| Coronary heart disease (CHD) remains a major global public health problem. Platelets play an important role in hemostasis but are also responsible for the formation of pathogenic thrombi underlying acute clinical manifestations of vascular atherothrombotic disease. Multiple pathways, including adenosine diphosphate (ADP), thromboxane A2(TXA2), and thrombin are capable of activating platelets. Deregulated platelet activation can lead to the formation of platelet-rich thrombi that occlude the arterial lumen and are capable of causing ischemia and cardiovascular events. Oral antiplatelet drugs are a milestone in the therapy of cardiovascular atherothrombotic diseases. The efficacy of antiplatelet drugs, such as aspirin and clopidogrel, in decreasing the risk of adverse events in CHD patients has been well studied in the past20years. Despite oral antiplatelet therapy, a number of adverse CHD events continue to occur. In recent years, many reports have shown a possible relationship between residual platelet activity, as measured by a variety of laboratory tests, and clinical outcomes; raising the possibility that "resistance" to oral antiplatelet drugs may underlie many such adverse events. These phenomena suggest that other pathways capable of stimulating platelet activation may exist. It is therefore meaningful to identify new therapeutic targets for anti-platelet therapy for CHD.Using differential proteomics in platelets, our previous data demonstrate that CHD, BSS (blood-stasis syndrome) and platelet activation have close relationship. We also study the correlation between BSS/non-BSS and platelet function proteins in CHD by the differential proteomics of platelet, and find that platelet gelsolin is the main differential protein of platelet between them, which indicates that platelet cytoskeleton may play an important role in the developing of BSS in CHD.During tissue injury and cell death, actin is released into the circulation where it can interact with components of the haemostatic and fibrinolytic systems, or polymerize and form F-actin, which has fatal toxic action for vascular endothelial cell and micrangium, it can plug smaller vessels and decrease blood flow to promote the formation of blood clots. Gelsolin, together with DBP (vitamin D binding protein), another extracellular actin-binding protein, functions as an "extracellular actin scavenger system"(EASS) responsible for the clearing of much more actin released by tissue injury and hold back the lengthen of F-actin. By severing F-actin, gelsolin reduces blood viscosity by promoting polymer disassembly. We speculate that abnormal increased platelet gelsolin as a result of reactive restruction of platelet cytoskeleton because of the vast depletion of plasma gelsolin in the development of BSS in CHD. These modifications promote the platelet activation and transformation, which involve in the progress of BSS in CHD.This study attempt to confirm the scientific hypothesis which has close relationship between gesolin in the PRP (Plasma rich platelet)and PPP(Plasma poor platelet)and platelet activation level, meanwhile, study on the role of platelet gelsolin in the development of BSS in CHD and intervention mechanism of effective components of Chuanxiong rhizome and Red peony root(XiongShao Capsule, XSC) by building the experimental system of platelet activation in Vivo and in Vitro which provide evidence of anti-platelet therapy of Chinese herbs for promoting blood circulation and removing blood stasis.This study is divided into three parts:literature review, clinical research and experimental research.1. Literature reviews:Including the following two reviews:Gelsolin and Cardiovascular disease, and Chinese Herb and Formulas of promoting blood circulation and removing blood stasis and antiplatelet therapies.2. Clinical researches:Including the following two parts.Study I:Identification of platelet Gelsolin in CHD patients with BSSObjective:To identify the platelet differential protein gelsolin in CHD patients with BSS by ELISA (enzyme-linked immuno sorbent assay) and WB (western blotting).Methods:All cases were divided into CHD patients with BSS, CHD patients with non-BSS and the healthy control,30in each group. CHD patients were diagnosed according to the1999ACC/AHA/ACP-ASIM Guidelines for the Management of Patients with Chronic Stable Angina and the2002ACC/AHA Guidelines for the Management of Patients with Unstable Angina and Non-ST-Segment Elevation Myocardial Infarction. A diameter stenosis of at least50%was diagnosed by visible estimation in a major coronary artery from standard selective coronary angiography. Patients with BSS were diagnosed according to guidelines for the diagnosis of BSS of Chinese medicine. Gelsolin in PRP and PPP was determined in blood samples taken following fasting from CHD patients with BSS, CHD patients with non-BSS and the healthy control by ELISA and WB respectively.Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels, medical therapy between CHD patients with BSS and CHD patients with non-BSS.Compared with the control group, gelsolin in PRP of the BSS group increased significantly (P<0.01),while that in PPP of the BSS and non-BSS groups decreased markedly (P<0.05)and gelsolin in PRP of non-BSS groups has no statistic deference (P>0.05).Compared with the non-BSS groups, gelsolin in PRP of the BSS groups increased significantly(P<0.05),and gelsolin in PPP has no statistic deference (P>0.05).Conclusion:Gelsolin, as the important component of cytoskeletal protein, is distributing in PRP and PPP differentially in the processing of BSS in CHD, which has consistent with the previous results of platelet proteomic.Study II Correlation between platelet gelsolin and BSS in CHD.Objective:To determine the correlation between platelet gelsolin and BSS in CHD.Methods:All cases were divided into CHD patients with BSS and CHD patients with non-BSS,57in each group and45healthy individuals were selected as the healthy control. Gelsolin concentration in PRP and PPP, F-actin and DBP of PPP were determined by ELISA. The fluorescence intensity of CD62p and cytoplasmic calcium ([Ca2+]i) in human platelets of patients and healthy people was measured with Flow cytometry.Results:There were no statistically significant differences of age, sex and body mass index among CHD patients with BSS, CHD patients with non-BSS and the healthy control. Additionally, there were no significant differences of subtypes of CHD, degree of coronary lesion vessels and medical therapy between CHD patients with BSS and CHD patients with non-BSS.Compared with the control group, gelsolin in PRP of the BSS group increased significantly (P<0.01),while that in PPP of the BSS and non-BSS groups decreased markedly (P<0.05), the CD62p,[Ca2+]i of platelet, F-actin and DBP of the BSS and non-BSS groups increased significantly (P<0.01). Compared with the non-BSS group, the gelsolin concentration in PRP and [Ca+]i of platelet of BSS group increased significantly (P<0.01), the CD62p expression of the BSS group increased markedly (P <0.05), F-actin and DBP of the BSS group have no statistic deference (P>0.05).Abnormally increased platelet gelsolin levels of BSS in CHD show high positive correlation with the level of platelet activity (P<0.01)Conclusion:Platelet gelsolin and platelet activation level of BSS in CHD have high positive correlation. During the platelet activation of BSS in CHD, cytoskeletal protein contents have different variation, which focus on platelet gelsolin and F-actin. We speculate that abnormal increased platelet gelsolin as a result of reactive restruction of platelet cytoskeleton because of the vast depletion of plasma gelsolin or increased calcium influx of platelet in the development of BSS in CHD. These modifications promote the platelet activation and transformation, which involve in the progress of BSS in CHD. So platelet gelsolin may as a new potential biomarker and/or therapeutic target of BSS in CHD.3Experimental researches:Including the following two parts.Study I Effect of Paeoniflorin and Ligustrazine phosphate on the F-actin-induced platelet activation level and platelet gelsolin in vitro.Objective:To investigate the effect of Paeoniflorin and Ligustrazine phosphate on the F-actin-induced platelet activation level and platelet gelsolin in vitro.Methods:Taken washing platelets of15healthy volunteers as the investigated subjects, and F-actin of different concentration as the platelet aggregation/activation inducers while AA, ADP and Thrombin as the positive control inducers. Meanwhile, we take combination of Paeoniflorin and Ligustrazine phosphate as the intervention drugs while aspirin as the positive control drug. The platelet CD62p expression is detected by FCM (flow cytometry), platelet aggregation rate is detected by platelet aggregation analyzer, and the platelet gelsolin is detected by ELISA.Results:①Compare with control group, F-actin of different concentrations, AA, ADP and Thrombin groups all can induce platelet aggregation and activation in vitro markedly (P<0.05or P<0.01),and concentration-dependent of F-actin. F-actin of lOμmol/L has the similar induced platelet aggregation/activation with AA,ADP and Thrombin at the same concentration (P>0.05)②Compare with the control group, F-actin of2.5μmol/L,ADP and Thrombin have no marked difference on platelet gelsolin level (P>0.05),while F-actin of5μmol/L and10μmol/L and AA can increase platelet gelsolin significantly (P<0.05), and F-actin of10μmol/L has the similar effect on the platelet gelsolin level with AA at the same concentration (P>0.05)③Paeoniflorin and ligustrazine phosphate can inhibit F-actin and AA induced platelet aggregation/activation in vitro (P<0.05),which have the similar effect with aspirin (P>0.05).Pretreatment of paeoniflorin and ligustrazine phosphate on the washing platelet can reduce the platelet gelsolin level of after F-acin and AA-induced platelet aggregation/activation in vitro(P<0.05),but pretreatment of aspirin has no similar effect (P>0.05)Conclusion:F-actin can induce platelet aggregation/activation in vitro, and high concentration of F-actin can increase the platelet gelsolin level of activated platelet, which has the similar effect with AA. Paeoniflorin and ligustrazine phosphate can inhibit platelet aggregation/activation in vitro and also reduce the platelet gelsolin level of activated platelet. But aspirin has no such effect in vitro.Study II Effect of Xiongshao Capsule on the platelet activation level and platelet gelsolin in rat model of Acute Myocardial infarction in vivo.Objective:To investigate the effects of active ingredients of Chuanxiong rhizome and red peony root (Chuangxiongol and paeoniflorin) on the platelet activation level and platelet gelsolin in rat model of acute myocardial infarction in vivo.Methods:The rat model of AMI is prepared by ligation of left anterior descending (LAD) artery. Taken Xiongshao Capsule (XSC) as the intervention drug, while aspirin and verapamil (calcium antagonist) as the positive control drugs. Got PRP and PPP by density gradient centrifugation, then gelsolin, F-actin, DBP, P-selection, CK-MB and cTnI were detected by ELISA, the [Ca2+]i of Platelet was detected by FCM, gelsolin expression of infracted myocardium was detected by WB.Results:①Compare with sham group, the concentration of CK-MB and cTnI of model group increased significantly after ligation of LAD for3hours(P<0.05),and P-selection level increased markedly (P<0.05).High dose of XSC can reduce CK-MB, cTnI, P-selection, COX-1and TXB2level markedly (P<0.05),which has similar effect with aspirin in vivo.②Compare with the sham group, the plasma gelsolin, DBP of Model group reduced significantly (P<0.05) while platelet gelsolin increased markedly (P<0.05).High dose of XSC can reduce platelet gelsolin and F-actin (P<0.05),while increased plasma gelsolin and DBP significantly (P<0.05).Platelet gelsolin of Model and Xscd group has high positive correlation with platelet activation level (P<0.05)③Compare with sham group,the fluorescence intensity of Ca2+of model group increased markedly (P<0.05),and high dose of XSC can inhibit plateletcalcium influx (P<0.05), which has similar effect of verapamil (P>0.05)④Compare with sham group, the gelsolin expression of infracted myocardium of model group increased markedly, and XSC can inhibit gelsolin expression of infracted myocardium, but verapamil has no such effect.Conclusion:The active ingredients of Chuanxiong rhizome and red peony root (Chuangxiongol and paeoniflorin) can reduce the platelet gelsolin level, enhance the activity of EASS, inhibit platelet activation and has the effect of calcium antagonist while it also can inhibit COX-1and TXB2which has the similar effect of aspirin in vivo. |
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