重楼皂苷H诱导血小板聚集效应及其机制的研究

目的 确定从中药重楼中分离的重楼皂苷H(PARG)对血小板聚集的直接诱导效应,并对其诱导血小板聚集的机制进行探索.方法 雄性Wistar大鼠心脏取血,离心分离获得富血小板血浆,样品中加入不同浓度重楼皂苷H,比浊法检测血小板聚集情况.分别用不同浓度的血小板膜糖蛋白 Ⅱb/Ⅲa受体拮抗剂(RGDS)、ADP抑制剂腺苷三磷酸双磷酸酶(Apyrase)、PKC抑制剂Ro31-8820和TXA2受体阻断剂SQ29548孵育血小板3min,加入重楼皂苷H诱导血小板聚集,观察各抑制剂对血小板聚集的影响.结果 PARG能直接诱导大鼠血小板聚集,呈现剂量效应,高浓度PARG(30μmol/L)可诱导血小板发生不可逆聚集.1mmol/L RGDS和0.5μmol/L SQ29548几乎完全抑制PARG诱导的血小板聚集;Apyrase(0.05～1U/ml)和Ro31-8220(20～50μmol/L)均呈剂量依赖效应抑制PARG诱导的血小板聚集.结论 PARG诱导血小板聚集依赖于血小板激活后ADP的释放和TXA2的生成.     

知母皂苷AⅢ抗血栓作用研究

目的:确定知母皂苷AⅢ体内、外给药对大鼠血小板聚集和血栓形成的抑制作用。方法:比浊法测定血小板聚集,动-静脉旁路血栓模型测定大鼠在体血栓的形成。结果与结论:体外血小板聚集模型研究证明,知母皂苷AⅢ可以抑制由ADP、胶原和凝血酶诱导的大鼠血小板的聚集,并呈剂量效应。体内给药同样可以有效抑制血小板的聚集率,当给药剂量达到40 mg/kg.d-1时对血小板聚集的抑制率可以达到26.7%。动-静脉旁路血栓形成实验结果显示,知母皂苷AⅢ体内给药可减小血栓的湿重和干重,说明知母皂苷AⅢ体内给药具有抗血栓的作用。知母皂苷AⅢ体内给药不影响出凝血时间。推测其抗血栓形成的功效可能是通过抑制血小板的黏附、聚集和活化而实现的。     

PLA2/AA信号途径与重楼皂苷诱导大鼠子宫平滑肌收缩活动的关系研究

目的:探讨宫血宁活性成分--重楼总皂苷(TSSP)诱导大鼠离体子宫肌条收缩的机制,考察PLA2/AA信号途径与TSSP引起的大鼠离体子宫收缩的关系.方法:应用离体子宫张力测定法,观察不同PLA2/AA信号途径抑制剂对TSSP引起的大鼠离体子宫收缩作用的影响.结果:非特异性前列腺素合成酶抑制剂可明显抑制TSSP和米索前列醇引起的子宫收缩,提示二者可能存在相似机制.环氧酶1(COX-1)抑制剂SC560,环氧酶2(COX-2)抑制剂NS-398,EP受体抑制剂AH6809和脂氧酶(5-LO)抑制剂REV5901均可对TSSP缩宫作用产生明显抑制;磷脂酶A2(PLA2)抑制剂AACOCF3也可明显抑制TSSP的缩宫作用,提示PLA2/AA信号途径与TSSP的缩宫作用密切相关.结论:TSSP对大鼠子宫平滑肌收缩活动的调节与PLA2/AA信号途径的激活有关.     

应用流式细胞术研究剪切力诱导的血小板聚集

目的　建立流式细胞术方法检测剪切力诱导的血小板聚集 (SIPA) ,研究不同剪切力作用后血小板聚集率的变化规律 ,并初步探讨SIPA测定的临床意义。方法　用锥板黏度计对全血标本施加剪切力作用 ,使血小板产生聚集反应 ,再以荧光抗体CD6 1PerCP标记血小板 ,然后进行流式细胞术分析 ,测定血小板聚集率的大小 ;同时在显微镜下直接观察血小板聚集体形态。结果　不同的剪切力作用后 ,血小板产生聚集反应程度不同。在 10 0S-1剪切作用下 ,只有少量的血小板聚集 ;5 0 0S-1时 ,血小板聚集增加 ;在高于 2 0 0 0S-1的剪切作用下 ,30s即可发生明显的血小板聚集 ,30 0 0S-1剪切作用 3min时血小板聚集率为 (5 8 4± 5 3) % ,已接近最大值 ,明显高于 10 0S-1的(9 4± 1 3) %和 5 0 0S-1的 (2 6 4±3 2 ) %。随着剪切力的增大和作用时间的延长 ,血小板聚集体逐渐增大 ,结构由松散到致密。AMI、脑梗死、TIA、糖尿病等动脉血栓相关性疾病患者的高剪切力诱导的血小板聚集明显高于正常。结论　直接的剪切力作用能够诱导血小板发生聚集反应 ,聚集程度与剪切力的大小和作用时间成正相关 ;高剪切力诱导的血小板聚集的测定对于动脉血栓相关性疾病的临床辅助诊断有一定的意义     

知母皂苷抑制血小板聚集的活性成分筛选及构效关系分析

目的 筛选从知母中分离的甾体皂苷抑制血小板聚集的活性成分并对其进行构效关系分析.方法 雄性Wistar大鼠心脏取血,离心分离获得富血小板血浆,分别加入呋甾烷型或螺甾烷型甾体皂苷化合物孵育3min后,以二磷酸腺苷(ADP)诱导大鼠血小板聚集,采用比浊法检测血小板聚集情况,通过比较不同甾体皂苷化合物对ADP诱导血小板聚集的抑制作用分析化合物的构效关系.结果 8个呋甾烷型甾体皂苷均无抑制血小板聚集的活性;4个螺甾烷型甾体皂苷在不同程度上抑制了血小板聚集,其中知母皂苷AⅢ作用最强,随后依次是知母皂苷AⅠ、知母皂苷AⅢ异构体和知母皂苷Ⅲ,而萨尔萨皂苷元没有活性;在苷元结构C-3位连接不同类型糖基的甾体皂苷抑制血小板聚集的作用不同,其中C-3位糖基为葡萄糖(D-Glc)时能完全抑制血小板聚集,而连接其他糖基如半乳糖(D-Gal)、阿拉伯糖(D-Ara)、核糖(D-Rib)、鼠李糖(L-Rha)或甘露糖(D-Man)时,抑制活性有不同程度的减弱.结论 知母皂苷AⅢ是知母抑制血小板聚集的主要有效活性成分之一;甾体皂苷抑制血小板聚集的活性与其结构密切相关;第3、15、22位上的基团类型可影响甾体皂苷抑制血小板聚集的生物活性.     

白及不同提取部位对家兔血小板聚集的影响

目的探讨白及不同提取部位对家兔血小板聚集的影响.方法比浊法测定血小板聚集率.结果白及正丁醇提取部位和水溶性部位可显著升高腺苷二磷酸（ADP）诱导的血小板最大聚集率,而乙酸乙酯提取部位可显著抑制ADP诱导的血小板聚集,石油醚提取部位对ADP诱导的血小板聚集无显著影响.各提取部位对血小板数无显著影响.结论白及正丁醇提取部位和水溶性部位是白及止血作用的主要有效部位,其止血作用与其促进血小板聚集作用有关.     

RGD肽类血小板聚集抑制剂的研究Ⅰ.N─乙酰─精氨酰─甘氨酰─天冬氨酸─苯乙酰胺的合成

活化的血小板暴露膜糖蛋白复合物（GPⅡb／Ⅲa），作为受体识别结合纤维蛋白原（Pg）等粘附蛋白共有的精氨酰─甘氨酰─天冬氨酸（Arg－Gly－Asp，即RGD）序列，导致血小板聚集和血栓形成。人工合成的含RGD多肽可阻断GPⅡb／Ⅲa与Fg等结合，从而抑制血小板聚集和血栓形成。为了开展肽类血小板聚集抑制剂的研究，我们用液相法合成了Ac－Arg－Gly一Asp－NHCH2CH2Ph。初步体外活性实验结果表明，该RGD肽衍生物对ADP诱导的血小板聚集具有较强的抑制作用，在血浆中3h作用强度基本不变。     

泽兰有效部位对血小板聚集和血栓形成的影响

目的：研究泽兰有效部位L．F04对血小板聚集和血栓形成的影响,以探讨其活血化瘀作用机理。方法：用高分子右旋糖酐静脉推注造成大鼠血瘀证动物模型,观察泽兰L．F04对ADP诱导的大鼠体内血小板聚集以及体内动静脉旁路血栓、体外旋转环内血栓形成的影响。结果：L．F040．408g／kg、0．204g／kg对模型组大鼠ADP诱导的体内血小板最大聚集率明显增加皆有显著的抑制,且呈剂量依赖关系;与对照组相比,血瘀模型大鼠体外血栓重量明显增加,长度仅有增加趋势,L．F040．408g／kg、0．204g／kg皆有抗血栓形成作用,L．F040．408g／kg对血栓干重、湿重的减轻尤为明显;L．F040．408g／kg、0．204g／kg对实验性动静脉旁路血栓形成均有明显的抑制作用,抑制率分别为27．41％、27．14％。结论：泽兰L．F04可显著抑制血小板聚集及体内、外血栓形成。     

血栓性疾病血小板聚集实验的临床意义

 目的 研究血栓性疾病及其影响因素对患者的血小板聚集指数变化情况及临床意义.方法 采用比浊法,应用血小板聚集仪,分别检测正常对照组,动脉硬化、冠心病、高血压、脑梗死、糖尿病患者血小板聚集指数,并观察阿司匹林治疗后的变化.结果 (1)与对照组比较,动脉硬化、冠心病、高血压、脑梗死、糖尿病患者的血小板聚集指数均明显升高(P<0.05);(2)阿司匹林治疗后各组患者的血小板聚集指数明显降低(P<0.05),与正常对照组比较无明显差异.结论 血小板聚集实验对血栓性疾病患者的诊断、治疗监测具有重要作用.     

猪血小板膜糖蛋白的纯化与鉴定

目的：分离纯化猪血小板膜糖蛋白并对其进行鉴定。方法：差速离心法分离猪血小板，1％TritonX-100溶解膜糖蛋白，麦胚凝集索亲和层析纯化糖蛋白(0．3mol／L N-乙酰葡萄糖胺洗脱)，用鼠抗人GPⅠb单抗PHN89作点印迹，聚丙烯酰胺凝胶电泳，考马斯亮蓝(Coomassie blue，CB)和过碘酸-Schiff(PAS)染色鉴定糖蛋白，腺苷二磷酸钠盐(ADP)和瑞斯托菌素(ristocetin)诱导的人血小板聚集测定其体外活性。结果：用GPⅠb单抗PHN89为一抗的点印迹表明0．3mol／L N-乙酰葡萄糖胺能充分洗脱结合到凝集素上的GPⅠb。PAS法糖定性有2条显色条带，SDS-PAGE纯化后有2条蛋白区带。ADP阳性对照血小板聚集率为74％，纯化前后的样品血小板聚集率分别为42％和4％，ristocetin阳性对照血小板聚集率为100％，纯化前后的样品血小板聚集率分别为25％和3％，表明制备的血小板糖蛋白具有生物学活性。结论：得到了较纯的血小板膜糖蛋白，为血小板膜糖蛋白的进一步研究及应用奠定基础。     

Effect of a low-osmolality nonionic contrast medium (iopromide) on blood platelet membrane structure and aggregation

Nonionic contrast media (NICM) used in radiology exhibit weaker anticoagulant properties than ionic media. This study was intended to evaluate the effect of iopromide NICM upon the platelet aggregation and their membrane structure (investigated by electron paramagnetic resonance method with a spin label). Blood was collected directly from the left ventricle during coronary angiography prior to and following the application of the contrast medium. These results were compared with in vitro examinations. Based on the spectral curves parameters that reflect conformational changes in membrane proteins were determined. The iopromide-induced decrease in platelet aggregation examined in vitro with adenosine diphosphate (ADP), collagen and ristocetine was statistically significant. In vitro platelet membrane structure was modified significantly with regard to both protein conformation and the accessibility of sulfhydryl (- SH) and amino (- NH₂) groups. In ex vivo studies the changes in platelet aggregation was only observed with ADP, whereas in the membrane-structure modifications were only noted regarding accessibility of - SH and - NH₂ groups. We conclude that iopromide-induced platelet aggregation changes are most probably due to their membrane-structure modifications, whereas the less-pronounced contrast effect ex vivo is due to its rapid dilution in the inflowing blood. The results confirm weak antiplatelet and anticoagulant influence of NICM in vivo. Correspondence to: J. Górski     

三种止血材料止血活性的体外实验研究

目的比较明胶纤维网(gelatin fleece ,GF)、氧化纤维素(oxidized cellulose ,OC)和明胶海绵(gelfoam ,GL)的止血效果,探讨GF的止血机制.方法使3种止血材料分别与富血小板血浆(PRP)接触0.5,1,1.5,2,2.5 min,然后通过灌注黏附实验及血小板聚集实验检测血小板黏附率及聚集率;使3种止血材料分别与PRP接触1,5 min,然后用酶联免疫吸附法检测血小板颗粒膜糖蛋白-140(GMP-140)释放的改变;用扫描电镜观察3种止血材料的结构和血小板黏附激活的形态.结果 PRP与GF接触后血小板的黏附率及聚集率较与OC和GL接触后高(P<0.05).3种止血材料均能促进血小板释放GMP-140,与未加止血材料时比较,差异均有非常显著性意义(P<0.01),其中GF促进血小板释放GMP-140的能力大于OC和GL(P<0.01).扫描电镜显示,GF呈网络致密状结构,而OC和GL的结构间均有较大网眼;GF间黏附有较多激活的血小板,有伪足形成,而GL仅黏附有少量激活的血小板.结论 GF的止血效果优于OC和GL,它主要是通过促进血小板黏附、聚集,并使激活的血小板释放促凝物来加快血液凝固.     

Inhibition of platelet aggregation by contrast media.

The effect of 30% Urografin on platelet aggregation induced by adenosine diphosphate (ADP), epinephrine, and arachidonic acid was examined. In vivo and in vitro experiments in 10 nondiabetic subjects and in vitro experiments in 7 diabetic-uremic patients showed a statistically significant decrease in platelet aggregation 20 minutes after in vitro incubation or in vivo infusion with Urografin. Urografin concentration was about 2.4-2.5%. The possibility of bleeding induced by contrast media and the connection with acute renal failure in diabetic-uremic patients are discussed.     

Platelet activation and aggregation after endothelial injury. Assessment with indium-111-labeled platelets and angiography

Although platelet activation and aggregation after endothelial injury are well documented, the time course of platelet deposition and the relationship between platelet aggregation and the release of vasoactive products have not been fully clarified in vivo. To study the effect of platelet vasoactive products, a collateral blood supply was induced by ligating the superficial femoral artery in male New Zealand white rabbits. Two weeks later, endothelial injury to the distal abdominal aorta was produced by cytologic brush or mimicked with a metal coil embolus. Platelet aggregation was assessed with indium-111 (111In)-labeled platelets, and scintigraphy demonstrated significant, progressive platelet deposition up to 3 hours after injury and evidence of residual activity 24 hours later. Angiography showed that the time course of peripheral vasoconstriction matched closely that of platelet deposition, indicating release of vasoactive substances from the aggregating platelets. These pathophysiologic changes secondary to endothelial injury may have significant implications for intravascular interventional procedures.     

两种前列腺素抑制二磷酸腺苷诱导血小板聚集强弱的体外对比

 目的 比较两种前列腺素依前列醇钠、前列地尔在体外抑制二磷酸腺苷(adenosine diphosphate, ADP)诱导的血小板聚集的强弱。方法 SD大鼠腹腔静脉取血,制得血小板数400×10⁹个/L的血小板混悬液。血小板混悬液中加入不同浓度的依前列醇钠和前列地尔(50、125、250、500、2500 ng/ml)在最佳ADP浓度和最佳孵育时间,分别测定5 min内最大聚集率,各浓度检测4次取平均值,从而得到依前列醇钠、前列地尔量效关系并进行对比。结果 ADP最佳诱导浓度为20 μM,最佳孵育时间为5 min,随着依前列醇钠、前列地尔浓度的增加,血小板聚集率依次降低。依前列醇钠5个浓度血小板抑制率依次为19.61%、48.13%、88.25%、93.71%和100.00%,前列地尔5个浓度血小板抑制率依次为5.20%、11.70%、23.16%、28.10%和65.09%。结论 依前列醇钠、前列地尔均具有血小板聚集抑制作用,并呈浓度依赖性,且依前列醇钠的血小板抑制作用远远强于前列地尔。     

In vitro study of the action of DOTA and DTPA-gadolinium complexes on platelet functions: aggregation and calcium excretion

Interaction of 2 complexes of Gadolinium (DOTA Gd meglumin and DTPA Gd meglumin) was studied in vitro with regard to platelet function: aggregation and excretion of ATP and of ionised calcium in response to collagen (2.5 Ig/ml). Excretory functions were measured simultaneously with aggregation by bioluminescent techniques using luciferin for the measurements of ATP and aequorin for measurements of ionised calcium. Platelet aggregation and excretion functions, investigated in vitro, were moderately inhibited in the presence of gadolinium complexes. The greatest effects were seen with DTPA Gd meglumin. Complexation of extra-cellular ionised calcium by NMR products in partially responsible for the inhibition of platelet aggregation.     

酸枣仁总皂甙对小鼠缺氧的保护作用和体外对家兔血小板聚集及产生血栓素B_2的影响

酸枣仁总皂甙(ZS 100mg/kg IP)对小鼠常压缺氧和异丙肾上腺素加重的缺氧及亚硝酸钠所致的携氧障碍均能显著延长存活时间。用比浊法和放射免疫测定法研究ZS对家兔凝血酶诱导的血小板聚集和产生血栓素B_2(TXB_2)的影响。ZS(25-660mg/L)显著抑制血小板聚集和TXB_2的产生。上述结果提示:ZS对缺氧的保护作用与抗血小板聚集和减少TXB_2生成有关。     

Effect of various contrast media on platelet aggregation--an in vivo study

Recently, some radiologists using non-ionic contrast media for angiography have noted the increased tendency of thrombus formation in the injection syringe or angiographic catheter contaminated with blood. In vitro studies by some authors have shown that non-ionic contrast media had only a slight anticoagulative effect as compared with ionic contrast media. But the in vivo studies comparing the anticoagulative effect of both ionic and non-ionic contrast media have not been performed previously. We investigated the effect of non-ionic and ionic contrast media on platelet aggregation in 40 patients undergoing angiography. The in vivo study revealed a negligible influence of both non-ionic and ionic contrast media on systemic platelet aggregation. The dose of contrast media also showed no significant correlation with platelet aggregation. Our study suggests that the data from in vitro experiments cannot be extended to in vivo study as for the relationship between contrast media and their anticoagulant effect.     

Iopiperidol: Nonionic iodinated contrast medium with promising anticoagulant and antiplatelet properties

RATIONALE AND OBJECTIVES: The purpose of this study was to determine the anticoagulant and antiplatelet characteristics of iopiperidol, a nonionic, triiodinated contrast agent. MATERIALS AND METHODS: Anticoagulant effects of iopiperidol were assessed both in vitro and in vivo after single or repeated intravenous administrations to rats. To this aim, results of prothrombin time, activated partial thromboplastin time, thrombin time, and fibrinogen tests were evaluated. To define better the mechanism of action of iopiperidol and of the contrast media used for comparison, in vitro tests to study the effects on thrombin activity and on thrombin generation were performed. In addition, the effect of iopiperidol was studied on adenosine diphosphate- and collagen-induced platelet aggregation both in vitro and in vivo after single or repeated intravenous administrations in the rat. RESULTS: In vitro, iopiperidol showed anticoagulant properties similar or superior to those of the ionic ioxaglate. Iopiperidol also inhibited collagen-induced platelet aggregation statistically significantly more than iodixanol and ioxaglate (P < .05). In vivo, no significant differences between iopiperidol and ioxaglate were observed after single or repeated administrations. CONCLUSION: The in vitro anticoagulant effect of iopiperidol is similar or even superior to that of ioxaglate; the in vivo effect is similar to that of reference nonionic contrast media.