血小板骨架蛋白Gelsolin在冠心病血瘀证中的作用及赤芍川芎有效组分的干预效应研究
|
摘要
本课题组前期研究结果表明冠心病、血瘀证、血小板功能状态三者之间存在极其密切的内在联系,我们采用血小板差异蛋白组学的研究方法,筛查了冠心病血瘀证、冠心病非血瘀证患者和健康对照人群的血小板差异功能蛋白,建立了冠心病血瘀证血小板差异蛋白表达谱。研究发现,血小板骨架蛋白凝溶胶蛋白(Gelsolin)是冠心病血瘀证与非血瘀证患者的血小板差异蛋白之一,其含量在冠心病血瘀证患者血小板中明显增多,提示血小板骨架蛋白的异常表达在冠心病血瘀证发生发展中可能起到关键作用。
梗死后心肌组织释放大量肌动蛋白微丝(F-actin)对内皮细胞和微血管产生毒害,血浆Gelsolin可切害(?)F-actin解聚成肌动蛋白单体(G-actin),并通过肝脏内皮网状系统清除体外而阻止这种细胞毒。前期课题组差异蛋白组学研究发现血小板Gelsolin与冠心病血瘀证明显相关,血小板无转录本信息,故我们推测血小板Gelsolin含量升高是由于血浆Gelsolin(?)肖耗而引起血小板骨架蛋白反应性重组,这种病理性升高进一步加速血小板活化、变形而参与冠心病血瘀证形成。
本研究拟通过冠心病血瘀证贫、富血小板血浆Gelsolin含量与血小板活化相关性对这一假说进行验证,并借助F-actin诱导血小板活化体外和急性心肌梗死大鼠体内血小板活化实验体系,研究Gelsolin参与血瘀证形成过程及赤芍川芎有效组分配伍对血小板Gelsolin重组的调控及对血小板聚集、活化的影响。本研究将为活血化瘀中药抗血小板活化提供一定实验依据。
本课题研究分为三部分：文献综述、临床研究和实验研究。
1.文献综述：本部分分别从凝溶胶蛋白和心血管疾病、活血化瘀方药和抗血小板治疗两个方面进行综述。
2.临床研究：包括以下两个部分。
研究一冠心病血瘀证血小板差异蛋白Gelsolin的临床鉴定
目的：分别应用酶联免疫吸附法(ELISA)和蛋白免疫印迹法(WB),对冠心病血瘀证血小板差异蛋白Gelsolin进行临床鉴定。
方法：冠心病诊断参照1999年及2002年美国心脏病学会(ACC)／美国心脏协会(AHA)／美国医师学会及美国内科学会(ACP-ASIM)联合协定关于《慢性稳定性心绞痛诊疗指南》、《不稳定性心绞痛、无ST段抬高心肌梗死诊疗指南》。同时,参考中国中西医结合学会活血化瘀专业委员会制定的血瘀证诊断标准。筛选符合冠心病、血瘀证、健康人入选标准的冠心病血瘀证组、冠心病非血瘀证组和健康对照组各30例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血7mL,前2mL弃用,梯度离心法分别获取富血小板血浆(PRP)和贫血小板血浆(PPP)。分别应用酶联免疫吸附法和蛋白免疫印迹法检测各组人群PRP和PPP的Gelsolin含量。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、合并疾病和用药情况方面无显著性差异,具有可比性。与健康对照组相比,冠心病血瘀证组患者富血小板血浆中的Gelsolin含量显著性升高(P<0.01),贫血小板血浆中的Gelsolin含量明显降低(P<0.05),冠心病非血瘀证组患者贫血小板血浆中Gelsolin含量亦明显降低(P<0.05),富血小板血浆中Gelsolin含量无明显差异(P>0.05)；与冠心病非血瘀证组相比,冠心病血瘀证患者富血小板血浆中Gelsolin含量明显升高(P<0.05),贫血小板血浆中Gelsolin含量无明显差异(P>0.05)。酶联免疫吸附法和蛋白免疫印迹法检测结果一致。
结论：Gelsolin作为细胞骨架蛋白的重要组成部分,在冠心病血瘀证状态下,其在富血小板血浆和贫血小板血浆中的含量分布存在差异性,与前期血小板蛋白质组学研究结果相一致,可能参与了冠心病血瘀证的形成过程。
研究二血小板Gelsolin水平与冠心病血瘀证相关性研究
目的：研究血小板Gelsolin水平与冠心病血瘀证的相关性。
方法：纳入冠心病血瘀证、冠心病非血瘀证患者各57例和健康对照组45例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血12mL,前2mL弃用,梯度离心法分别获取富血小板血浆(PRP)和贫血小板血浆(PPP)。采用ELISA法分别测定PRP中Gelsolin含量及PPP中Gelsolin、肌动蛋白微丝(F-actin)及维生素D结合蛋白(DBP)的含量；采用流式细胞术(FCM)检测血小板胞浆Ca2+浓度及反映血小板活化程度的CD62p的表达。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、合并疾病和用药情况方面无显著性差异,具有可比性。与健康对照组相比,冠心病血瘀证患者PRP中Gelsolin含量显著升高(P<0.01),冠心病血瘀证及非血瘀证患者PPP中Gelsolin含量明显降低(P<0.05), F-actin及DBP含量明显升高(P<0.01),血小板CD62p表达及胞浆钙离子浓度显著升高(P<0.01)。与冠心病非血瘀证组相比,冠心病血瘀证组患者PRP中Gelsolin含量及胞浆钙离子浓度明显升高(P<0.01),血小板CD62p表达明显增高(P<0.05),PPP中Gelsolin、F-actin及DBP含量无明显差异(P>0.05)。对冠心病血瘀证组患者血小板Gelsolin含量与血小板活化标志物CD62p的表达进行相关性分析,结果显示冠心病血瘀证组患者血小板Gelsolin含量与血小板活化标志物CD62p高度正相关(P<0.01)。
结论：血小板Gelsolin水平与冠心病血瘀证血小板活化水平高度相关,在冠心病血瘀证血小板活化过程中,细胞骨架蛋白含量也出现了不同程度的变化,集中体现在血小板Gelsolin和其靶蛋白F-actin的异常变化上,推测血浆Gelsolin含量降低与其因切割、清除F-actin而大量消耗有关,这种消耗引起血小板上的Gelsolin病理性重组,同时引起血小板钙离子内流增加,导致血小板变形能力增强而参与冠心病血瘀证的形成,因此血小板Gelsolin可能是冠心病血瘀证的特异性分子靶标之一。
3.实验研究：包括以下两个部分。
研究一赤芍川芎有效组分对F-actin刺激的血小板活化及血小板Gelsolin水平的体外干预效应研究
目的：研究F-actin体外刺激对血小板活化及血小板Gelsolin含量的影响,同时观察赤芍、川芎有效组分的体外干预效应
方法：以人水洗血小板作为研究对象,以不同浓度的F-actin作为体外血小板活化诱导剂,同时以花生四烯酸(AA)、二磷酸腺苷(ADP)以及凝血酶(Thrombin)作为阳性对照诱导剂。同时以赤芍川芎有效组分芍药苷和川芎嗪联合作为干预药物,以阿司匹林作为阳性对照药物。采用流式细胞术检测血小板活化标志物CD62p的表达,使用血小板聚集仪测定最大血小板聚集率,采用ELISA法测定血小板Gelsolin的含量。
结果：①低(2.5μmol/L)、中(5μmol/L)、高(10μmol/L)不同浓度的F-actin以及AA、ADP和凝血酶(Thrombin)均可引起血小板聚集率和血小板活化水平明显升高(P<0.05及P<0.01),且F-actin随浓度的不断增加其致血小板聚集和活化程度不断增高,呈现浓度依赖性。F-actin最大浓度(10μmol/L)时刺激的最大血小板聚集率与血小板活化水平与同浓度的AA、ADP及Thrombin刺激组无明显差异(P>0.05)。
②低浓度(2.5μmol/L)的F-actin、ADP及Thrombin的刺激对血小板gelsolin含量无明显影响(P>0.05),中(5μmol/L)、高(10μmol/L)农度的F-actin以及AA均可刺激血小板Gelsolin含量明显增高(P<0.05)。F-actin最大浓度(10μmol/L)时刺激的血小板gelsolin含量与同浓度的AA无明显差异(P>0.05)。
③川芎嗪和芍药苷联合体外可以明显抑制由同浓度的F-actin和AA诱导的血小板聚集和活化反应(P<0.05),与阿司匹林相比无明显差异(P>0.05)。水洗血小板预先用川芎嗪和芍药苷联合孵育后,再用F-actin或AA诱导,可明显降低血小板Gelsolin含量(P<0.05),但预先用阿司匹林体外孵育无此效应(P>0.05)。
结论：F-actin体外能够激活血小板导致其聚集、活化,且高浓度能够刺激血小板大量分泌Gelsolin,其效应与AA相似。赤芍、川芎有效组分芍药苷、川芎嗪体外联合应用对聚集、活化后的血小板具有明显抑制作用,同时能够明显降低活化后血小板Gelsolin的含量,而阿司匹林体外干预无此效应。
研究二赤芍川芎有效组分对急性心肌梗死大鼠模型血小板活化及血小板Gelsolin水平的干预效应研究
目的：研究赤芍、川芎有效组分对急性心肌梗死大鼠模型血小板活化及血小板Gelsolin水平的体内干预机制
方法：通过在体结扎前降支方法建立急性心肌梗死大鼠模型,以赤芍川芎有效组分配伍药物芎芍胶囊(XSC)作为干预药物,同时以阿司匹林及钙离子拮抗剂维拉帕米作为阳性对照药物,观察芎芍胶囊对模型大鼠血小板活化及血小板Gelsolin含量及其相关指标的干预效应。血浆梯度离心法获得大鼠PRP和PPP,使用ELISA法测定Gelsolin、F-actin、DBP、P-选择素以及CK-MB、cTnl的含量,使用流式细胞术测定大鼠血小板钙离子浓度,使用WB法测定梗死区心肌组织Gelsolin的蛋白表达。
结果：①模型大鼠结扎前降支3小时后心肌损伤标志物血清CK-MB、cTnI含量均显著增高(P<0.05),同时血小板活化特异性指标血浆P-选择素含量亦较假手术组显著升高(P<0.05)；XSC高剂量组能够明显降低模型组大鼠血清CK-MB、cTnI水平(P<0.05),同时能够降低血浆P-选择素、COX-1及TXB2水平(P<0.05),其效应与阿司匹林组无明显差异；
②模型组大鼠血浆F-actin含量显著增高(P<0.05)而血浆Gelsolin、DBP含量明显降低(P<0.05),同时血小板Gelsolin含量明显增高(P<0.05)；XSC高剂量能够明显降低模型组大鼠血小板Gelsolin以及血浆F-actin水平(P<0.05),同时能够使血浆Gelsoin以及DBP含量显著升高(P<0.05)；模型组及XSC高剂量组大鼠血小板Gelsolin与血小板活化标志物P-选择素水平均呈正相关(P<0.05)。
③模型组大鼠血小板钙离子荧光强度明显增强(P<0.05),XSC高剂量能够明显抑制模型大鼠血小板钙离子内流(P<0.05),其效应与维拉帕米无明显差异(P>0.05)。
④模型大鼠梗死区心肌组织Gelsolin蛋白表达明显增强,而XSC可以明显抑制其表达,但钙拮抗剂维拉帕米无此效应。
结论：赤芍川芎有效组分能明显降低血小板Gelsolin水平,增强血浆肌动蛋白清除系统活性,抑制血小板活化,同时具有一定的钙拮抗效应,且在抑制COX-1活性、降低TXB2水平方面与阿司匹林相似。同时,经相关性分析发现,赤芍川芎有效组分抑制血小板活化程度与降低血小板Gelsolin含量有关。
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 BSS
Objective: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.
梗死后心肌组织释放大量肌动蛋白微丝(F-actin)对内皮细胞和微血管产生毒害,血浆Gelsolin可切害(?)F-actin解聚成肌动蛋白单体(G-actin),并通过肝脏内皮网状系统清除体外而阻止这种细胞毒。前期课题组差异蛋白组学研究发现血小板Gelsolin与冠心病血瘀证明显相关,血小板无转录本信息,故我们推测血小板Gelsolin含量升高是由于血浆Gelsolin(?)肖耗而引起血小板骨架蛋白反应性重组,这种病理性升高进一步加速血小板活化、变形而参与冠心病血瘀证形成。
本研究拟通过冠心病血瘀证贫、富血小板血浆Gelsolin含量与血小板活化相关性对这一假说进行验证,并借助F-actin诱导血小板活化体外和急性心肌梗死大鼠体内血小板活化实验体系,研究Gelsolin参与血瘀证形成过程及赤芍川芎有效组分配伍对血小板Gelsolin重组的调控及对血小板聚集、活化的影响。本研究将为活血化瘀中药抗血小板活化提供一定实验依据。
本课题研究分为三部分：文献综述、临床研究和实验研究。
1.文献综述：本部分分别从凝溶胶蛋白和心血管疾病、活血化瘀方药和抗血小板治疗两个方面进行综述。
2.临床研究：包括以下两个部分。
研究一冠心病血瘀证血小板差异蛋白Gelsolin的临床鉴定
目的：分别应用酶联免疫吸附法(ELISA)和蛋白免疫印迹法(WB),对冠心病血瘀证血小板差异蛋白Gelsolin进行临床鉴定。
方法：冠心病诊断参照1999年及2002年美国心脏病学会(ACC)／美国心脏协会(AHA)／美国医师学会及美国内科学会(ACP-ASIM)联合协定关于《慢性稳定性心绞痛诊疗指南》、《不稳定性心绞痛、无ST段抬高心肌梗死诊疗指南》。同时,参考中国中西医结合学会活血化瘀专业委员会制定的血瘀证诊断标准。筛选符合冠心病、血瘀证、健康人入选标准的冠心病血瘀证组、冠心病非血瘀证组和健康对照组各30例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血7mL,前2mL弃用,梯度离心法分别获取富血小板血浆(PRP)和贫血小板血浆(PPP)。分别应用酶联免疫吸附法和蛋白免疫印迹法检测各组人群PRP和PPP的Gelsolin含量。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、合并疾病和用药情况方面无显著性差异,具有可比性。与健康对照组相比,冠心病血瘀证组患者富血小板血浆中的Gelsolin含量显著性升高(P<0.01),贫血小板血浆中的Gelsolin含量明显降低(P<0.05),冠心病非血瘀证组患者贫血小板血浆中Gelsolin含量亦明显降低(P<0.05),富血小板血浆中Gelsolin含量无明显差异(P>0.05)；与冠心病非血瘀证组相比,冠心病血瘀证患者富血小板血浆中Gelsolin含量明显升高(P<0.05),贫血小板血浆中Gelsolin含量无明显差异(P>0.05)。酶联免疫吸附法和蛋白免疫印迹法检测结果一致。
结论：Gelsolin作为细胞骨架蛋白的重要组成部分,在冠心病血瘀证状态下,其在富血小板血浆和贫血小板血浆中的含量分布存在差异性,与前期血小板蛋白质组学研究结果相一致,可能参与了冠心病血瘀证的形成过程。
研究二血小板Gelsolin水平与冠心病血瘀证相关性研究
目的：研究血小板Gelsolin水平与冠心病血瘀证的相关性。
方法：纳入冠心病血瘀证、冠心病非血瘀证患者各57例和健康对照组45例为研究对象。冠心病血瘀证和非血瘀证患者于冠脉造影前采血,健康对照者于清晨空腹状态下抽取静脉血12mL,前2mL弃用,梯度离心法分别获取富血小板血浆(PRP)和贫血小板血浆(PPP)。采用ELISA法分别测定PRP中Gelsolin含量及PPP中Gelsolin、肌动蛋白微丝(F-actin)及维生素D结合蛋白(DBP)的含量；采用流式细胞术(FCM)检测血小板胞浆Ca2+浓度及反映血小板活化程度的CD62p的表达。
结果：冠心病血瘀证组、非血瘀证组和健康对照组在年龄、性别、体重指数且两疾病组在冠心病类型、冠脉病变支数、合并疾病和用药情况方面无显著性差异,具有可比性。与健康对照组相比,冠心病血瘀证患者PRP中Gelsolin含量显著升高(P<0.01),冠心病血瘀证及非血瘀证患者PPP中Gelsolin含量明显降低(P<0.05), F-actin及DBP含量明显升高(P<0.01),血小板CD62p表达及胞浆钙离子浓度显著升高(P<0.01)。与冠心病非血瘀证组相比,冠心病血瘀证组患者PRP中Gelsolin含量及胞浆钙离子浓度明显升高(P<0.01),血小板CD62p表达明显增高(P<0.05),PPP中Gelsolin、F-actin及DBP含量无明显差异(P>0.05)。对冠心病血瘀证组患者血小板Gelsolin含量与血小板活化标志物CD62p的表达进行相关性分析,结果显示冠心病血瘀证组患者血小板Gelsolin含量与血小板活化标志物CD62p高度正相关(P<0.01)。
结论：血小板Gelsolin水平与冠心病血瘀证血小板活化水平高度相关,在冠心病血瘀证血小板活化过程中,细胞骨架蛋白含量也出现了不同程度的变化,集中体现在血小板Gelsolin和其靶蛋白F-actin的异常变化上,推测血浆Gelsolin含量降低与其因切割、清除F-actin而大量消耗有关,这种消耗引起血小板上的Gelsolin病理性重组,同时引起血小板钙离子内流增加,导致血小板变形能力增强而参与冠心病血瘀证的形成,因此血小板Gelsolin可能是冠心病血瘀证的特异性分子靶标之一。
3.实验研究：包括以下两个部分。
研究一赤芍川芎有效组分对F-actin刺激的血小板活化及血小板Gelsolin水平的体外干预效应研究
目的：研究F-actin体外刺激对血小板活化及血小板Gelsolin含量的影响,同时观察赤芍、川芎有效组分的体外干预效应
方法：以人水洗血小板作为研究对象,以不同浓度的F-actin作为体外血小板活化诱导剂,同时以花生四烯酸(AA)、二磷酸腺苷(ADP)以及凝血酶(Thrombin)作为阳性对照诱导剂。同时以赤芍川芎有效组分芍药苷和川芎嗪联合作为干预药物,以阿司匹林作为阳性对照药物。采用流式细胞术检测血小板活化标志物CD62p的表达,使用血小板聚集仪测定最大血小板聚集率,采用ELISA法测定血小板Gelsolin的含量。
结果：①低(2.5μmol/L)、中(5μmol/L)、高(10μmol/L)不同浓度的F-actin以及AA、ADP和凝血酶(Thrombin)均可引起血小板聚集率和血小板活化水平明显升高(P<0.05及P<0.01),且F-actin随浓度的不断增加其致血小板聚集和活化程度不断增高,呈现浓度依赖性。F-actin最大浓度(10μmol/L)时刺激的最大血小板聚集率与血小板活化水平与同浓度的AA、ADP及Thrombin刺激组无明显差异(P>0.05)。
②低浓度(2.5μmol/L)的F-actin、ADP及Thrombin的刺激对血小板gelsolin含量无明显影响(P>0.05),中(5μmol/L)、高(10μmol/L)农度的F-actin以及AA均可刺激血小板Gelsolin含量明显增高(P<0.05)。F-actin最大浓度(10μmol/L)时刺激的血小板gelsolin含量与同浓度的AA无明显差异(P>0.05)。
③川芎嗪和芍药苷联合体外可以明显抑制由同浓度的F-actin和AA诱导的血小板聚集和活化反应(P<0.05),与阿司匹林相比无明显差异(P>0.05)。水洗血小板预先用川芎嗪和芍药苷联合孵育后,再用F-actin或AA诱导,可明显降低血小板Gelsolin含量(P<0.05),但预先用阿司匹林体外孵育无此效应(P>0.05)。
结论：F-actin体外能够激活血小板导致其聚集、活化,且高浓度能够刺激血小板大量分泌Gelsolin,其效应与AA相似。赤芍、川芎有效组分芍药苷、川芎嗪体外联合应用对聚集、活化后的血小板具有明显抑制作用,同时能够明显降低活化后血小板Gelsolin的含量,而阿司匹林体外干预无此效应。
研究二赤芍川芎有效组分对急性心肌梗死大鼠模型血小板活化及血小板Gelsolin水平的干预效应研究
目的：研究赤芍、川芎有效组分对急性心肌梗死大鼠模型血小板活化及血小板Gelsolin水平的体内干预机制
方法：通过在体结扎前降支方法建立急性心肌梗死大鼠模型,以赤芍川芎有效组分配伍药物芎芍胶囊(XSC)作为干预药物,同时以阿司匹林及钙离子拮抗剂维拉帕米作为阳性对照药物,观察芎芍胶囊对模型大鼠血小板活化及血小板Gelsolin含量及其相关指标的干预效应。血浆梯度离心法获得大鼠PRP和PPP,使用ELISA法测定Gelsolin、F-actin、DBP、P-选择素以及CK-MB、cTnl的含量,使用流式细胞术测定大鼠血小板钙离子浓度,使用WB法测定梗死区心肌组织Gelsolin的蛋白表达。
结果：①模型大鼠结扎前降支3小时后心肌损伤标志物血清CK-MB、cTnI含量均显著增高(P<0.05),同时血小板活化特异性指标血浆P-选择素含量亦较假手术组显著升高(P<0.05)；XSC高剂量组能够明显降低模型组大鼠血清CK-MB、cTnI水平(P<0.05),同时能够降低血浆P-选择素、COX-1及TXB2水平(P<0.05),其效应与阿司匹林组无明显差异；
②模型组大鼠血浆F-actin含量显著增高(P<0.05)而血浆Gelsolin、DBP含量明显降低(P<0.05),同时血小板Gelsolin含量明显增高(P<0.05)；XSC高剂量能够明显降低模型组大鼠血小板Gelsolin以及血浆F-actin水平(P<0.05),同时能够使血浆Gelsoin以及DBP含量显著升高(P<0.05)；模型组及XSC高剂量组大鼠血小板Gelsolin与血小板活化标志物P-选择素水平均呈正相关(P<0.05)。
③模型组大鼠血小板钙离子荧光强度明显增强(P<0.05),XSC高剂量能够明显抑制模型大鼠血小板钙离子内流(P<0.05),其效应与维拉帕米无明显差异(P>0.05)。
④模型大鼠梗死区心肌组织Gelsolin蛋白表达明显增强,而XSC可以明显抑制其表达,但钙拮抗剂维拉帕米无此效应。
结论：赤芍川芎有效组分能明显降低血小板Gelsolin水平,增强血浆肌动蛋白清除系统活性,抑制血小板活化,同时具有一定的钙拮抗效应,且在抑制COX-1活性、降低TXB2水平方面与阿司匹林相似。同时,经相关性分析发现,赤芍川芎有效组分抑制血小板活化程度与降低血小板Gelsolin含量有关。
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 BSS
Objective: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.
引文
[1]Yin HL, Stossel TP. Control of cytop lasmic actin gelsol transformation by gelsolin, a calcium dependent regulatory protein. Nature,1979,281:583-586.
[2]Leven R. M. Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading. J.Cell.Physiol.1995,163(3):597-607.
[3]Zunino R, Li Q, Rose SD,et al. Expression of scinderin in megakaryoblastic leukemia cells induces differentiation, maturation and apoptosis with release of plateletlike particles and inhibits proliferation and tumorigenesis. Blood,2001, 98(7):2210-2219.
[4]Witke W, Sharpe AH, Hartwig JH, et al. Hemostatic, inflammatory and fibroblast responses are blunted in mice lacking gelsolin. Cell,1995,81(1):41-51.
[5]Bearer EL, Prakash JM, Manchester RD, Allen PG.VASP protects actin filaments from gelsolin:an in vitro study with implications for platelet actin reorganizations. Cell Motil Cytoskeleton,2000,47(4):351-364.
[6]Casella JF, Flanagan MD, Lin S. Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature,1981,293(5830):302-305.
[7]Barkalow KL, Falet H, Italiano JE Jr, et al. Role phosphoin-ositide 3-kinase in Fc gamma RIIA-induced platelet shape change. Am J Physiol Cell Physiol,2003, 285(4):C797-805.
[8]Haddad JG, Harper KD, Guoth M, et al. Angiopathic consequences of saturating the plasma scavenger system for actin.Proc Natl Acad Sci USA, 1990,87(4):1381-1385.
[9]Carol A. Vasconcellos, Stuart E. Lind.Coordinated Inhibition of Actin-Induced Platelet Aggregation by Plasma Gelsolin and Vitamin D-Binding Protein. Blood, 1997,82(12):3648-3657.
[10]Suhler E, Lin W, Yin HL, Lee WM. Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock, and myonecrosis. Crit Care Med.1997,25(4):594-598.
[11]Petra J. Mateos-Caceres, Carlos Macaya, Luis Azcona, et al.Different expression of proteins in platelets from aspirin-resistant and aspirin-sensitive patients. Thrombosis and Haemostasis,2010,103:160-170.
[12]Lopez-Farre AJ, Mateos-Cdceres PJ, Sacristan D,et al. Relationship between Vitamin D Binding Protein and Aspirin Resistance in Coronary Ischemic Patients: A Proteomic Study. J Proteome Res,2007,6(7):2481-2487.
[13]Xue mei, Chen keji, Yin huijun. Relationship between platelet activation related factors and polymorphism of related genes in patients with coronary heart disease of blood-stasis syndrome. Chin J Integr Med,2008,14(4):267-273.
[14]李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究.中国分子心脏病学杂志,2009,09(6)：326-331
[15]Kwiatkowski DJ. Functions of gelsolin:motility, signaling, apoptosis, cancer. Curr Opin Cell Biol,1999,11(1):103-108.
[16]Silacci P, Mazzolai L, Gauci C, et al. Gelsolin superfamily proteins:key regulators of cellular functions. Cell Mol Life Sci,2004,61:2614-2623.
[17]Philchenkov AA. Caspases as regulators of apoptosis and other cell functions. Biochemistry (Mosc),2003,68(4):365-376.
[18]Chhabra D, Nosworthy NJ, dos Remedios CGThe N-terminal fragment of gelsolin inhibits the interaction of DNase I with isolated actin, but not with the cofilin-actin comples. Proteomics,2005,5(12):3131-3136.
[19]Yang J, Moravec CS, Sussman MA, et al. Decreased SLIM1 expression and increased gelsolin expression in failing human hearts measured by high-density oligonucleotide arrays. Circulation,2000,102:3046-3052.
[20]Li GH, Shi Y, Chen Y, et al. Gelsolin regulates cardiac remodeling after myocardial infarction through DNase I-mediated apoptosis. Circ Res,2009,104:896-904.
[21]Yue L, Feng J,Gaspo R,et al.Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation.Circ Res,1997,81(4):512-515.
[22]Morillo CA, Klein GJ, JonesDL, et al.Chronic rapid atrialpacing Structural, functional, and electrophysiological characteristics of a new model of sustained atrial fibrillation.Circulation,1995,91 (5):1588-1595.
[23]Lader AS, Kwiatkowski DJ, Cantiello HF. Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels. Am J Physiol,1999,277: C1277-C1283.
[24]Schrickel JW, Fink K, Meyer R, et al. Lack of gelsolin promotes perpetuation of atrial fibrillation in the mouse heart. J Interv Card Electrophysiol,2009, 26(l):3-10.
[25]Janmey PA,Bucki R.Methods of using gelsolin to treat or prevent bacterial sepsis:USPA·20070238668.2006-03-28.
[26]王佳,赵卫国,闵锐.凝溶胶蛋白的生物学功能及临床意义.中国危重病急救医学,2009,21(4)：253-255.
[27]Osborn TM, Dahlgren C, Hartwig JH, et al. Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin. Am J Physiol Cell Physiol,2007,292(4):C1323-C1330.
[1]Antiplatelet trialists'collaboration. Collaborative overview of randomized trials of antiplatelet therapy--Ⅰ:Prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients [J]. BMJ,1994, 308(6921):81-106.
[2]Antithrombotic trialists'collaboration. Collaborate meta-analysis of randomized trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high-risk patients [J].BMJ,2002,324(7329):71-86.
[3]Gergely F, Andrea F, Gabriella P, et al. Clinical importance of aspirin and clopidogrel resistance[J]. World Journal of Cardiology,2010,2(7):171-186.
[4]Pena A, Collet JP, Hulot JS. Can we override clopidogrel resistance? [J]. Circulation, 2009,119:2854-2857.
[5]李永健,党群,陈康寅,李广平.抗血小板药物抵抗真的缺乏临床意义吗?[J].实用心脑肺血管病杂志,2010,18(9)：1352-1354.
[6]David N. Juurlink DN, Gomes T, et al. A population-based study of the drug interaction between protonpump inhibitors and clopidogrel [J]. CMAJ,2009,180: 713-718.
[7]王宇玫.常用抗血小板药物临床应用中值得关注的问题[J].医学研究杂志.2010, 39(2)：115-119.
[8]严晓伟.抗血小板药物的研发进展[J].中国循环杂志.2011,26(1)：4-5.
[9]陈可冀,李连达,翁维良,等.血瘀证与活血化瘀研究[J].中西医结合心脑血管病杂志.2005,3(1)：1-2.
[10]陈可冀.活血化瘀方药降低心血管风险可能性的探索[J].中国中西医结合杂志.2008,28(5)：389.
[11]陈可冀,薛梅,殷惠军.血小板活化与冠状动脉粥样硬化性心脏病和血瘀证的关系[J].首都医科大学学报.2008,29(3)：266-269.
[12]Xue mei, Chen keji, Yin huijun. Relationship between platelet activation related factors and polymorphism of ralated genes in patients with coronary heart disease of blood-stasis syndrome [J]. Chin J Integr Med 2008; 14(4):267-273.
[13]汪钟.活血化瘀中药对血小板功能调节的机理[J].中国中西医结合杂志.1992,12(9)：567-570.
[14]李家增,贺石林,王鸿利.血栓病学[M].北京：科学出版社,1998.P24-27,P169.
[15]徐凤芹,陈可远,马晓昌,等.芎芍胶囊治疗冠心病心绞痛的临床观察[J].中国中西医结合杂志,2003,23(1)：16-18.
[16]冯洁,王嗣岑.复方丹参滴丸对大鼠血小板聚集功能的影响[J].中国误诊学杂志,2006,6(12)：2261-2263.
[17]江劲波,杨静,邓常青.补阳还五汤及有效组分生物碱和苷对大鼠血小板聚集及血小板环核苷酸的影响[J].中南药学,2008,6(4)：388-391.
[18]王阶,许军,衷敬柏,等.三七总苷对高黏血症患者血小板活化分子表达和血小板聚集的影响[J].中国中西医结合杂志,2004,24(4)：312-316.
[19]王东生,陈方平,贺石林,等.大黄蜇虫丸抗血小板活化的机制研究[J].中国中医药杂志,2008,23(9)：818-821.
[20]刘芳,李金,王新德.通心络胶囊对脑梗死患者血小板聚集功能的影响[J].中国中西医结合杂志,2008,28(4)：304-306.
[21]李家明,赵永海,钟国琛,等.阿魏酸衍生物的合成及抗血小板聚集活性[J].药学学报,2011,46(3)：305-310.
[22]舒冰,周重建,马迎辉,等.中药川芎中有效成分的药理作用研究进展[J].中国药理学通报,2006,22(9)：1043-1047.
[23]陈可冀.桔丙酯的抗栓作用.中国处方药,2003,9：38-39.
[24]陈鹏,杨丽川,雷伟亚,沈志强.白藜芦醇甙对血小板聚集功能及内钙水平的影响[J].天然产物研究与开发,2005,17(1)：21-25.
[25]夏泉,董婷霞,詹华强,等.莪术二酮对ADP诱导的兔血小板聚集的抑制作用[J].中国药理学通报,2006,22(9)：1151-1152.
[26]马建建,宋艳,贾敏,等.血竭总黄酮对血小板聚集、血栓形成及心肌缺血的影响[J].中草药,2002,33(11)：1008-1010.
[27]Yao Y, Wu WY, Liu AH, et al. Interaction of salvianolic acids and notoginsengnosides in inhibition of ADP-induced platelet aggregation [J].Am J Chin Med.2008,36(2):313-328.
[28]姜宗文,赵丽娟,张海,等.水蛭肽注射液抗大鼠血栓形成作用[J].吉林大学学报(医学版),2003,29(4)：417-418.
[29]秦晓娟,魏宗德.红花黄色素对心血管作用的研究进展[J].临床荟萃,2009,24(3)：263-265.
[30]Hsu-Lin S, Berman CL, Furie BC, et al. A platelet membrane protein expressed during platelet activation and secretion:studies using a monoclonal antibody specific for thrombin-activated platelets [J]. J Biol Chem,1984,259:9121-9126.
[31]Michelson AD, Furman MI. Laboratory markers of platelet activation and their clinical significance [J]. Curr Opin Hematol,1999,6:342-348.
[32]Ikeda H, Takajo Y, Ichiki K, et al. Increased soluble form of P-selectin in patients with unstable angina [J]. Circulation,1995,92:1693-1696.
[33]Shimomura H, Ogawa H, Arai H, et al. Serial changes in plasma levels of soluble P-selectin in patients with acute myocardial infarction [J]. Am J Cardiol,1998, 81:397-400.
[34]Furman MI, Benoit SE, Barnard MR, et al. Increased platelet reactivity and circulating monocyte-platelet aggregates in patients with stable coronary artery disease [J]. J Am Coll Cardiol,1998,31:352-358.
[35]Liu Yue, Yin Huijun, Jiang Yuerong, et al. Research on the correlation between platelet gelsolin and blood-stasis syndrome of coronary heart disease [J]. Chin J Integr Med 2011; 17(8):587-592.
[36]何勇,谭智艳,胡大军,等.丹红注射液对急性冠脉综合征患者介入术后血小板活化的干预[J].中国现代药物应用,2008,2(13)：43-44.
[37]陈章强,洪浪,王洪,等.川芎嗪对急性冠脉综合征患者介入术后血小板活化因子及血管内皮功能影响[J].中国中西医结合杂志,2007,27(12)：1078-1081.
[38]熊攀,周莉.复方丹参滴丸对不稳定型心绞痛患者血浆内皮素和血小板A2膜颗粒蛋白的影响[J].中西医结合心脑血管病杂志,2009,7(5)：510-511.
[39]韩岚,彭代银,许钒,等.桃红四物汤抗血小板活化作用及机制研究[J].中国中药杂志,2010,35(19)：2609-2612.
[40]罗海明,符德玉,任敏之,等.通心络胶囊对冠心病人血小板GPⅡb/Ⅲa复合物活性影响的临床研究[J].中成药,2005,27(2)：181-183.
[41]Ana Kasier-Friede, Maria Rita Cozzi, Mario Mazzucato, et al. Signaling through GP-IX-V activates αⅡβ3 independently of other receptors [J]. Blood,2004,103 (9):3247-3606.
[42]王东生,陈方平,贺石林,等.大黄蛰虫丸血浆药理学与血清药理学作用的比较研究[J].血栓与止血学,2005,11(1)：5-8.
[43]李艳丽.血府逐瘀汤治疗心血管疾病的研究进展[J].北京中医药,2008,27(3)：228-230.
[44]Yacoub, D., J.F. Theoret, L. Villeneuve, H. Abou-Saleh, W. Mourad, B.G. Allen, Y. Merhi. Essential role of protein kinase C in platelet signaling, aⅡβ3 activation, and thromboxane A2 release [J]. J. Biol. Chem.2006,281(40):30024-30035.
[45]Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C [J]. Science,1992,258 (5082):607-614.
[46]Yu-Min Yang, Xing-Xiang Wang, Jun-Zhu Chen, Shi-Jun Wang, Hu Hu, Hong-Qiang Wang. Resveratrol attenuates adenosine diphosphate-induced platelet activation by reducing protein kinase C activity [J]. The American Journal of Chinese Medicine,2008,36(3):603-613.
[47]Kaplan KL, OWEN J. Plasma levels of (3-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo [J]. Blood,1981,57(2):199-202.
[48]Pumphrey CW, Dawes J. Plasma beta-thromboglobulin as a measure of platelet activity. Effect of risk factors and findings in ischemic heart disease and after acute myocardial infarction [J]. Am J Cardial,1982,50(6):1258-1261.
[49]Slungaard A. Platelet factor 4:a chemokine enigma [J]. Int J Biochem Cell Biol, 2005,37(6):1162-1167.
[50]Hope W, Martin TJ, Chesterman CN, Morgan FJ. Human β-thromboglobulin inhibits PGI2 production and binds to specific site in bovine aortic endothelial cells [J]. Nature,1979,282(5735):210-212.
[51]孔月琼,姚震,云美玲,等.丹参粉针治疗冠心病心绞痛患者的临床疗效及对 血小板功能的影响[J].高血压杂志,2002,10(5)：451-453.
[52]Kato M, Kambe M, Kajiyama G. Increased cytosolic free Mg2+ and Ca2+ in Platelets of patients with vasospastic. Am J Physiol 1998; 274:548-554.
[53]Fujinishi A, Takahara K,Ohba C, et al.Effects of nisoldipine on cytosolic calcium, platelet aggregation, and coagulation/fibrinolysis in patients with coronary artery disease [J]. Angiology,1997; 48:515-521.
[54]张荣新,连秀峰,连娜.中药钙拮抗剂治疗心血管病的研究进展[J].陕西中医学院学报,1999,22(4)：52-54.
[55]陈文梅,金鸣,吴伟,等.红花黄色素抑制血小板激活因子介导的血小板活化作用的研究[J].中国药学杂志,2000,35(11)：741.
[56]陈春,杨天伦TXA2/PGI2与心血管疾病[J].现代生物医学进展.2008,8(11)：2166-2172.
[57]徐红梅,刘清云,戴敏,等.赤芍总苷对大鼠血小板功能的影响.合肥工业大学学报(自然科学版).2003,26(1)：141-144.
[58]高会丽,李贻奎,仝燕,等.冠心Ⅱ号系列组方对犬急性心肌缺血保护作用的比较研究[J].中药药理与临床,2007,23(5)：1-4.
[59]蓝肇熙,王万智,马忆南,等.桃红四物汤对大鼠损伤血淤证中TXB2、6-keto-PGF1α的影响[J].华西药学杂志,2008,23(6)：687-688.
[60]武源,郭宏宝,王铁军,等.几种中药组分对家兔体外血小板聚集作用的比较[J].中国临床药理学与治疗学,2007,12(9)：1047-1051.
[61]吁文贵,徐理纳.乙酰丹酚酸A对血小板功能的影响[J].药学学报,1994,29(6)：412.
[62]袁淑娟,张志伟,高天红,朴晋华.红花注射液抗血栓作用机制研究[J].中国中药杂志,2011,36(11)：1528-1529.
[63]王振义,李家增,阮长耿.血栓与止血基础理论与临床[M].上海：上海科学技术出版社,2004：63-71.
[64]许澍淮.环核苷酸与血小板功能[J].生理科学进展,1992,23(4)：318-322.
[65]祝国光,罗瑞芝,郭治听.复方丹参滴丸抗血小板活化及聚集性研究进展[J].中国心血管杂志,2007,12(2)：149-151.
[66]杨佳,秦彩玲.复方丹参方及丹参、三七对血小板功能影响的研究概况[J].中国实验方剂学杂志,2003,9(2)：59-62.
[67]裴海云,韩颖.信号转导与血小板激活[J].中国实验血液学杂志,2004,12(5)： 704-707.
[68]卢建,余应年,徐仁宝.受体信号转导系统与疾病.济南：山东科学技术出版社,1999.
[69]Cosemans JM, Munnix IC, Wetzker R, et al. Continuous signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization [J]. Blood.2006,108 (9):3045-3052.
[70]Li Z, Zhang G, Le Breton GC, et al. Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases [J]. The Journal of Biological Chemistry.2003,278(33):30725-30731.
[71]Kroner C, Eybrechts K, Akkerman JW. Dual regulation of platelet protein kinase B. The Journal of biological chemistry.2000,275 (36):27790-27798.
[72]Huang ZS, Zeng CL, Zhu LJ, Jiang L, Li N, Hu H.Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3-kinase [J]. J Thromb Haemost,2010,8(6):1383-1393.
[73]CHEN Ke-ji, SHI Da-zhuo, XU Hao, et al. XS0601 reduces the incidence of restenosis:a prospective study of 335 patients undergoing percutaneous coronary intervention in China [J]. Chinese Medical Journal,2006,119(1):6-13.
[74]Garci'a A. Clinical proteomics in platelet research:challenges ahead [J]. J Thromb Haemost 2010; 8:1784-1785.
[75]Thiele T, Steil L, Gebhard S, Scharf C, et al. Profiling of alterations in platelet proteins during storage of platelet concentrates [J]. Transfusion 2007; 47:1221-1233;
[76]Cristina Banfi, Maura Brioschi, Giancarlo Marenzi, De Metrio M, Camera M, Mussoni L, et al. Proteome of platelets in patients with coronary artery disease [J]. Experimental Hematology 2010; 38:341-350.
[77]Senzel L, Gnatenko DV, Bahou WF. The platelet proteome [J]. Curr Opin Hematol 2009; 16:329-333.
[78]李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究[J].中国分子心脏病学杂志,2009,09(6)：326-331.
1. Lena Brydon, Kesson Magid, Andrew Steptoe. Platelets, coronary heart disease, and stress. Brain, Behavior, and Immunity 2006; 20(2):113-119.
2.张京春,陈可冀.病证结合是中西医结合临床的最佳模式[J].世界中医药,2006,1：14-15.
3. Faxon DP, Freedman JE. Facts and controversies of aspirin and clopidogrel therapy. Am Heart J.2009,157(3):412-22.
4. Wang TH, BhattDL, Topol EJ. Aspirin and clopidogrel resistance:an emerging clinical entity. Eur Heart J,2006,27:647-654.
5. Frelinger AL, Furman MI, Linden MD, et al. Residual Arachidonic Acid-Induced Platelet Activation via an Adenosine iphosphate-Dependent but cyclooxygenase-1-and Cyclooxygenase-2 -Independent Pathway A 700-Patient Study of Aspirin Resistance. Circulation.2006,113:2888-2896.
6.徐浩,文川,陈可冀,等.川芍、赤芍及其有效部位配伍对载脂蛋白E基因缺陷小鼠动脉粥样硬化斑块稳定性影响的研究[J].中国中西医结合杂志2007,27(6)：513-518
7.李立志,刘剑刚,马鲁波,等.芎芍胶囊对兔动脉粥样硬化模型脂质代谢及血小板聚集的影响[J].中国中西医结合杂志,2008,28(12)：1100-1103.
8.严萍,林久茂,陈宇宁,等.芎芍胶囊对心肌梗死大鼠缺血心肌mRNA表达水平的影响[J].中国中医急症,2011,20(9)：1426-1427.
9.徐凤芹,徐浩,刘剑刚,等.芎芍胶囊对动脉粥样硬化兔血管平滑肌细胞增殖的影响[J].中国中西医结合杂志,2008,28(10)：912-917.
10.鹿小燕,史大卓,徐浩,等.芎芍胶囊干预冠心病介入治疗后再狭窄的研究[J].中国中西医结合杂志2006,26(1)：13-17
11. CHEN KJ, SHI DZ, XU H, et al. XS0601 reduces the incidence of restenosis:a prospective study of 335 patients undergoing percutaneous coronary intervention in China [J]. Chinese Medical Journal,2006,119(1):6-13.
12.李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究[J].中国分子心脏病学杂志2009；9(6)：326-331.
1. Gibbons RJ, Chatterjee K, Daley J, et al. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina:a report of the American college of cardiology/American Heart Association task force on practice guidelines (Committee on management of patients with chronic stable angina). J Am Coll Cardio,1999,33(7):2092-2197.
2. Braunwald E, Antman EM, Kupersmith JW, et al. ACC/AHA 2002 guideline update for the management of patients with unstable angina and non-ST-segment elevation myocaridal infarction-summary article:A report of the American college of cardiology/American Heart Association task force on practice guidelines (committee on the management of patients with unstable angina) J Am Coll Cardio,2002,40(7): 1266-1374.
3. Davi G, Patrono C. Platelet activation and atherothrombosis [J]. N Engl J Med 2007; 357(24):2482-2494.
4. Moliterno DJ. Advances in antiplatelet therapy for ACS and PCI [J]. J Interv Cardiol 2008;21(S11):S18-24.
5. Garci'a A. Clinical proteomics in platelet research:challenges ahead [J]. J Thromb Haemost 2010; 8:1784-1785.
6. Thiele T, SteilL, Gebhard S, Scharf C, et al. Profiling of alterations in platelet proteins during storage of platelet concentrates [J]. Transfusion 2007; 47:1221-1233;
7. Cristina Banfi, Maura Brioschi, Giancarlo Marenzi, De Metrio M, Camera M, Mussoni L,et al. Proteome of platelets in patients with coronary artery disease [J]. Experimental Hematology 2010; 38:341-350.
8. Senzel L, Gnatenko DV, Bahou WF. The platelet proteome [J]. CurrOpin Hematol 2009; 16:329-333.
9.陈可冀,薛梅,殷惠军.血小板活化与冠状动脉粥样硬化性心脏病和血瘀证的关系[J].首都医科大学学报,2008,29(3)：266-269.
10. Xue mei,Chen keji,Yin huijun. Relationship between platelet activation related factors and polymorphism of related genes in patients with coronary heart disease of blood-stasis syndrome [J]. Chin J Integr Med 2008;14(4):267-273.
11.李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究[J].中国分子心脏病学杂志2009；9(6)：326-331.
12. Yin HL, Stossel TP. Control of cytop lasmic actin gelsol transformation by gelsolin, a calcium dependent regulatory protein [J]. Nature 1979; 281(5732):583-586.
13. Snabes MC, Boyd AE 3rd, Bryan J. Detection of actin-binding proteins in human platelets by 1251-actin overlay of polyacrylamide gels [J]. J Cell Biol 1981; 90(3):809-812.
14. Harris HE, Bamburg JR, Weeds AG. Actin filament disassembly in blood plasma [J]. FEBS Lett.1980; 121(1):175-177.
15. Lee WM, Galbraith RM. The extracellular actin scavenger system and actin toxicity [J]. N Engl J Med 1992; 326(20):1335-1341.
16.倪晓光,赵平.凝溶胶蛋白的结构和功能及其抑制肿瘤的作用[J].实用癌症杂志2007；22(1)：105-107.
17.刘玥,蒋跃绒,殷惠军,陈可冀.凝溶胶蛋白与心血管疾病[J].中国分子心脏病学杂志2011；11(1)：50-53.
18. de la Chapelle A, Tolvanen R, Boysen G, Santavy J, Bleeker-Wagemakers L, Maury CP, Kere J.Gelsolin-derived familial amyloidosis caused by asparagine or tyrosine substitution for aspartic acid at residue 187 [J].Nat Genet 1992;2(2):157-160.
19. Suhler E, Lin W, Yin HL. Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock and myonecrosis [J]. Crit Care Med 1997; 25(4):594-598.
1. Gibbons RJ, Chatterjee K, Daley J, et al. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina:a report of the American college of cardiology/American Heart Association task force on practice guidelines (Committee on management of patients with chronic stable angina). J Am Coll Cardio,1999,33(7):2092-2197.
2. Braunwald E, Antman EM, Kupersmith JW, et al. ACC/AHA 2002 guideline update for the management of patients with unstable angina and non-ST-segment elevation myocaridal infarction-summary article:A report of the American college of cardiology/American Heart Association task force on practice guidelines (committee on the management of patients with unstable angina) J Am Coll Cardio,2002,40(7): 1266-1374.
3.庄明明,温奕欣,刘少列,等.流式细胞仪测定血小板胞浆游离钙浓度.西安交通大学学报(医学版),2005；26(5)：508-510.
4. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem,1985,260(6):3440-3450.
5. Antoniades C, Bakogiannis C, Tousoulis D, Demosthenous M, Marinou K, Stefanadis C. Platelet activation in atherogenesis associated with low-grade inflammation. Inflamm Allergy Drug Targets,2010,12,9(5):334-45.
6. Michelson A D, Furman M I. Laboratory markers of platelet activation and their clinical significance. Curr Opin Hematol,1999,6(5):342-348.
7. Oestreich JH, Holt J, Dunn SP. Considerable variability in platelet activity among patients with coronary artery disease in response to an increased maintenance dose of clopidogrel. Coronary Artery Disease 2009,20(3):207-213
8. Xue mei, Chen keji, Yin huijun. Relationship between platelet activation related factors and polymorphism of related genes in patients with coronary heart disease of blood-stasis syndrome. Chin J Integr Med,2008,14(4):267-273.
9.李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究.中国分子心脏病学杂志,2009,9(6)：326-331.
10. Spinardi L, Witke W. Gelsolin and diseases. Subcell Biochem 2007; 45:55-69.
11. Carol A. Vasconcellos, Stuart E. Lind. Coordinated Inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein. Blood,1997, 82(12):3648-365.
12. Lee WM, Galbraith RM. The extracellular actin scavenger system and actin toxicity. N Engl J Med,1992,326(20):1335-1341.
13. Suhler E, Lin W, Yin HL. Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock and myonecrosis. Crit Care Med,1997, 25(4):594-598.
14. Lopez-Farre A J, Mateos-Caceres P J, Sacristan D, et al. Relationship between vitamin D binding protein and aspirin resistance in coronary ischemic patients:A proteomic study. J Proteome Res,2007,6(7):2481-2487
15. Kato M, Kambe M, Kajiyama G.Increased cytosolic free Mg2+ and Ca2+ in Platelets of patients with vasospastic. Am J Physiol,1998,274:548-554.
16. Fujinishi A, Takahara K, Ohba C, et al. Effects of nisoldipine on cytosolic calcium, platelet aggregation, and coagulation/fibrinolysis in patients with coronary artery disease. Angiology,1997,48(6):515-521.
1. Carol A. Vasconcellos, Stuart E. Lind. Coordinated Inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein. Blood,1997, 82(12):3648-3657.
2. Martin Quinn, Desmond Fitzgerald/卢瑾,郭如华(译).血小板功能：评估、诊断和治疗.北京：科学出版社,2008：3-4.
3. Jennings LK. Mechanisms of platelet activation:need for new strategies to protect against platelet-mediated atherothrombosis. Thromb Haemost 2009,102(2):248-257
4. Gladding P, Webster M, Ormiston J, et al. Antiplatelet drug nonresponseness. Am Heart J.2008,155 (4):591-599.
5. Navarro-Nunez L, Lozano ML, Palomo M, et al. Apigenin inhibits platelet adhesion and thrombus formation and synergizes with aspirin in the suppression of the arachidonic acid pathway. J Agric Food Chem 2008,56(9):2970-2976
6. Mousa SA, Jeske WP, Fareed J. Prasugrel:a novel platelet ADP P2Y (12) receptor antagonist. Methods Mol Biol,2010,663:221-228.
7.石鑫,宋丽,苏琦,刘雅文,程熠.凝血酶GPIb-Ⅸ-Ⅴ受体信号转导通路研究进展.中国老年学杂志,2012,3(2)：648-650.
8. Haddad JG, Harper KD, Guoth M, et al. Angiopathic consequences of saturating the plasma scavenger system for actin. Proc Nail Acad Sci USA,1990,87(4):1381-1385.
9.项耀祖,商洪才,张伯礼.抗血小板中药研究进展.中草药,2008,39(2)：290-294
10.史秀丽,傅佳,李光武.血小板靶向中药有效成分及单体研究进展.中国中药杂志,2006,31(5)：361-365.
11.刘剑刚,徐凤芹,史大卓,董国菊.川芎赤芍提取物不同配比的活血化瘀作用研究.中药新药与临床药理,2005,16(5)：315-317.
12.刘超,王静,杨军.赤芍总苷活血化瘀作用的研究.中药材,2000,23(9)：557-560.
13.管昌益.川芎嗪在心血管领域的应用现状与存在问题.中国中西医结合杂志,2003,23(5)：378.
1.徐叔云,卞如濂,陈修.药理实验方法学(第3版).北京：人民卫生出版社,2002.
2.庄明明,温奕欣,刘少列,等.流式细胞仪测定血小板胞浆游离钙浓度[J].西安交通大学学报(医学版),2005,26(5)：508-510.
3. Oestreich JH, Holt J, Dunn SP. Considerable variability in platelet activity among patients with coronary artery disease in response to an increased maintenance dose of clopidogrel [J]. Coronary Artery Disease,2009,20(3):207-213
4. Terpening C. An appraisal of dual antiplatelet therapy with clopidogrel and aspirin for prevention of cardiovascular events [J]. J Am Board Fam Med,2009,22(1):51-56
5. Reaume KT, Regal RE, Dorsch MP.Indications for dual antiplatelet therapy with aspirin and clopidogrel:evidence-based recommendations for use [J]. Ann Pharmacother,2008,42(4):550-557.
6. Wang TH, BhattDL, Topol EJ. Aspirin and clopidogrel resistance:an emerging clinical entity [J]. Eur Heart J,2006,27:647-654.
7.王玲玲,沈薇,Doffoel Michel抗凝和/或抗血小板药物致消化道出血的临床特点及影响因素[J].胃肠病学和肝病学杂志,2011,20(6)：542-545.
8. Angiolillo DJ, Guzman LA, Bass TA. Current antiplatelet therapies:benefits and limitations [J]. Am Heart J,2008,156(2 Suppl):3-9
9.徐浩,文川,陈可冀,等.川芍、赤芍及其有效部位配伍对载脂蛋白E基因缺陷小鼠动脉粥样硬化斑块稳定性影响的研究[J].中国中西医结合杂志2007,27(6)：513-518
10.李立志,刘剑刚,马鲁波,等.芎芍胶囊对兔动脉粥样硬化模型脂质代谢及血小板聚集的影响[J].中国中西医结合杂志,2008,28(12)：1100-1103.
11.严萍,林久茂,陈宇宁,等.芎芍胶囊对心肌梗死大鼠缺血心肌mRNA表达水平的影响[J].中国中医急症,2011,20(9)：1426-1427.
12.徐凤芹,徐浩,刘剑刚,等.芎芍胶囊对动脉粥样硬化兔血管平滑肌细胞增殖的影响[J].中国中西医结合杂志,2008,28(10)：912-917.
13.鹿小燕,史大卓,徐浩,等.芎芍胶囊干预冠心病介入治疗后再狭窄的研究[J].中国中西医结合杂志2006,26(1)：13-17
14. CHEN KJ, SHI DZ, XU H, et al. XS0601 reduces the incidence of restenosis:a prospective study of 335 patients undergoing percutaneous coronary intervention in China [J]. Chinese Medical Journal,2006,119(1):6-13.
15. Lee WM, Galbraith RM. The extracellular actin scavenger system and actin toxicity [J]. N EngI J Mcd,1992,326(20):1335-1341.
16. Messaris E. Actin-binding plasma gelsolin:A potential future ally in the fight against sepsis [J].Crit Care Med.2007,35(3):970-971.
17. Yang J, Moravec CS, Sussman MA, et al. Decreased SLIM1 expression and increased gelsolin expression in failing human hearts measured by high-density oligonucleotide arrays[J]. Circulation.2000,102(25):3046-3052.
[2]Leven R. M. Differential regulation of integrin-mediated proplatelet formation and megakaryocyte spreading. J.Cell.Physiol.1995,163(3):597-607.
[3]Zunino R, Li Q, Rose SD,et al. Expression of scinderin in megakaryoblastic leukemia cells induces differentiation, maturation and apoptosis with release of plateletlike particles and inhibits proliferation and tumorigenesis. Blood,2001, 98(7):2210-2219.
[4]Witke W, Sharpe AH, Hartwig JH, et al. Hemostatic, inflammatory and fibroblast responses are blunted in mice lacking gelsolin. Cell,1995,81(1):41-51.
[5]Bearer EL, Prakash JM, Manchester RD, Allen PG.VASP protects actin filaments from gelsolin:an in vitro study with implications for platelet actin reorganizations. Cell Motil Cytoskeleton,2000,47(4):351-364.
[6]Casella JF, Flanagan MD, Lin S. Cytochalasin D inhibits actin polymerization and induces depolymerization of actin filaments formed during platelet shape change. Nature,1981,293(5830):302-305.
[7]Barkalow KL, Falet H, Italiano JE Jr, et al. Role phosphoin-ositide 3-kinase in Fc gamma RIIA-induced platelet shape change. Am J Physiol Cell Physiol,2003, 285(4):C797-805.
[8]Haddad JG, Harper KD, Guoth M, et al. Angiopathic consequences of saturating the plasma scavenger system for actin.Proc Natl Acad Sci USA, 1990,87(4):1381-1385.
[9]Carol A. Vasconcellos, Stuart E. Lind.Coordinated Inhibition of Actin-Induced Platelet Aggregation by Plasma Gelsolin and Vitamin D-Binding Protein. Blood, 1997,82(12):3648-3657.
[10]Suhler E, Lin W, Yin HL, Lee WM. Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock, and myonecrosis. Crit Care Med.1997,25(4):594-598.
[11]Petra J. Mateos-Caceres, Carlos Macaya, Luis Azcona, et al.Different expression of proteins in platelets from aspirin-resistant and aspirin-sensitive patients. Thrombosis and Haemostasis,2010,103:160-170.
[12]Lopez-Farre AJ, Mateos-Cdceres PJ, Sacristan D,et al. Relationship between Vitamin D Binding Protein and Aspirin Resistance in Coronary Ischemic Patients: A Proteomic Study. J Proteome Res,2007,6(7):2481-2487.
[13]Xue mei, Chen keji, Yin huijun. Relationship between platelet activation related factors and polymorphism of related genes in patients with coronary heart disease of blood-stasis syndrome. Chin J Integr Med,2008,14(4):267-273.
[14]李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究.中国分子心脏病学杂志,2009,09(6)：326-331
[15]Kwiatkowski DJ. Functions of gelsolin:motility, signaling, apoptosis, cancer. Curr Opin Cell Biol,1999,11(1):103-108.
[16]Silacci P, Mazzolai L, Gauci C, et al. Gelsolin superfamily proteins:key regulators of cellular functions. Cell Mol Life Sci,2004,61:2614-2623.
[17]Philchenkov AA. Caspases as regulators of apoptosis and other cell functions. Biochemistry (Mosc),2003,68(4):365-376.
[18]Chhabra D, Nosworthy NJ, dos Remedios CGThe N-terminal fragment of gelsolin inhibits the interaction of DNase I with isolated actin, but not with the cofilin-actin comples. Proteomics,2005,5(12):3131-3136.
[19]Yang J, Moravec CS, Sussman MA, et al. Decreased SLIM1 expression and increased gelsolin expression in failing human hearts measured by high-density oligonucleotide arrays. Circulation,2000,102:3046-3052.
[20]Li GH, Shi Y, Chen Y, et al. Gelsolin regulates cardiac remodeling after myocardial infarction through DNase I-mediated apoptosis. Circ Res,2009,104:896-904.
[21]Yue L, Feng J,Gaspo R,et al.Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation.Circ Res,1997,81(4):512-515.
[22]Morillo CA, Klein GJ, JonesDL, et al.Chronic rapid atrialpacing Structural, functional, and electrophysiological characteristics of a new model of sustained atrial fibrillation.Circulation,1995,91 (5):1588-1595.
[23]Lader AS, Kwiatkowski DJ, Cantiello HF. Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels. Am J Physiol,1999,277: C1277-C1283.
[24]Schrickel JW, Fink K, Meyer R, et al. Lack of gelsolin promotes perpetuation of atrial fibrillation in the mouse heart. J Interv Card Electrophysiol,2009, 26(l):3-10.
[25]Janmey PA,Bucki R.Methods of using gelsolin to treat or prevent bacterial sepsis:USPA·20070238668.2006-03-28.
[26]王佳,赵卫国,闵锐.凝溶胶蛋白的生物学功能及临床意义.中国危重病急救医学,2009,21(4)：253-255.
[27]Osborn TM, Dahlgren C, Hartwig JH, et al. Modifications of cellular responses to lysophosphatidic acid and platelet-activating factor by plasma gelsolin. Am J Physiol Cell Physiol,2007,292(4):C1323-C1330.
[1]Antiplatelet trialists'collaboration. Collaborative overview of randomized trials of antiplatelet therapy--Ⅰ:Prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients [J]. BMJ,1994, 308(6921):81-106.
[2]Antithrombotic trialists'collaboration. Collaborate meta-analysis of randomized trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high-risk patients [J].BMJ,2002,324(7329):71-86.
[3]Gergely F, Andrea F, Gabriella P, et al. Clinical importance of aspirin and clopidogrel resistance[J]. World Journal of Cardiology,2010,2(7):171-186.
[4]Pena A, Collet JP, Hulot JS. Can we override clopidogrel resistance? [J]. Circulation, 2009,119:2854-2857.
[5]李永健,党群,陈康寅,李广平.抗血小板药物抵抗真的缺乏临床意义吗?[J].实用心脑肺血管病杂志,2010,18(9)：1352-1354.
[6]David N. Juurlink DN, Gomes T, et al. A population-based study of the drug interaction between protonpump inhibitors and clopidogrel [J]. CMAJ,2009,180: 713-718.
[7]王宇玫.常用抗血小板药物临床应用中值得关注的问题[J].医学研究杂志.2010, 39(2)：115-119.
[8]严晓伟.抗血小板药物的研发进展[J].中国循环杂志.2011,26(1)：4-5.
[9]陈可冀,李连达,翁维良,等.血瘀证与活血化瘀研究[J].中西医结合心脑血管病杂志.2005,3(1)：1-2.
[10]陈可冀.活血化瘀方药降低心血管风险可能性的探索[J].中国中西医结合杂志.2008,28(5)：389.
[11]陈可冀,薛梅,殷惠军.血小板活化与冠状动脉粥样硬化性心脏病和血瘀证的关系[J].首都医科大学学报.2008,29(3)：266-269.
[12]Xue mei, Chen keji, Yin huijun. Relationship between platelet activation related factors and polymorphism of ralated genes in patients with coronary heart disease of blood-stasis syndrome [J]. Chin J Integr Med 2008; 14(4):267-273.
[13]汪钟.活血化瘀中药对血小板功能调节的机理[J].中国中西医结合杂志.1992,12(9)：567-570.
[14]李家增,贺石林,王鸿利.血栓病学[M].北京：科学出版社,1998.P24-27,P169.
[15]徐凤芹,陈可远,马晓昌,等.芎芍胶囊治疗冠心病心绞痛的临床观察[J].中国中西医结合杂志,2003,23(1)：16-18.
[16]冯洁,王嗣岑.复方丹参滴丸对大鼠血小板聚集功能的影响[J].中国误诊学杂志,2006,6(12)：2261-2263.
[17]江劲波,杨静,邓常青.补阳还五汤及有效组分生物碱和苷对大鼠血小板聚集及血小板环核苷酸的影响[J].中南药学,2008,6(4)：388-391.
[18]王阶,许军,衷敬柏,等.三七总苷对高黏血症患者血小板活化分子表达和血小板聚集的影响[J].中国中西医结合杂志,2004,24(4)：312-316.
[19]王东生,陈方平,贺石林,等.大黄蜇虫丸抗血小板活化的机制研究[J].中国中医药杂志,2008,23(9)：818-821.
[20]刘芳,李金,王新德.通心络胶囊对脑梗死患者血小板聚集功能的影响[J].中国中西医结合杂志,2008,28(4)：304-306.
[21]李家明,赵永海,钟国琛,等.阿魏酸衍生物的合成及抗血小板聚集活性[J].药学学报,2011,46(3)：305-310.
[22]舒冰,周重建,马迎辉,等.中药川芎中有效成分的药理作用研究进展[J].中国药理学通报,2006,22(9)：1043-1047.
[23]陈可冀.桔丙酯的抗栓作用.中国处方药,2003,9：38-39.
[24]陈鹏,杨丽川,雷伟亚,沈志强.白藜芦醇甙对血小板聚集功能及内钙水平的影响[J].天然产物研究与开发,2005,17(1)：21-25.
[25]夏泉,董婷霞,詹华强,等.莪术二酮对ADP诱导的兔血小板聚集的抑制作用[J].中国药理学通报,2006,22(9)：1151-1152.
[26]马建建,宋艳,贾敏,等.血竭总黄酮对血小板聚集、血栓形成及心肌缺血的影响[J].中草药,2002,33(11)：1008-1010.
[27]Yao Y, Wu WY, Liu AH, et al. Interaction of salvianolic acids and notoginsengnosides in inhibition of ADP-induced platelet aggregation [J].Am J Chin Med.2008,36(2):313-328.
[28]姜宗文,赵丽娟,张海,等.水蛭肽注射液抗大鼠血栓形成作用[J].吉林大学学报(医学版),2003,29(4)：417-418.
[29]秦晓娟,魏宗德.红花黄色素对心血管作用的研究进展[J].临床荟萃,2009,24(3)：263-265.
[30]Hsu-Lin S, Berman CL, Furie BC, et al. A platelet membrane protein expressed during platelet activation and secretion:studies using a monoclonal antibody specific for thrombin-activated platelets [J]. J Biol Chem,1984,259:9121-9126.
[31]Michelson AD, Furman MI. Laboratory markers of platelet activation and their clinical significance [J]. Curr Opin Hematol,1999,6:342-348.
[32]Ikeda H, Takajo Y, Ichiki K, et al. Increased soluble form of P-selectin in patients with unstable angina [J]. Circulation,1995,92:1693-1696.
[33]Shimomura H, Ogawa H, Arai H, et al. Serial changes in plasma levels of soluble P-selectin in patients with acute myocardial infarction [J]. Am J Cardiol,1998, 81:397-400.
[34]Furman MI, Benoit SE, Barnard MR, et al. Increased platelet reactivity and circulating monocyte-platelet aggregates in patients with stable coronary artery disease [J]. J Am Coll Cardiol,1998,31:352-358.
[35]Liu Yue, Yin Huijun, Jiang Yuerong, et al. Research on the correlation between platelet gelsolin and blood-stasis syndrome of coronary heart disease [J]. Chin J Integr Med 2011; 17(8):587-592.
[36]何勇,谭智艳,胡大军,等.丹红注射液对急性冠脉综合征患者介入术后血小板活化的干预[J].中国现代药物应用,2008,2(13)：43-44.
[37]陈章强,洪浪,王洪,等.川芎嗪对急性冠脉综合征患者介入术后血小板活化因子及血管内皮功能影响[J].中国中西医结合杂志,2007,27(12)：1078-1081.
[38]熊攀,周莉.复方丹参滴丸对不稳定型心绞痛患者血浆内皮素和血小板A2膜颗粒蛋白的影响[J].中西医结合心脑血管病杂志,2009,7(5)：510-511.
[39]韩岚,彭代银,许钒,等.桃红四物汤抗血小板活化作用及机制研究[J].中国中药杂志,2010,35(19)：2609-2612.
[40]罗海明,符德玉,任敏之,等.通心络胶囊对冠心病人血小板GPⅡb/Ⅲa复合物活性影响的临床研究[J].中成药,2005,27(2)：181-183.
[41]Ana Kasier-Friede, Maria Rita Cozzi, Mario Mazzucato, et al. Signaling through GP-IX-V activates αⅡβ3 independently of other receptors [J]. Blood,2004,103 (9):3247-3606.
[42]王东生,陈方平,贺石林,等.大黄蛰虫丸血浆药理学与血清药理学作用的比较研究[J].血栓与止血学,2005,11(1)：5-8.
[43]李艳丽.血府逐瘀汤治疗心血管疾病的研究进展[J].北京中医药,2008,27(3)：228-230.
[44]Yacoub, D., J.F. Theoret, L. Villeneuve, H. Abou-Saleh, W. Mourad, B.G. Allen, Y. Merhi. Essential role of protein kinase C in platelet signaling, aⅡβ3 activation, and thromboxane A2 release [J]. J. Biol. Chem.2006,281(40):30024-30035.
[45]Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C [J]. Science,1992,258 (5082):607-614.
[46]Yu-Min Yang, Xing-Xiang Wang, Jun-Zhu Chen, Shi-Jun Wang, Hu Hu, Hong-Qiang Wang. Resveratrol attenuates adenosine diphosphate-induced platelet activation by reducing protein kinase C activity [J]. The American Journal of Chinese Medicine,2008,36(3):603-613.
[47]Kaplan KL, OWEN J. Plasma levels of (3-thromboglobulin and platelet factor 4 as indices of platelet activation in vivo [J]. Blood,1981,57(2):199-202.
[48]Pumphrey CW, Dawes J. Plasma beta-thromboglobulin as a measure of platelet activity. Effect of risk factors and findings in ischemic heart disease and after acute myocardial infarction [J]. Am J Cardial,1982,50(6):1258-1261.
[49]Slungaard A. Platelet factor 4:a chemokine enigma [J]. Int J Biochem Cell Biol, 2005,37(6):1162-1167.
[50]Hope W, Martin TJ, Chesterman CN, Morgan FJ. Human β-thromboglobulin inhibits PGI2 production and binds to specific site in bovine aortic endothelial cells [J]. Nature,1979,282(5735):210-212.
[51]孔月琼,姚震,云美玲,等.丹参粉针治疗冠心病心绞痛患者的临床疗效及对 血小板功能的影响[J].高血压杂志,2002,10(5)：451-453.
[52]Kato M, Kambe M, Kajiyama G. Increased cytosolic free Mg2+ and Ca2+ in Platelets of patients with vasospastic. Am J Physiol 1998; 274:548-554.
[53]Fujinishi A, Takahara K,Ohba C, et al.Effects of nisoldipine on cytosolic calcium, platelet aggregation, and coagulation/fibrinolysis in patients with coronary artery disease [J]. Angiology,1997; 48:515-521.
[54]张荣新,连秀峰,连娜.中药钙拮抗剂治疗心血管病的研究进展[J].陕西中医学院学报,1999,22(4)：52-54.
[55]陈文梅,金鸣,吴伟,等.红花黄色素抑制血小板激活因子介导的血小板活化作用的研究[J].中国药学杂志,2000,35(11)：741.
[56]陈春,杨天伦TXA2/PGI2与心血管疾病[J].现代生物医学进展.2008,8(11)：2166-2172.
[57]徐红梅,刘清云,戴敏,等.赤芍总苷对大鼠血小板功能的影响.合肥工业大学学报(自然科学版).2003,26(1)：141-144.
[58]高会丽,李贻奎,仝燕,等.冠心Ⅱ号系列组方对犬急性心肌缺血保护作用的比较研究[J].中药药理与临床,2007,23(5)：1-4.
[59]蓝肇熙,王万智,马忆南,等.桃红四物汤对大鼠损伤血淤证中TXB2、6-keto-PGF1α的影响[J].华西药学杂志,2008,23(6)：687-688.
[60]武源,郭宏宝,王铁军,等.几种中药组分对家兔体外血小板聚集作用的比较[J].中国临床药理学与治疗学,2007,12(9)：1047-1051.
[61]吁文贵,徐理纳.乙酰丹酚酸A对血小板功能的影响[J].药学学报,1994,29(6)：412.
[62]袁淑娟,张志伟,高天红,朴晋华.红花注射液抗血栓作用机制研究[J].中国中药杂志,2011,36(11)：1528-1529.
[63]王振义,李家增,阮长耿.血栓与止血基础理论与临床[M].上海：上海科学技术出版社,2004：63-71.
[64]许澍淮.环核苷酸与血小板功能[J].生理科学进展,1992,23(4)：318-322.
[65]祝国光,罗瑞芝,郭治听.复方丹参滴丸抗血小板活化及聚集性研究进展[J].中国心血管杂志,2007,12(2)：149-151.
[66]杨佳,秦彩玲.复方丹参方及丹参、三七对血小板功能影响的研究概况[J].中国实验方剂学杂志,2003,9(2)：59-62.
[67]裴海云,韩颖.信号转导与血小板激活[J].中国实验血液学杂志,2004,12(5)： 704-707.
[68]卢建,余应年,徐仁宝.受体信号转导系统与疾病.济南：山东科学技术出版社,1999.
[69]Cosemans JM, Munnix IC, Wetzker R, et al. Continuous signaling via PI3K isoforms beta and gamma is required for platelet ADP receptor function in dynamic thrombus stabilization [J]. Blood.2006,108 (9):3045-3052.
[70]Li Z, Zhang G, Le Breton GC, et al. Two waves of platelet secretion induced by thromboxane A2 receptor and a critical role for phosphoinositide 3-kinases [J]. The Journal of Biological Chemistry.2003,278(33):30725-30731.
[71]Kroner C, Eybrechts K, Akkerman JW. Dual regulation of platelet protein kinase B. The Journal of biological chemistry.2000,275 (36):27790-27798.
[72]Huang ZS, Zeng CL, Zhu LJ, Jiang L, Li N, Hu H.Salvianolic acid A inhibits platelet activation and arterial thrombosis via inhibition of phosphoinositide 3-kinase [J]. J Thromb Haemost,2010,8(6):1383-1393.
[73]CHEN Ke-ji, SHI Da-zhuo, XU Hao, et al. XS0601 reduces the incidence of restenosis:a prospective study of 335 patients undergoing percutaneous coronary intervention in China [J]. Chinese Medical Journal,2006,119(1):6-13.
[74]Garci'a A. Clinical proteomics in platelet research:challenges ahead [J]. J Thromb Haemost 2010; 8:1784-1785.
[75]Thiele T, Steil L, Gebhard S, Scharf C, et al. Profiling of alterations in platelet proteins during storage of platelet concentrates [J]. Transfusion 2007; 47:1221-1233;
[76]Cristina Banfi, Maura Brioschi, Giancarlo Marenzi, De Metrio M, Camera M, Mussoni L, et al. Proteome of platelets in patients with coronary artery disease [J]. Experimental Hematology 2010; 38:341-350.
[77]Senzel L, Gnatenko DV, Bahou WF. The platelet proteome [J]. Curr Opin Hematol 2009; 16:329-333.
[78]李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究[J].中国分子心脏病学杂志,2009,09(6)：326-331.
1. Lena Brydon, Kesson Magid, Andrew Steptoe. Platelets, coronary heart disease, and stress. Brain, Behavior, and Immunity 2006; 20(2):113-119.
2.张京春,陈可冀.病证结合是中西医结合临床的最佳模式[J].世界中医药,2006,1：14-15.
3. Faxon DP, Freedman JE. Facts and controversies of aspirin and clopidogrel therapy. Am Heart J.2009,157(3):412-22.
4. Wang TH, BhattDL, Topol EJ. Aspirin and clopidogrel resistance:an emerging clinical entity. Eur Heart J,2006,27:647-654.
5. Frelinger AL, Furman MI, Linden MD, et al. Residual Arachidonic Acid-Induced Platelet Activation via an Adenosine iphosphate-Dependent but cyclooxygenase-1-and Cyclooxygenase-2 -Independent Pathway A 700-Patient Study of Aspirin Resistance. Circulation.2006,113:2888-2896.
6.徐浩,文川,陈可冀,等.川芍、赤芍及其有效部位配伍对载脂蛋白E基因缺陷小鼠动脉粥样硬化斑块稳定性影响的研究[J].中国中西医结合杂志2007,27(6)：513-518
7.李立志,刘剑刚,马鲁波,等.芎芍胶囊对兔动脉粥样硬化模型脂质代谢及血小板聚集的影响[J].中国中西医结合杂志,2008,28(12)：1100-1103.
8.严萍,林久茂,陈宇宁,等.芎芍胶囊对心肌梗死大鼠缺血心肌mRNA表达水平的影响[J].中国中医急症,2011,20(9)：1426-1427.
9.徐凤芹,徐浩,刘剑刚,等.芎芍胶囊对动脉粥样硬化兔血管平滑肌细胞增殖的影响[J].中国中西医结合杂志,2008,28(10)：912-917.
10.鹿小燕,史大卓,徐浩,等.芎芍胶囊干预冠心病介入治疗后再狭窄的研究[J].中国中西医结合杂志2006,26(1)：13-17
11. CHEN KJ, SHI DZ, XU H, et al. XS0601 reduces the incidence of restenosis:a prospective study of 335 patients undergoing percutaneous coronary intervention in China [J]. Chinese Medical Journal,2006,119(1):6-13.
12.李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究[J].中国分子心脏病学杂志2009；9(6)：326-331.
1. Gibbons RJ, Chatterjee K, Daley J, et al. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina:a report of the American college of cardiology/American Heart Association task force on practice guidelines (Committee on management of patients with chronic stable angina). J Am Coll Cardio,1999,33(7):2092-2197.
2. Braunwald E, Antman EM, Kupersmith JW, et al. ACC/AHA 2002 guideline update for the management of patients with unstable angina and non-ST-segment elevation myocaridal infarction-summary article:A report of the American college of cardiology/American Heart Association task force on practice guidelines (committee on the management of patients with unstable angina) J Am Coll Cardio,2002,40(7): 1266-1374.
3. Davi G, Patrono C. Platelet activation and atherothrombosis [J]. N Engl J Med 2007; 357(24):2482-2494.
4. Moliterno DJ. Advances in antiplatelet therapy for ACS and PCI [J]. J Interv Cardiol 2008;21(S11):S18-24.
5. Garci'a A. Clinical proteomics in platelet research:challenges ahead [J]. J Thromb Haemost 2010; 8:1784-1785.
6. Thiele T, SteilL, Gebhard S, Scharf C, et al. Profiling of alterations in platelet proteins during storage of platelet concentrates [J]. Transfusion 2007; 47:1221-1233;
7. Cristina Banfi, Maura Brioschi, Giancarlo Marenzi, De Metrio M, Camera M, Mussoni L,et al. Proteome of platelets in patients with coronary artery disease [J]. Experimental Hematology 2010; 38:341-350.
8. Senzel L, Gnatenko DV, Bahou WF. The platelet proteome [J]. CurrOpin Hematol 2009; 16:329-333.
9.陈可冀,薛梅,殷惠军.血小板活化与冠状动脉粥样硬化性心脏病和血瘀证的关系[J].首都医科大学学报,2008,29(3)：266-269.
10. Xue mei,Chen keji,Yin huijun. Relationship between platelet activation related factors and polymorphism of related genes in patients with coronary heart disease of blood-stasis syndrome [J]. Chin J Integr Med 2008;14(4):267-273.
11.李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究[J].中国分子心脏病学杂志2009；9(6)：326-331.
12. Yin HL, Stossel TP. Control of cytop lasmic actin gelsol transformation by gelsolin, a calcium dependent regulatory protein [J]. Nature 1979; 281(5732):583-586.
13. Snabes MC, Boyd AE 3rd, Bryan J. Detection of actin-binding proteins in human platelets by 1251-actin overlay of polyacrylamide gels [J]. J Cell Biol 1981; 90(3):809-812.
14. Harris HE, Bamburg JR, Weeds AG. Actin filament disassembly in blood plasma [J]. FEBS Lett.1980; 121(1):175-177.
15. Lee WM, Galbraith RM. The extracellular actin scavenger system and actin toxicity [J]. N Engl J Med 1992; 326(20):1335-1341.
16.倪晓光,赵平.凝溶胶蛋白的结构和功能及其抑制肿瘤的作用[J].实用癌症杂志2007；22(1)：105-107.
17.刘玥,蒋跃绒,殷惠军,陈可冀.凝溶胶蛋白与心血管疾病[J].中国分子心脏病学杂志2011；11(1)：50-53.
18. de la Chapelle A, Tolvanen R, Boysen G, Santavy J, Bleeker-Wagemakers L, Maury CP, Kere J.Gelsolin-derived familial amyloidosis caused by asparagine or tyrosine substitution for aspartic acid at residue 187 [J].Nat Genet 1992;2(2):157-160.
19. Suhler E, Lin W, Yin HL. Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock and myonecrosis [J]. Crit Care Med 1997; 25(4):594-598.
1. Gibbons RJ, Chatterjee K, Daley J, et al. ACC/AHA/ACP-ASIM guidelines for the management of patients with chronic stable angina:a report of the American college of cardiology/American Heart Association task force on practice guidelines (Committee on management of patients with chronic stable angina). J Am Coll Cardio,1999,33(7):2092-2197.
2. Braunwald E, Antman EM, Kupersmith JW, et al. ACC/AHA 2002 guideline update for the management of patients with unstable angina and non-ST-segment elevation myocaridal infarction-summary article:A report of the American college of cardiology/American Heart Association task force on practice guidelines (committee on the management of patients with unstable angina) J Am Coll Cardio,2002,40(7): 1266-1374.
3.庄明明,温奕欣,刘少列,等.流式细胞仪测定血小板胞浆游离钙浓度.西安交通大学学报(医学版),2005；26(5)：508-510.
4. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem,1985,260(6):3440-3450.
5. Antoniades C, Bakogiannis C, Tousoulis D, Demosthenous M, Marinou K, Stefanadis C. Platelet activation in atherogenesis associated with low-grade inflammation. Inflamm Allergy Drug Targets,2010,12,9(5):334-45.
6. Michelson A D, Furman M I. Laboratory markers of platelet activation and their clinical significance. Curr Opin Hematol,1999,6(5):342-348.
7. Oestreich JH, Holt J, Dunn SP. Considerable variability in platelet activity among patients with coronary artery disease in response to an increased maintenance dose of clopidogrel. Coronary Artery Disease 2009,20(3):207-213
8. Xue mei, Chen keji, Yin huijun. Relationship between platelet activation related factors and polymorphism of related genes in patients with coronary heart disease of blood-stasis syndrome. Chin J Integr Med,2008,14(4):267-273.
9.李雪峰,蒋跃绒,吴彩凤,陈可冀,殷惠军.冠心病血小板功能蛋白与证侯相关性研究.中国分子心脏病学杂志,2009,9(6)：326-331.
10. Spinardi L, Witke W. Gelsolin and diseases. Subcell Biochem 2007; 45:55-69.
11. Carol A. Vasconcellos, Stuart E. Lind. Coordinated Inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein. Blood,1997, 82(12):3648-365.
12. Lee WM, Galbraith RM. The extracellular actin scavenger system and actin toxicity. N Engl J Med,1992,326(20):1335-1341.
13. Suhler E, Lin W, Yin HL. Decreased plasma gelsolin concentrations in acute liver failure, myocardial infarction, septic shock and myonecrosis. Crit Care Med,1997, 25(4):594-598.
14. Lopez-Farre A J, Mateos-Caceres P J, Sacristan D, et al. Relationship between vitamin D binding protein and aspirin resistance in coronary ischemic patients:A proteomic study. J Proteome Res,2007,6(7):2481-2487
15. Kato M, Kambe M, Kajiyama G.Increased cytosolic free Mg2+ and Ca2+ in Platelets of patients with vasospastic. Am J Physiol,1998,274:548-554.
16. Fujinishi A, Takahara K, Ohba C, et al. Effects of nisoldipine on cytosolic calcium, platelet aggregation, and coagulation/fibrinolysis in patients with coronary artery disease. Angiology,1997,48(6):515-521.
1. Carol A. Vasconcellos, Stuart E. Lind. Coordinated Inhibition of actin-induced platelet aggregation by plasma gelsolin and vitamin D-binding protein. Blood,1997, 82(12):3648-3657.
2. Martin Quinn, Desmond Fitzgerald/卢瑾,郭如华(译).血小板功能：评估、诊断和治疗.北京：科学出版社,2008：3-4.
3. Jennings LK. Mechanisms of platelet activation:need for new strategies to protect against platelet-mediated atherothrombosis. Thromb Haemost 2009,102(2):248-257
4. Gladding P, Webster M, Ormiston J, et al. Antiplatelet drug nonresponseness. Am Heart J.2008,155 (4):591-599.
5. Navarro-Nunez L, Lozano ML, Palomo M, et al. Apigenin inhibits platelet adhesion and thrombus formation and synergizes with aspirin in the suppression of the arachidonic acid pathway. J Agric Food Chem 2008,56(9):2970-2976
6. Mousa SA, Jeske WP, Fareed J. Prasugrel:a novel platelet ADP P2Y (12) receptor antagonist. Methods Mol Biol,2010,663:221-228.
7.石鑫,宋丽,苏琦,刘雅文,程熠.凝血酶GPIb-Ⅸ-Ⅴ受体信号转导通路研究进展.中国老年学杂志,2012,3(2)：648-650.
8. Haddad JG, Harper KD, Guoth M, et al. Angiopathic consequences of saturating the plasma scavenger system for actin. Proc Nail Acad Sci USA,1990,87(4):1381-1385.
9.项耀祖,商洪才,张伯礼.抗血小板中药研究进展.中草药,2008,39(2)：290-294
10.史秀丽,傅佳,李光武.血小板靶向中药有效成分及单体研究进展.中国中药杂志,2006,31(5)：361-365.
11.刘剑刚,徐凤芹,史大卓,董国菊.川芎赤芍提取物不同配比的活血化瘀作用研究.中药新药与临床药理,2005,16(5)：315-317.
12.刘超,王静,杨军.赤芍总苷活血化瘀作用的研究.中药材,2000,23(9)：557-560.
13.管昌益.川芎嗪在心血管领域的应用现状与存在问题.中国中西医结合杂志,2003,23(5)：378.
1.徐叔云,卞如濂,陈修.药理实验方法学(第3版).北京：人民卫生出版社,2002.
2.庄明明,温奕欣,刘少列,等.流式细胞仪测定血小板胞浆游离钙浓度[J].西安交通大学学报(医学版),2005,26(5)：508-510.
3. Oestreich JH, Holt J, Dunn SP. Considerable variability in platelet activity among patients with coronary artery disease in response to an increased maintenance dose of clopidogrel [J]. Coronary Artery Disease,2009,20(3):207-213
4. Terpening C. An appraisal of dual antiplatelet therapy with clopidogrel and aspirin for prevention of cardiovascular events [J]. J Am Board Fam Med,2009,22(1):51-56
5. Reaume KT, Regal RE, Dorsch MP.Indications for dual antiplatelet therapy with aspirin and clopidogrel:evidence-based recommendations for use [J]. Ann Pharmacother,2008,42(4):550-557.
6. Wang TH, BhattDL, Topol EJ. Aspirin and clopidogrel resistance:an emerging clinical entity [J]. Eur Heart J,2006,27:647-654.
7.王玲玲,沈薇,Doffoel Michel抗凝和/或抗血小板药物致消化道出血的临床特点及影响因素[J].胃肠病学和肝病学杂志,2011,20(6)：542-545.
8. Angiolillo DJ, Guzman LA, Bass TA. Current antiplatelet therapies:benefits and limitations [J]. Am Heart J,2008,156(2 Suppl):3-9
9.徐浩,文川,陈可冀,等.川芍、赤芍及其有效部位配伍对载脂蛋白E基因缺陷小鼠动脉粥样硬化斑块稳定性影响的研究[J].中国中西医结合杂志2007,27(6)：513-518
10.李立志,刘剑刚,马鲁波,等.芎芍胶囊对兔动脉粥样硬化模型脂质代谢及血小板聚集的影响[J].中国中西医结合杂志,2008,28(12)：1100-1103.
11.严萍,林久茂,陈宇宁,等.芎芍胶囊对心肌梗死大鼠缺血心肌mRNA表达水平的影响[J].中国中医急症,2011,20(9)：1426-1427.
12.徐凤芹,徐浩,刘剑刚,等.芎芍胶囊对动脉粥样硬化兔血管平滑肌细胞增殖的影响[J].中国中西医结合杂志,2008,28(10)：912-917.
13.鹿小燕,史大卓,徐浩,等.芎芍胶囊干预冠心病介入治疗后再狭窄的研究[J].中国中西医结合杂志2006,26(1)：13-17
14. CHEN KJ, SHI DZ, XU H, et al. XS0601 reduces the incidence of restenosis:a prospective study of 335 patients undergoing percutaneous coronary intervention in China [J]. Chinese Medical Journal,2006,119(1):6-13.
15. Lee WM, Galbraith RM. The extracellular actin scavenger system and actin toxicity [J]. N EngI J Mcd,1992,326(20):1335-1341.
16. Messaris E. Actin-binding plasma gelsolin:A potential future ally in the fight against sepsis [J].Crit Care Med.2007,35(3):970-971.
17. Yang J, Moravec CS, Sussman MA, et al. Decreased SLIM1 expression and increased gelsolin expression in failing human hearts measured by high-density oligonucleotide arrays[J]. Circulation.2000,102(25):3046-3052.