血管内皮下层纤维连接素在血小板粘附中的作用

本文利用扫描电镜和荧光分光光度计技术,研究了血管内皮下层Fn在血小板与血管壁粘附中的作用.当内皮细胞剥脱后血管壁上可见大量血小板粘附:当裸露的内皮下层失与抗Fn抗体反应后,血小板的相对粘附率降低了70.1％(P<0.01),而正常血清对照组无明显变化,说明Fn在血小板粘附中起重要作用.     

vWF的分子生物学

vWF(von Willehrand Factor)是一种与止血过程有关的糖蛋白。它由血管内皮细胞和巨核细胞合成,可在血小板的α颗粒、血浆和内皮下层发现。在血浆中,vWF以多聚体形式存在。多聚体由几乎完全相同的成熟亚基组成。每多聚体中亚基数目可从2个到20余个不等。vWF至少有2个与止血机制相关的生物学功能:①介导血小板粘附于受损的血管壁;②在循环中与FⅧ:C结合并稳定其活性。vWF的分子缺陷或生成不足可导致vWD(von Wille-     

血小板与血管内皮下层粘附的体外实验模型

报道了一种研究血小板与血管壁内皮下层粘附的体外实验模型。血管壁经1.0％NP-40处理5min,300mmo/L氯化钾处理10min,内皮剥脱。血小板用吖啶橙标记,血小板内可见橙黄色颗粒。血管壁与血小板反应后,扫描电镜观察可见内皮下层表面有大量血小板粘附。用2.0％SDS洗脱粘附的血小板,用荧光分光光度计测定洗脱血小板的荧光强度,结果表明,吖啶橙标记后血小板浓度与其荧光强度成正比。粘附血小板个数用下式计算: 每块标本粘附的血小板个数-标准管血小板浓度/标准管荧光强度×测定管体积×测定管荧光强度     

血小板与血管壁粘附的扫描电镜观察

利用血小板粘附体外实验模型和扫描电镜技术,研究了内皮细胞不同程度损伤后粘附血小板的形态特征和内皮下层的纤维连接素在血小板粘附中的作用。粘附血小板表现为接触型、粘合型、铺展型和血小板聚集体4种类型。当内皮细胞剥脱后,内皮下层可见大量接触型、粘合型、铺展型血小板和少量血小板聚集体。当内皮下层的纤维连接素被去除或其活性被阻断后,粘附血小板减少,来见铺展型血小板,说明纤维连接素是参与血小板粘附的重要因素之一,主要是参与血小板在内皮下层表面的铺展。     

血小板-血管壁的相互作用和遗传性血小板病

在受损血管处刹住流血的最初阶段,包括一个血管收缩降低局部血压的过程和血小板跟血管壁相互作用的过程。后者亦称为止血机制:血小板粘附于受损血管,随之释放血小板成份,形成内过氧化物(endoperoxide)和血栓素(thromboxane),同时活化血液凝固机制,而血小板的凝聚反应(aggregation)又加速凝血过程,最终形成纤维蛋白。血管壁的组成包括内皮、介质和外膜;血管内皮是对抗血栓形成的,而内皮下成份则促进血栓形成。在电子显微镜下血管内皮     

2型糖尿病中内皮细胞损伤及血小板功能状态的改变

目的:观察2型糖尿病患者内皮细胞损伤及血小板功能状态的变化及临床意义. 方法:利用双抗体夹心( ELISA)法测定血浆血管性血友病因子抗原(vWF:Ag)水平;采用玻珠柱法测定血小板黏附率(PAdT). 结果:vWF:Ag(160.8±35.6)%、PAdT(89.5±7.8)%水平显著高于无血管病变组,差异有显著意义(P<0.05～0.01). 结论:2型糖尿病患者均存在血管内皮损伤,并由此引起血小板功能状态的改变;血浆vWF:Ag及PAdT水平在一定程度上可反映2型糖尿病患者血管病变的情况.     

内皮细胞损伤、血小板活化及功能状态与2型糖尿病的相关性

目的: 观察2型糖尿病患者内皮细胞损伤、血小板活化及功能状态的变化及临床意义. 方法: 利用双抗体夹心 ELISA法测定血浆血管性血友病因子抗原vWF∶ Ag,血小板膜表面α-颗粒膜蛋白GMP-140水平;采用玻珠柱法测定血小板黏附率PAdT. 结果: 糖尿病组血浆vWF∶ Ag(145.6±25.6)%,GMP-140(16.8±3.5)μg/L,PAdT(80.3±5.3)%水平显著高于对照组(P<0.05);有血管病变的患者血浆vWF∶ Ag(160.8±35.6)%,GMP-140(18.9±5.5)μg/L,PAdT(89.5±7.8)%水平显著高于无血管病变组(P<0.05);相关分析显示,GMP-140,PAdT水平与血浆vWF∶ Ag水平显著正相关(r=0.446,0.413,P均<0.05). 结论: 2型糖尿病患者均存在血管内皮损伤,并由此引起血小板活化及血小板功能状态的改变;血浆vWF:Ag,GMP-140及PAdT水平在一定程度上可反映2型糖尿病患者血管病变的情况.     

血小板的止血功能与出血性疾病

在初期止血和血栓形成过程中血小板起着重要作用。当血管内皮损害暴露内皮下组分时血小板就会被活化 ,发生血小板的粘附和聚集。血小板的粘附、聚集和活化反应是通过血小板膜糖蛋白的功能而实现的。目前进行深入研究的有下列 3类血小板膜糖蛋白 :( 1 )糖蛋白 (ＧＰ)Ｉｂ -ＩＸ作为粘附蛋白ｖＷＦ受体参与血小板粘附于受损血管壁的反应。 ( 2 )ＧＰＩＩｂ -ＩＩＩａ作为粘附蛋白纤维蛋白原 (Ｆｂｇ)的受体在血小板聚集和血栓形成中起重要作用。 ( 3)ＧＭＰ - 1 40 (亦称Ｐ -Ｓｅｌｅｃｔｉｎ)在血小板活化后暴露在血小板膜表面 ,并作为ＰＳＧ…     

血小板膜糖蛋白多态性与血栓形成的关系

血管内皮损伤时,血小板通过以下3个分子机制粘附到内皮下结缔组织:血小板膜上的糖蛋白Ⅰ b-Ⅸ-Ⅴ复合物(Gp Ⅰ b-Ⅸ-Ⅴ)和已结合在胶原上的vWF结合;血小板上的糖蛋白IaⅡa(GP Ⅰ a-Ⅱa)直接与胶原结合;血小板上的糖蛋白Ⅰc-Ⅱa(GP Ⅰ c-Ⅱa)和纤维连接蛋白结合。血小板粘附后发生一系列生物化学反应如:释放ADP、在血小板表面生成凝血酶。ADP和凝血酶通过纤维蛋白原及其血小板膜糖蛋白受体(如GPⅡ     

氯沙坦、福辛普利和硝苯地平控释剂对原发性高血压患者血管内皮功能的保护作用

为评价和比较氯沙坦、福辛普利和硝苯地平控释剂对原发性高血压 (简称高血压病 )患者血管内皮功能的保护作用 ,分别应用以上 3种药物对轻中度高血压病患者进行治疗 ,并与对照组比较。应用放射免疫分析法、酶法及酶联免疫吸附法观察比较治疗前及治疗 4周末对血管内皮功能的影响。结果发现高血压病患者血浆内皮素 (ET)、血小板α 颗粒膜蛋白 (GMP1 4 0 )、血管性假血友病因子抗原 (vWF)显著高于正常对照组 (P <0 .0 5,P <0 .0 1 ) ,一氧化氮(NO)与对照组相比无显著性差异 ,但NO/ET显著低于对照组 (P <0 .0 5)。 3种药物治疗 4周后 ,血浆ET、vWF均较治疗前显著降低 (P <0 .0 5,P <0 .0 1 ) ,但vWF仍高于正常对照组 (P <0 .0 5) ;血浆NO及NO/ET较治疗前及正常对照组显著升高 (P <0 .0 5,P <0 .0 1 ) ;与氯沙坦和福辛普利组相比 ,硝苯地平控释剂组血浆GMP1 4 0治疗后显著降低(P <0 .0 5)。提示以上 3种药均能有效改善高血压病早期的血管内皮功能损害 ,硝苯地平控释剂对GMP1 4 0的改善作用优于氯沙坦组和福辛普利组     

体外血小板流动腔研究方法的建立和应用

目的建立模拟体内血液流动环境下，实时观察、研究血小板与血管表面血管性假性血友病因子（vWF）相互作用的模型。方法利用包被vWF的细玻璃管模拟血管，分别采用负压装置、蠕动泵和恒流注射泵产生不同流态和精确剪切率的流体环境，结合倒置显微镜和电荷耦合器件图像传感器（CCD，Charge—coupled device）实时观察和记录血小板或细胞在流体作用下的一系列反应。结果血小板和转染了血小板糖蛋白Ⅰb-Ⅸ的中国仓鼠卵巢细胞（Chinese hamster ovary，CHO），在不同流态下都可与玻璃管壁上的vWF相互作用，从而介导血小板或CHO细胞与玻璃管壁的瞬时结合（滚动），并最终牢固结合在玻璃管壁上。结论本研究方法可以在精确剪切率和不同流态的流体环境条件下，实时地观察、记录血小板与vWF相互作用的情况，为血小板各种生理功能的研究以及相关药物的研究应用创造条件。     

vWF与GPⅡb/Ⅲa在血小板黏附、聚集中的作用及与PCI术并发症的关系

遗传性假血友病因子(vWF)是一种黏附、多聚糖蛋白,分布在血浆、血小板和内皮下,在血小板黏附到内皮下的过程中起着重要作用。GPⅡb/Ⅲa在凝血过程中的血小板黏附和聚集中也起关键作用,血小板激活导致GPⅡb/Ⅲa构型发生变化,继而使GPⅡb/Ⅲa受体与配体纤维蛋白原的结合加强,纤维蛋白原在相邻血小板分子间形成桥梁,导致血小板聚集的加速。经皮冠状动脉介入治疗术后并发症的发生与血小板的激活、血栓形成有关,本文旨在简述vWF和Ⅱb/Ⅲ在血小板黏附和聚集中的作用及其与经皮冠状动脉介入治疗(PCI)术后并发症发生的关系。     

The Role of Fibronectin in Arterial Subendothelium in Platelet Adhesion

The role of fibronectin in vascular subendothelium in platelet adhesion after the removal ofthe endothelium of aorta was studied in normal rabbits. The thoracic aortae were treated with 1%Nonidet P-40 for 5 min and with 0. 3 mol/L KCl for 10 min. The whole endothelium was re-moved and a large amount of fibronectin in the subendothelium was exposed. Autogenous platelet-rich plasma was isolated and platelets were labeled with 1:1000 acridine orange. The adheredplatelets were washed away with 2% SDS solution. Finally, the intensity of fluorescence of the wash-ing fluid was measured with a spectrofluorophotometer. The animals were divided into 4 groups:(1) normal endothelium group. (2) lysed endothelium group, (3) lysed endothelium and guineapig anti-rabbit fibronectin antisera inhibition group, (4) lysed endothelium and guinea pig normalsera control group. The adhesion of platelets in the 2nd group was taken as 100%. The resultsshowed that after the treatment of anti-rabbit fibronectin antisera the platelet adhesion onsubendothelium was inhibited up to 70. 1%. It is suggested that the fibronectin in subendotheliumplays an important role in platelet adhesion.     

Application of a monoclonal antibody SZ-51 specific for activated human platelets in canine thrombosis imaging]

To detect specifically the localization of thrombus in vivo, we prepared a monoclonal antibody (McAb) SZ-51 specific for an alpha-granule membrane protein (GMP-140) exposed on the surface of activated human platelets. 131I labeled SZ-51 antibody combined preferentially with thrombus induced by thrombin in vitro. Owing to McAb SZ-51 crossreaction with the activated platelets of dogs, thrombi in both femoral artery and vein were prepared and imaged with SPECT in dogs. The ratio of radioactivity between thrombi and blood pool was significantly increased in both arterial and venous thrombus after injection of 131I-SZ-51, especially at the 4th hour. Moreover, the imaging of arterial thrombus is better than that of venous thrombus. However, both arterial and venous thrombi could not be imaged by the injection of 131I-nonimmune IgG. These findings were in agreement with the radioactivity ratios between the removed thrombi and blood 24 hours after injection. Therefore, these results indicate that McAb SZ-51 has the power of detecting thrombi in vivo.     

抗血小板膜糖蛋白Ⅰba／Ⅲa双特异单链抗体的构建及其功能研究

目的：构建、表达抗血小板膜糖蛋白（GP）Ibα/GPⅢa双特异单链抗体,为其进一步研究、应用奠定基础。方法：应用PCR技术,扩增出接头片段和SZ-21单链抗体基因,克隆入pUm-T载体,测序分析。应用基因重组技术将接头片段、SZ-21单链抗体基因和SZ-2单链抗体基因重组拼接,构建SZ-2/SZ-21双特异单链抗体表达载体pET22-21-L-2scFv,并测序验证。导入大肠杆菌BL21(DE3)Plys,诱导表达。流式细胞术、ELISA、Western印迹检测SZ-2/SZ-21双特异单链抗体与血小板结合能力,瑞斯托霉素、凝血酶和ASP诱导血小板聚集试验对表达产物功能进行研究。结果：表达载体pET22-21-L-2scFv构建拼接正确;表达产物以包涵体形式为主,表达量占菌体总蛋白的14％;表达产物具有与血小板以及血小板GP Ibα和Ⅲa结合的活性,可抑制瑞斯托霉素、凝血酶和ADP诱导的血小板聚集,此作用呈剂量依赖性。结论：成功表达了SZ-2/SZ-21双特异单链抗体,该抗体具有与相应2相靶抗原结合的活性,并可抑制瑞斯托霉素、凝血酶和ADP诱导的血小板聚集。     

Specific inhibiting effects of Ilexonin A on von Willebrand factor-dependent platelet aggregation under high shear rate

Background Ilexonin A (IA), purified from the Chinese herbal medicine Maodongqing (Ilex pubescens Hook, et Arn) has been commonly used in south China to treat thrombotic disorders. In this study, we aimed to study the inhibiting effects and mechanism of IA on von Willebrand factor (vWF)-dependent high shear-induced platelet aggregation. Methods vWF-dependent high shear (10800 s-1) induced aggregation of platelets obtained from normal donors in the presence or absence of IA was measured by a modified cone-plate viscometer and shear-induced vWF binding was measured by quantitative flowcytometry with monoclonal antibody known to bind exclusively to the C-terminal domain of vWF (LJ-C3) directly labeled with fluorescein isothiocyanate (FITC). P-selectin surface expression was also measured by a similar method with FITC conjugated anti-P-selectin monoclonal antibody (WGA1). Results Shear-induced platelet aggregation was inhibited by IA in a dose-dependent manner. The extent of aggregation decreased from (78.6±4.6)% in the absence of IA to (36.5±2.1)% in the presence of IA (3.3 mmol/L) (P&lt;0.0001, n=9) with a high shear rate of 10800 s-1. vWF binding and P-selectin expression were also inhibited by IA in a dose dependent manner. The number of binding FITC-LJ-C3 molecules increased after exposure of platelet-rich plasma to a high shear rate of 10800 s-1 for 6 minutes, but this shear-induced increased binding platelet surface vWF molecules and P-selectin expression can be decreased in the presence of IA.Conclusion vWF binding and vWF mediated platelet activation, aggregation occurring under high shear rate were inhibited by IA. IA may be a unique antithrombotic drug inhibiting the vWF-GP Ibα interaction, and may thus facilitate drug design targeting arterial thrombosis.     

老年大鼠血小板反应性及血小板膜LDL受体活性的改变

本文利用受体放射配基分析方法观察不同年龄大鼠血小板聚集性、血浆脂质水平及血小板膜LDL受体活性的改变。结果表明,老年大鼠血小板对ADP诱导的聚集反应性明显增强;血浆总胆固醇、低密度脂蛋白胆固醇水平增高;血小板膜LDL受体活性增强,解离常数显著低于对照组。本文作者认为血小板膜受体活性的增强及血浆LDL水平的增高可促进LDL与血小板的结合,提高了LDL对血小板功能的影响,与老年大鼠血小板高反应性改变可能有关。     

THE IMPAIRMENT OF PLATELET FUNCTION IN FIBRINOLYSIS AND PRESERVING EFFECT OF APROTININ

Platelet adhesion depends on the platelet membrane glycoprotein Ib (GPIb) and plasma von Willebrand Factor (vWF), which can be reflected by ristocetin-induced aggregation. Here we report damage effect of fibrinolysis and preserving effect of aprotinin on platelet function. Addition of 40 U/ml urokinase and 0.3 U/ml plasmin to PRP or washed platelets made the ristocetin-induced aggregation decline to 31.6% and 38.5% of control value respectively. The extent of declining was positively correlated with the concentration of urokinase and plasmin. Meanwhile, the platelet GPIb decreased to 76.4% of control value. The results showed that the fibrinolysis impaired the platelet function and this effect may be associated with the hydrolysis of GPIb. Further research found that by adding the same dose of urokinase or plasmin to aprotinin-pretreated PRP or washed platelets, the aggregation did not change statistically and decrement of GPIb is much less marked. We concluded that the aprotinin could relieve the platelet dsfunction effectively by its inhibitory effect on fibrinolytic activity.