剪切应力作用下血小板活化反应对血栓形成的影响

目的：通过剪切应力对血小板细胞骨架和细胞内游离钙离子浓度([Ca^2+]i)及血栓形成的影响，探讨物理作用下血小板活化机制。方法：利用锥板式血小板聚集仪、SDS-PAGE方法及Fura-2／AM法观察剪切应力对血小板聚集、细胞支架和胞浆内[Ca^2+]i变化。结果：剪切前肌动蛋白纤维含量为(40．16&;#177;15．03)％，细胞内[Ca^2+]i浓度为(29．88&;#177;8．89)nmol／L，低剪切与高剪切作用1min后肌动蛋白纤维含量分别达(47．42&;#177;18．00)％与(48．62&;#177;18．04)％，而细胞内[Ca^2+]i浓度升高达(57．32&;#177;18．32)nmol／L及(128．28&;#177;56．28)nmol／L，与剪切前相比差异均有显著性意义(P&;lt;0．05或0．01)，此外，钙调蛋白抑制剂阻碍血小板肌动蛋白纤维的形成及血小板聚集。结论：剪切应力作用下血小板发生了肌动蛋白纤维含量及细胞内游离钙水平的增高，并与剪切诱导的血小板聚集存在着平行关系，物理作用对体内血栓形成的机制有助于对血栓形成和一级康复干预的进一步认识。     

血小板活化在炎症中的作用研究进展

炎症时存在复杂的白细胞与内皮、血小板与内皮、白细胞与血小板之间的反应,血小板及其活化产物广泛参与了炎症反应,并在其中发挥重要作用。现就近年来相关研究成果进行综述。     

血栓活化血小板放射性核素显像研究进展

血栓活化血小板放射性核素显像能够显示血栓,确定血栓部位,鉴别新鲜与陈旧血栓。本文对血小板活化机制、血栓活化血小板放射性核素显像的研究进展进行综述。     

刺激型血小板单克隆抗体的作用及其机制

刺激型血小板单克隆抗体的作用及其机制彭林综述李家增审校早在８０年代初期，人们在利用单克隆抗体（单抗）对不同类型的血细胞进行研究的过程中，发现某些血小板单抗能够引发血小板聚集、释放等激活现象。这以后，国内外文献又陆续报道了十几个可引起血小板激活的单抗，...     

心内膜功能异常和血小板活化在左心房血栓形成中作用的研究进展

风湿性二尖瓣狭窄是导致左心房血栓形成最为常见的原 因,血栓脱落常可导致脑及外周动脉的栓塞,引起重要脏器的缺 血甚至梗死,有极高的致死、致残率.研究表明,二尖瓣狭窄患 者左心房内膜组织存在损伤和结构异常[1] ,血小板活化、凝血功 能亢进及纤溶功能紊乱,左心房内血液呈高凝状态,血浆P-选择 素、血管性血友病因子(Vwf)等多种内皮源性因子在二尖瓣狭 窄左心房血栓形成患者左心房血中浓度明显升高,可能是左心 房血栓形成的重要分子机制[1-2] .本文对此方面的研究现状进 行综述.     

短暂预缺血对血小板活化的影响

缺血 /再灌注 (Ischemia/Reperfusion ,I/R)是造成组织严重损伤甚至坏死的重要原因 ,其中血栓形成是影响整个病理过程的重要环节。缺血预适应 (IschemicPreconditioning ,IP)的研究显示[1] :短暂预缺血通过多种保护机制削弱了I/R的损伤效应并改善预后 ,其中的机制之一是减轻I/R中血小板介导的血栓形成。由于只有活化的血小板才能介导血栓形成 ,因此短暂预缺血对I/R中血小板活化是否有直接影响值得研究。材料和方法1　动物模型　雄性SD大鼠 2 7只 ,体重 30 0～ 35 0 g ,随机分为 3组 (n =9) :①假手术组 (ShamOperation ,SO) ;②缺血…     

血小板活化的研究进展

血小板活化发生在各种缺血性心脑血管疾病如脑梗死、高血压、急性冠脉综合征等,有血栓形成倾向的疾病如弥散性血管内凝血(DIC),急性早幼粒细胞白血病,阵发性睡眠性血红蛋白尿症,急性肺栓塞,膜性增生性肾小球肾炎,房室瓣或主动     

木贼提取物对大鼠血小板聚集与血栓形成的影响

目的:研究木贼提取物对血小板聚集和血栓形成的影响,以期为脑卒中预防ntg药物干预提供实验基础。方法:实验于2004-06/07在锦州医学院药理学教研室完成。选用SD大鼠30只,以二磷酸腺苷(ADP)、胶原和凝血酶作诱导剂诱导血小板聚集,按比浊法用TYXN-96系列多功能智能血液凝集仪测定大鼠血小板聚集率;用血栓法测定大鼠血栓重量。结果:按1.0g/kg,3.0g/kg,10.0g/kg的木贼提取物给大鼠灌胃,均能抑制ADP、胶原和凝血酶诱导的大鼠血小板聚集,并能减轻血栓的重量。以上两种作用均呈现明显的剂量依赖性。结论:木贼提取物具有明显的抗血小板聚集及抗血栓形成的作用。     

不同剂量氯化镁对血小板聚集和血栓形成的影响

目的:观察不同剂量氯化镁对血小板聚集和血栓形成的影响,并与阿司匹林作用相比较。方法:实验于2004-06/07在锦州医学院药理学实验室完成。选用雄性SD大鼠50只,随机分为5组,生理盐水对照组;阿司匹林对照组;氯化镁低剂量组;氯化镁中剂量组;氯化镁高剂量组。给药方法均为经尾静脉给药,1次/d,连续3d。以二磷酸腺苷、胶原和凝血酶作诱导剂诱导血小板聚集,按比浊法用TYXN-96系列多功能智能血液凝集仪测定大鼠血小板聚集率;用血栓法测定大鼠血栓重量。结果:50只大鼠均进入结果分析。①对血小板聚集率抑制率:氯化镁高、中、低剂量组对二磷酸腺苷、胶原及凝血酶诱导的血小板聚集的抑制率,高剂量组优于中剂量组,优于低剂量组(55.5%,46.1%,42.4%;64.3%,59.3%,51.2%;49.5%,30.4%,20.0%),高、中、低剂量3组抑制率优于阿司匹林组(39.0%,55.3%,53.2%)。②对血栓形成的影响:氯化镁高、中剂量组使血栓质量减少65.8%和55.7%,小剂量组与阿司匹林组基本一致(50.7%,49.1%)。结论:小剂量氯化镁抑制血栓形成的作用与阿司匹林相当,随着剂量增大氯化镁抑制血栓形成的作用强于阿司匹林,说明氯化镁具有很好的抗血栓形成作用。     

莴笋提取物对大鼠血小板聚集与血栓形成的影响

目的:研究莴笋提取物对实验性大鼠血小板聚集和血栓形成的影响。方法:以APD、胶原和凝血酶作诱导剂诱导大鼠血小板聚集,按比浊法用TYXN-96系列多功能智能血液凝集仪测定大鼠血小板聚集率并计算聚集抑制率;用血栓法测定大鼠血栓形成重量。结果:按1.0g/kg,3.0g/kg,10.0g/kg的莴笋提取物给大鼠灌胃,均能抑制APD、胶原和凝血酶诱导的大鼠血小板聚集,并能减少血栓形成的重量。以上两种作用均呈现明显的剂量依赖性。结论:莴笋提取物具有明显的抗血小板聚集及抗血栓形成的作用。     

高剪切力活化血小板钙离子反应的研究

目的　检测细胞外钙离子 (Ca2 )浓度对高剪切力诱导血小板聚集的影响 ,并测定高剪切力活化血小板时血小板内Ca2 浓度的变化 ,探讨Ca2 在剪切力活化血小板中的作用。方法　用锥板黏度计施加高剪切力作用 ,以荧光抗体CD6 1PerCP标记血小板 ,流式细胞术 (FCM )测定血小板聚集率。以荧光染料Fluo 3AM标记血小板 ,FCM测定高剪切力作用后血小板内Ca2 浓度 ;并测定二磷酸腺苷 (ADP)对高剪切力作用下Ca2 反应的影响。结果　洗涤全血标本或加入钙离子螯合剂乙二醇四乙酸 (EGTA) ,造成细胞外低钙或无钙环境 ,高剪切力诱导的血小板聚集率由 5 9 6 %±5 1%下降到很低水平 ,而加入外源性Ca2 在一定程度上增强这种聚集。全血标本受到高剪切力作用 ,血小板内Ca2 浓度发生明显的升高 ;增加细胞外Ca2 浓度能增强这种反应 ,降低细胞外Ca2 浓度或阻断血小板膜糖蛋白GPⅠb/Ⅸ与血浆血管假性血友病因子 (vWF)之间的相互作用 ,使血小板Ca2 反应消失 ,而阻断GPⅡb/Ⅲa与血浆vWF之间的相互作用则无明显影响。低浓度的ADP与高剪切力对血小板钙离子反应有协同作用。结论　细胞外Ca2 是高剪切力诱导血小板聚集的必需条件 ,血小板内Ca2 反应可能是剪切力活化血小板时信息传递的重要物质基础。     

Role of cytoplasmic and releasable ADP in platelet aggregation induced by laminar shear stress

We examined the extent of lytic and sublytic platelet injury after exposure of platelets to shear stress and the role in shear-induced PAG of ADP liberated from platelets as a result of shear-induced platelet dense body release and/or platelet damage. Platelets in C-PRP or TAS were subjected to well-defined, laminar shear stress in a rotational viscometer, and PAG (loss of single, nonaggregated platelets), 14C-serotonin release, and loss from platelets of LDH and 51Cr were determined. Increased PAG with increasing shear stresses was associated with progressive loss of LDH and 51Cr. Loss of 51Cr was consistently in excess of that of LDH, indicating sublytic platelet injury, which was confirmed by electron microscopy. At the lowest shear stress used (50 dynes/cm2), PAG in C-PRP was observed in the absence of detectable loss of 51Cr or LDH. When platelets in TAS were sheared in the presence of CP/CPK, an enzyme system capable of removing extracellular ADP, PAG was only partially (approximately 40%) inhibited. However, when platelets were preincubated with CP/CPK and ATP (to saturate platelet ADP receptors), shear-induced PAG was almost completely suppressed. Similar results were obtained with PAG induced by collagen in the aggregometer. The findings indicate that (1) shear-induced PAG in this system may occur without measurable lytic or sublytic platelet damage and (2) ADP liberated from platelets as a result of shear-induced release or damage may represent the major if not sole mediator of shear-induced PAG.     

桂皮醛对抗血小板聚集和血栓形成的特点

目的：观察桂皮醛对血小板聚集和血栓形成的影响。方法：实验于2005—07／11在第四军医大学药学系药物研究所实验室完成。④体外血小板聚集实验：制备大鼠洗涤血小板，观察0．15，0．30和0．60mmol／L桂皮醛对胶原蛋白（100mg／L）和凝血酶（3U／mL）诱导的大鼠体外血小板聚集的抑制作用。桂皮醛购自中国医药集团上海化学试剂公司，纯度〉98％。②出、凝血时间测定及胶原蛋白-肾上腺素诱发体内血栓形成实验：取BALB／c小鼠60只，随机分成6组（n=10），生理盐水组灌胃生理盐水；阿司匹林组灌胃阿司匹林100mg／kg为阳性对照；还设置桂皮醛灌胃（250，500mg／kg）和腹腔注射（50，100mg／kg）4个剂量组。所有动物每天给药1次，10μL/g，连续11d。第10天给药后1h采用断尾法和玻片法测定小鼠出血、凝血时间；第11天给药后1h观察小鼠尾静脉注射胶原蛋白（3．57mg／kg）-肾上腺素（0．143mg／kg）混合血栓诱导剂后5min内的存活率。⑧动-静脉旁路血栓形成实验：SD大鼠48只，随机分为6组（n=8）。生理盐水组灌胃生理盐水；阿司匹林组灌胃阿司匹林80mg／kg为阳性对照；桂皮醛分为灌胃200，400mg／kg组和腹腔注射40，80mg/kg4个剂量组。所有动物每天给药1次，10μL／g，连续10d。末次给药后1h测定动-静脉旁路丝线上的血栓湿质量。结果外鼠60只和大鼠48只全部进入结果分析。①桂皮醛能够明显抑制胶原蛋白和凝血酶诱导的大鼠体外血小板聚集，并呈剂量依赖性，与对照组比较差异显著（P〈0．05，0．01）。②桂皮醛显著延长小鼠出、凝血时间，与对照组比较差异非常显著（P〈0．01）。⑧胶原蛋白-肾上腺素诱发性小鼠肺动脉栓塞存活率：生理盐水组为10％，阿司匹林组为80％，桂皮醛各剂量组为70％～100％。④动-静脉旁路丝线上的血栓湿质量：阿司匹林组和桂皮醛各剂量组均低于生理盐水组（P〈0．01）；桂皮醛各剂量组对大鼠血栓的抑制率范围为30％～43．4％。结论：桂皮醛体外能够明显抑制胶原蛋白和凝血酶诱导的大鼠血浆中血小板的聚集；体内能够显著延长小鼠断尾后的出、凝血时间，减轻大鼠动-静脉旁路丝线上血栓的质量。提示桂皮醛具有明显抗血小板聚集和体内抗血栓作用。     

桂皮醛对抗血小板聚集和血栓形成的特点

目的:观察桂皮醛对血小板聚集和血栓形成的影响。方法:实验于2005-07/11在第四军医大学药学系药物研究所实验室完成。①体外血小板聚集实验:制备大鼠洗涤血小板,观察0.15,0.30和0.60mmol/L桂皮醛对胶原蛋白(100mg/L)和凝血酶(3U/mL)诱导的大鼠体外血小板聚集的抑制作用。桂皮醛购自中国医药集团上海化学试剂公司,纯度>98%。②出、凝血时间测定及胶原蛋白-肾上腺素诱发体内血栓形成实验:取BALB/c小鼠60只,随机分成6组(n=10),生理盐水组灌胃生理盐水;阿司匹林组灌胃阿司匹林100mg/kg为阳性对照;还设置桂皮醛灌胃(250,500mg/kg)和腹腔注射(50,100mg/kg)4个剂量组。所有动物每天给药1次,10μL/g,连续11d。第10天给药后1h采用断尾法和玻片法测定小鼠出血、凝血时间;第11天给药后1h观察小鼠尾静脉注射胶原蛋白(3.57mg/kg)-肾上腺素(0.143mg/kg)混合血栓诱导剂后5min内的存活率。③动-静脉旁路血栓形成实验:SD大鼠48只,随机分为6组(n=8),生理盐水组灌胃生理盐水;阿司匹林组灌胃阿司匹林80mg/kg为阳性对照;桂皮醛分为灌胃200,400mg/kg组和腹腔注射40,80mg/kg4个剂量组。所有动物每天给药1次,10μL/g,连续10d。末次给药后1h测定动-静脉旁路丝线上的血栓湿质量。结果:小鼠60只和大鼠48只全部进入结果分析。①桂皮醛能够明显抑制胶原蛋白和凝血酶诱导的大鼠体外血小板聚集,并呈剂量依赖性,与对照组比较差异显著(P<0.05,0.01)。②桂皮醛显著延长小鼠出、凝血时间,与对照组比较差异非常显著(P<0.01)。③胶原蛋白-肾上腺素诱发性小鼠肺动脉栓塞存活率:生理盐水组为10%,阿司匹林组为80%,桂皮醛各剂量组为70%～100%。④动-静脉旁路丝线上的血栓湿质量:阿司匹林组和桂皮醛各剂量组均低于生理盐水组(P<0.01);桂皮醛各剂量组对大鼠血栓的抑制率范围为30%～43.4%。结论:桂皮醛体外能够明显抑制胶原蛋白和凝血酶诱导的大鼠血浆中血小板的聚集;体内能够显著延长小鼠断尾后的出、凝血时间,减轻大鼠动-静脉旁路丝线上血栓的质量。提示桂皮醛具有明显抗血小板聚集和体内抗血栓作用。     

Prothrombotic effects of diclofenac on arteriolar platelet activation and thrombosis in vivo

Summary. Background: Diclofenac, like selective cyclooxygenase‐2 inhibitors, which alter vascular levels of platelet active prostaglandins, has been reported to increase rates of acute myocardial infarction. Objective: The study was performed to investigate, in an animal model of arterial thrombosis in vivo, whether diclofenac differentially influences platelet activation and thrombosis in vessels under non‐stimulated conditions or during acute systemic inflammation, such as induced by tumor necrosis factor‐α (TNF‐α). Methods: Platelet–vessel wall interaction (PVWI), firm platelet adhesion and arterial thrombosis following vessel injury were analyzed by intravital microscopy in arterioles of hamsters in the dorsal skinfold chamber model. Prostacyclin [prostaglandin I₂ (PGI₂)] and thromboxane A₂ (TxA₂) metabolites were measured. In vitro, endothelial adhesion molecule expression in cultured human microvascular endothelial cells was analyzed. Results: Under non‐stimulated conditions, diclofenac (1 mg kg^?1) enhanced PVWI, which was not mediated by increased adhesion molecule expression, but by decreased systemic PGI₂ levels. Following ferric chloride‐induced endothelial injury, diclofenac accelerated thrombotic vessel occlusion time, an effect that was reversed by the stable PGI₂ analog iloprost. TNF‐α, through induction of endothelial adhesion molecule expression, also enhanced PVWI, firm adhesion, and arterial thrombosis, but simultaneous treatment with TNF‐α and diclofenac did not have an additive effect. Conclusions: By decreasing levels of PGI₂ without, at the same time, altering prothrombotic TxA₂ levels, diclofenac can exert prothrombotic effects. However, this is not the case when an inflammatory situation is created by TNF‐α treatment. These data may explain the enhanced risk of acute myocardial infarction observed in patients taking diclofenac.     

银杏内酯B对胶原或二磷酸腺苷刺激血小板活化后一氧化氮含量及血管舒张因子刺激磷酸蛋白磷酸化的影响

背景:研究表明,增龄机体血小扳一氧化氮合成能力下降,导致环磷鸟苷水平降低,进而使血小扳功能亢进,成为血栓易感状态.目的:探讨胶原或二磷酸腺苷不同血小板刺激剂诱导后,银杏内酯B对增龄机体血小板一氧化氮含量、血管舒张因子刺激的磷酸蛋白磷酸化的影响.设计、时间及地点:细胞学体外观察,于2006-08/2007-12在卫生部老年医学重点实验室完成.材料:13月龄Wistar大鼠5只,36月龄新西兰白兔5只,均由中吲科学院动物所提供.银杏内酯B购自徐州大观园有限公司,批号BN52021,其中银杏内酯B含量为92%,其他8%为银杏内酯A,C,M,J成分.方法:用3.8%枸橼酸抗凝抽取全血,离心分离富血小板血浆.分别将不同浓度的银杏内酯B加入到富血小板血浆中孵育5 min,再加入10 mg/L胶原或5 μmol/L二磷酸腺苷刺激血小板5 min.主要观察指标:使用一氧化氮试剂盒测定一氧化氮含量,Western blot法分析血小板血管舒张因子刺激的磷酸蛋白磷酸化水平,血小板聚集仪测定银杏内酯B对胶原或二磷酸腺苷诱导血小板聚集的抑制效果.结果:800 mg/L银杏内酯B预处理的血小板经胶原刺激活化后,血小板内一氧化氮含量增加62.3%:经二磷酸腺苷刺激活化后一氧化氮含量增加24%.胶原刺激血小板活化时,银杏内酯B可明显提高血管舒张因子刺激的磷酸蛋白的磷酸化水平:二磷酸腺苷刺激血小板活化时,银杏内酯B亦可提高血管舒张因子刺激的磷酸蚩白磷酸化水平,但增加幅度不如前者明显.800 mg/L银杏内酯B几乎完全抑制了胶原诱导的血小板聚集,血小板聚集率仅为2%;但其对二磷酸腺苷诱导的血小板聚集抑制效果较弱,血小板聚集率达64%.结论:银杏内酯B对胶原刺激血小板活化的抑制作用与一氧化氮/环磷鸟苷通道相关,能够完全阻止胶原刺激的血小板聚集,对二磷酸腺苷刺激血小板活化的抑制效果较差,提示银杏内酯B对胶原、二磷酸腺苷诱导的血小板活化有不同的调节机制.     

Comparative effects of two synthetic oligosaccharides on platelet activation induced by plasma from HIT patients

Summary.  Heparin-induced thrombocytopenia (HIT) is a serious secondary event encountered in the clinical use of heparin. HIT results from the consumption of platelets that are immunologically activated by antibodies directed against complexes formed by platelet factor 4 (PF4) and sulfated polysaccharides that activate platelet aggregation, leading to paradoxical, life-threatening thrombosis. There is strong evidence that the ability of heparin and related compounds to induce HIT is closely linked to the structure of the polysaccharide, and particularly to its negative charge and to the length of the molecule. To test this hypothesis, we synthesized two sulfated oligosaccharides: SanOrg123781, a 16-mer, presenting two terminal charged domains separated by a 7-mer neutral linker, and SR121903, a highly sulfated 17-mer. Both of them displayed strong anti-factor (F) Xa and anti-FIIa activities but their affinities for PF4 were markedly different. SR121903 displaced PF4-bound heparin, whereas SanOrg123781 did not, underlining the importance of the charge of the molecule for the interaction with PF4. Platelet studies, in the presence of HIT serum, showed that SR121903 induced the secretion of platelet-dense granules (measured by the release of serotonin) whereas SanOrg123781 did not, a result in accordance with an absence of affinity of this molecule for PF4. These results were confirmed by measurements of platelet activation by flow cytometry (measured by annexin V binding, CD62 detection and activation of the GpIIb–IIIa complexes). In conclusion, we have demonstrated the importance of the charge of the polysaccharides in the HIT-induced platelet reactions measured by diverse methods, of which some are described for this purpose for the first time.