et al. ^〔⁴^〕, which may explain why enteric-coated aspirin is ineffective in some cerebral infarction patients. With additional administration of puerarin, PMA, PAC-1, CD42b and CD62p and CD61 levels were all decreased, suggesting that puerarin can inhibit the activation of platelets and formation of platelet-mononuclear aggregates, which may enhance the antiplatelet effect of enteric-coated aspirin when combined with puerarin, and further inhibit thrombosis progression.
The initiation process of platelet activation begins with vascular endothelial injury after cerebral arteriosclerosis, which leads to collagen exposure, subsequent platelet adhesion and the occurrence of aggregation after platelet adhesion. There are two types of aggregation, homologous and heterologous. Homologous aggregation is aggregation among platelets, while heterologous aggregation occurs between platelets and leukocytes and is closely associated with the development of cerebral infarction. After platelet activation, the expression of adhesion molecules on its surface increases and these adhesion molecules bind to corresponding receptors on the surface of activated white blood cells directly or through the bridging function of fibrinogen, forming platelet-leukocyte aggregates (PLA). PLA leads to an increase in the area of blood vessel obstruction and aggravation of neurological function, resulting in a worse outcome. The main mechanism for PLA promotion of thrombosis is the formation of microclots, which directly block small blood vessels. Another study found that PLA can release a variety of inflammatory mediators, cytokines and vasoactive substances to further shrink blood vessels and promote ischemia and hypoxia, resulting in a vicious cycle ^〔⁵^〕. Platelets can bind with monocytes to form PMA, bind with neutrophils resulting in platelet-neutrophil aggregation (PNA), or bind with lymphocytes to cause platelet-lymphocytic aggregation (PLyA). Previous studies ^〔⁶^〕have demonstrated that PMA was significantly increased in patients with cerebral infarction, while the PlyA and PNA level were not significantly changed; therefore, PMA was chosen as the testing index in our study.

Antiplatelet therapy is recommended by ischemic stroke treatment guidelines of many countries. As a classic antiplatelet drug, enteric-coated aspirin plays an irreplaceable role in treatment and prevention of ischemic stroke. However, the effects of aspirin as first or second preventive measures are not adequate in some patients. Our study showed that aspirin can only inhibit platelet adhesion and aggregation, and had no inhibitory effect on platelet activation and the formation of PMA, consistent with findings by Lukasik et al.^〔⁷^〕. These results indicate that enteric-coated aspirin antiplatelet therapy alone is neither comprehensive nor sufficient for acute cerebral infarction patients. However, when enteric-coated aspirin treatment was combined with puerarin, the adhesion, aggregation and activation of platelets, as well as formation of PMA were all significantly reduced. Thus, combined therapy of puerarin with enteric-coated aspirin may have improved antiplatelet effects, which can inhibit the progression of thrombosis.

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