丹参液在体外对血小板活化的抑制作用研究

目的：探讨丹参液在体外对血小板活化的抑制作用。方法：用流式细胞仪三色分析测定丹参液在体外对血小板活化膜表面纤维蛋白原受体(FIB-R)和P-选择素(CD42P)的表达。结果：丹参液有保护血小板FIB-R和CD42P被活化，同时部份抑制ADP活化血小板。结论：丹参液是一种较强的抗血小板活化药物，在血栓性疾病的防治中有积极作用。     

木瓜蛋白酶体外对血小板聚集的抑制作用

目的：以洗涤血小板为模型,观察木瓜蛋白酶在体外对血小板聚集的抑制作用,以探讨其抗血栓作用的可能机制。方法：将不同剂量木瓜蛋白酶与洗涤血小板作用,以血小板聚集分析仪检测ADP、花生四烯酸（AA）、胶原和凝血酶诱导的血小板最大聚集率,以流式细胞仪检测活化血小板膜纤维蛋白原受体（FIB-R）和P-选择素表达水平,以SDS-PAGE分析血小板肌动蛋白聚合体的变化。检测ADP诱导的原发性高血压（PH）及急性心肌梗死（AMI）患者血小板最大聚集率和FIB-R表达水平。结果：木瓜蛋白酶剂量依赖性地抑制血小板聚集,降低血小板最大聚集水平,血小板聚集水平与木瓜蛋白酶剂量呈负相关（P〈0.01）。木瓜蛋白酶剂量依赖性地抑制ADP诱导的血小板FIB-R表达,降低FIB-R表达水平（P〈0.01）。木瓜蛋白酶降低ADP诱导的血小板膜P-选择素表达水平和抑制肌动蛋白聚合体增加（P〈0.01）。木瓜蛋白酶抑制PH及AMI患者血小板聚集和FIB-R表达（P〈0.01）。结论：木瓜蛋白酶通过抑制活化血小板膜纤维蛋白原受体的表达,并抑制肌动蛋白聚合以及释放反应从而剂量依赖性地抑制血小板的聚集反应,有抗血栓形成作用。     

溃疡性结肠炎患者黏附分子CD62p表达与血小板功能的临床研究

目的探讨血小板活化和溃疡性结肠炎的关系。方法对28例活动期溃疡性结肠炎、15例缓解期溃疡性结肠炎患者和正常对照组30例患者用FACSCAN型流式细胞仪检测血小板表面纤维蛋白原受体（PAC-1）和血小板α-颗粒膜糖蛋白（CD62p），用酶联免疫法检测血栓素B2（TXB2）。结果活动期UC患者血PAC-1、CD62p均较缓解期和对照组高，差异有统计学意义（P〈0．05）；活动期UC患者血浆TXB2较缓解期UC患者和对照组高，差异有统计学意义（P〈0．01）；缓解期患者PAC-1、CD62p仍高于正常对照组（P〈0．05），缓解期TXB2与正常对照组无明显差异（P〉0．05）。CD62p及TXB2与病情程度有关（P〈0．05）。结论活动期溃疡性结肠炎体内存在血小板活化，PAC-1、CD62p是溃疡性结肠炎较特异的指标，TXB2影响血液高凝状态，抗血小板药物对溃疡性结肠炎有一定效果。     

急性心肌梗死患者血小板活化分子标记物血小板激活复合物1及CD62p与纤维蛋白原的相关性研究

目的 探讨急性心肌梗死（AMI）患者血小板活化分子标记物血小板激活复合物1（PAC-1）、CD62p的变化及其与纤维蛋白原（Fg）的关系.方法 用三色全血流式细胞术测定36例AMI患者（AMI组）外周血中血小板PAC-1、CD62p的表达水平,并检测患者Fg水平,与32例健康体检者（健康对照组）的相关项目进行比较.结果 AMI组PAC-1、CD62p、Fg均高于健康对照组[（79.53±11.23）%比（47.02±5.98）%,（44.76±6.85）%比（29.13±4.38）%,（4.72±0.98）g/L比（3.38±0.48）g/L,P＜0.01].AMI组PAC-1与Fg呈正相关（r=0.56,P＜0.01）,CD62p与Fg呈正相关（r=0.42,P＜0.05）.结论 AMI患者血清Fg增加、血小板明显活化,其血小板活化程度与Fg关系密切.     

AoGDW肽抑制血小板聚集的作用机制

目的研究RGD(精氨酸-甘氨酸-天门冬氨酸)肽衍生物-AoGDW(ω-氨基辛酸-甘氨酸-天门冬氨酸-色氨酸)抑制血小板聚集的作用机制。方法采用双色荧光标记以及流式细胞技术检测AoGDW肽对CD41抗体与血小板GPⅡb/Ⅲa受体结合的抑制作用以及对血小板膜上P-选择素表达的影响。结果随着AoGDW肽浓度的增加,CD41抗体与活化血小板GPⅡb/Ⅲa受体结合率呈现下降趋势,相对于阴性对照组,P<0.01。但CD41抗体与静止血小板GPⅡb/Ⅲa受体的结合率以及CD62p抗体与静止血小板P选择素的结合率与阴性对照组相比,P>0.05。结论AoG-DW肽是通过占据血小板上纤维蛋白原的结合位点即活化的GPⅡb/Ⅲa受体而发挥抑制血小板聚集的作用,并且不具有活化血小板的作用,是一种选择性的GPⅡb/Ⅲa受体拮抗剂。     

流式细胞术检测阿司匹林对冠心病患者血小板活化标志物的影响

目的：探讨冠心病患者及其服用阿司匹林前后血小板表面糖蛋白的变化及其意义。方法：采用流式细胞仪（flow cytometry，FCM）测定冠心病患者阿司匹林治疗前后的全血中血小板表面糖蛋白（CD62p／PAC-1）的表达率，采用自身对照分析阿司匹林对血小板表面糖蛋白的影响。结果：冠心病组治疗前的血小板表面糖蛋白PAC-1、CD62p的表达率分别为（10.4±6．2）％和（10．7±7．1）％，较健康对照组明显升高（P＜0．01）．经以阿司匹林为基础的抗血小板治疗后CD62p、PAC-1的表达率下降至（4．3±2．1）％和（4．9±2．4）％（P＜0．01），但仍高于健康对照组。结论：CD62p和PAC-1是血小板活化的敏感和特异指标，阿司匹林能够抑制血小板表面糖蛋白的表达．从而抑制血小板活化，抑制血栓形成。     

上消化道出血高危患者经皮冠状动脉介入术后应用西洛他唑氯吡格雷联合治疗对血小板膜糖蛋白的影响

目的观察合并上消化道出血（UGH）高危因素的冠心病患者经皮冠状动脉介入治疗（PCI）后应用西洛他唑联合氯吡格雷治疗对血小板活化的抑制效果、安全的影响。方法125例冠心病合并UGH高危因素PCI术后的患者分为：A组（n=62）：口服西洛他唑100mgBid＋氯吡格雷75mgQd;B组（n=63）：口服阿司匹林100mgQd＋氯吡格雷75mgQd。利用流式细胞术分别检测和比较2组治疗第7、14天纤维蛋白原受体（PAC-1）和P选择素（CD62P）抑制率,记录主要不良心脏事件（MACE）,出血、消化道事件。结果2组在同时间点对PAC-1、CD62P抑制率、MACE无统计学差异;A组的出血并发症、消化道事件显著少于B组（P〈0．05）。结论合并UGH高危因素的冠心病患者PCI术后应用西洛他唑联合氯吡格雷治疗对血小板活化抑制效果好,安全性好。     

西洛他唑与氯吡格雷联合治疗对非ST段抬高急性冠脉综合征血小板膜糖蛋白的影响

目的：评价联合抗血小板治疗方案的有效性及安全性,以探讨西洛他唑＋氯吡格雷联合治疗非ST段抬高急性冠脉综合征（NSTE-ACS）患者的应用价值。方法：将63例确诊为NSTE-ACS的患者,随机分为两组,A组（31例）：口服西洛他唑100mgbid＋氯吡格雷75mgqd;B组（32例）：口服阿司匹林100mgqd＋氯吡格雷75mgqd。利用流式细胞术分别检测治疗前、治疗第7、14天的血小板膜糖蛋白[纤维蛋白原受体（PAC-1）和P选择素（CD62P）]的表达率,计算并比较其抑制率,观察治疗过程中主要不良心脏事件（MACE）发生率、出血并发症。结果：两组PAC-1或CD62P的表达率在治疗第7、14天均较治疗前明显下降（P〈0.01）,第14天最显著。治疗前,治疗第7、14天A组和B组同期的PAC-1、CD62P表达率之间差异无统计学意义（P〉0.05）;两组在相同治疗时间点对PAC-1、CD62P抑制率差异无统计学意义（P〈0.05）;两组MACE发生率对比无统计学差异（P〈0.05）,A组的出血并发症明显少于B组（P〈0.05）。结论：对NSTE-ACS患者,西洛他唑＋氯吡格雷对血小板抑制的近期效果与阿司匹林＋氯吡格雷方案相似,且安全性更好。     

生物标志物检测法评价注射用丹参多酚酸对血小板活性的影响

目的：采用生物标志物检测法评价注射用丹参多酚酸对血小板活性的影响。方法：用检测生物标志物（血小板膜糖蛋白CD62p）的方法测定经二磷酸腺苷(ADP)激活的血小板活化率,判定受试物是否具有抑制血小板活化的作用。体内法比较大鼠给予受试物后和给药前抗凝血的血小板活化率,体外法比较添加受试物与未加受试物混合的大鼠抗凝血的血小板活化率。结果：在体内和体外模型中,注射用丹参多酚酸可明显抑制血小板上由ADP激活的CD62p高表达,血小板的活化受到抑制。结论：采用生物标志物检测的方法,简单快捷,可有效的评价药物对血小板活性的影响,体外法更适合建立检测标准。     

仙鹤草水提物体外对血小板聚集、凝血功能及血液流变学的影响

目的 观察仙鹤草水提物在体外对血小板聚集、凝血试验及血液流变学的影响,探讨其对止血与凝血功能的作用及机制。方法 将0、4、20、80g/L仙鹤草水提物分别与全血、富血小板血浆（PRP）和贫血小板血浆（PPP）作用;分别以血小板聚集仪测定ADP诱导的血小板聚集率,以流式细胞仪测定ADP诱导的血小板纤维蛋白原受体（Fg-R）和P-选择素表达水平;以血液凝固分析仪测定凝血酶原时间（PT）、活化部分凝血活酶时间（APTT）、凝血酶时间（TT）、纤维蛋白原（Fg）,以硅化试管法测定凝血时间（CT）,以电极法测定血浆Ca2＋浓度,以乏因子血浆纠正试验测定凝血因子Ⅶ、Ⅷ、Ⅸ和Ⅺ活性;以血液流变仪测定全血黏度、血浆黏度及红细胞聚集指数。结果 4g/L组PAR、Fg-R和P-选择素表达率显著低于0g/L组（P〈0.05）。4g/L组1s-1全血黏度、200s-1全血黏度、血浆黏度和红细胞聚集指数均显著高于0g/L组（P〈0.01）,4者水平均与浓度呈显著正相关（P〈0.01）。 结论 仙鹤草水提物可能通过抑制血小板Fg-R活化和释放反应途径而抑制血小板聚集以及可能通过抑制内源凝血途径而具有抗凝作用。仙鹤草水提物也可能通过活化外源凝血途径并增加血液黏度而具有促凝作用。     

流式细胞术预测冠心病患者阿司匹林抵抗的研究

目的 利用流式细胞术制定阿司匹林抵抗(AR)的诊断标准,预测冠心病患者阿司匹林抵抗的发生率.方法 采用流式细胞术测定127例冠心病(阿司匹林组103例和空白对照组24例)患者人院时及经治疗7 d后P选择素(CD62P)和纤维蛋白原受体(PAC-1)的表达率.利用ROC曲线法设定AR诊断标准,统计AR在住院冠心病患者中的发生率.结果 阿司匹林组治疗前CD62P(10.16±6.80)%、PAC-1(14.66±10.56)%,治疗后CD62P(5.70±4.28)%、PAC-1(8.93±7.08)%;空白对照组治疗前CD62P(9.14±6.52)%、PAC-1(17.67±11.53)%,治疗后CD62P(7.81±5.72)%、PAG-1(14.97±8.05)%,差异有统计学意义(P＜0.05).根据ROC曲线设定CD62P抑制率＜21.5%或PAC-1抑制率＜17.7%为诊断AR标准,AR在住院冠心病患者中的发生率为17.5%.结论 CD62P抑制率＜21.5%或PAC-1抑制率＜17.7%为AR诊断标准.AR发生率为17.5%.     

Parallel synthesis and biological evaluation of 837 analogues of procaspase-activating compound 1 (PAC-1)

Procaspase-Activating Compound 1 (PAC-1) is an ortho-hydroxy N-acyl hydrazone that enhances the enzymatic activity of procaspase-3 in vitro and induces apoptosis in cancer cells. An analogue of PAC-1, called S-PAC-1, was evaluated in a veterinary clinical trial in pet dogs with lymphoma and found to have considerable potential as an anticancer agent. With the goal of identifying more potent compounds in this promising class of experimental therapeutics, a combinatorial library based on PAC-1 was created, and the compounds were evaluated for their ability to induce death of cancer cells in culture. For library construction, 31 hydrazides were condensed in parallel with 27 aldehydes to create 837 PAC-1 analogues, with an average purity of 91%. The compounds were evaluated for their ability to induce apoptosis in cancer cells, and through this work, six compounds were discovered to be substantially more potent than PAC-1 and S-PAC-1. These six hits were further evaluated for their ability to relieve zinc-mediated inhibition of procaspase-3 in vitro. In general, the newly identified hit compounds are two- to four-fold more potent than PAC-1 and S-PAC-1 in cell culture, and thus have promise as experimental therapeutics for treatment of the many cancers that have elevated expression levels of procaspase-3.     

Differential effects of procaspase-3 activating compounds in the induction of cancer cell death

The evasion of apoptosis is a key characteristic of cancer, and thus strategies to selectively induce apoptosis in cancer cells hold considerable promise in personalized anticancer therapy. Structurally similar procaspase activating compounds PAC-1 and S-PAC-1 restore procaspase-3 activity through the chelation of inhibitory zinc ions in vitro, induce apoptotic death of cancer cells in culture, and reduce tumor burden in vivo. Ip or iv administrations of high doses of PAC-1 are transiently neurotoxic in vivo, while S-PAC-1 is safe even at very high doses and has been evaluated in a phase I clinical trial of pet dogs with spontaneously occurring lymphoma. Here we show that PAC-1 and S-PAC-1 have similar mechanisms of cell death induction at low concentrations (less than 50 μM), but at high concentrations PAC-1 displays unique cell death induction features. Cells treated with a high concentration of PAC-1 have a distinctive gene expression profile, unusual cellular and mitochondrial morphology, and an altered intracellular Ca(2+) concentration, indicative of endoplasmic reticulum (ER) stress-induced apoptosis. These studies suggest strategies for anticancer clinical development, specifically bolus dosing for PAC-1 and continuous rate infusion for S-PAC-1.     

Anti-platelet effects of GPIIb/IIIa and P-selectin antagonism,platelet activation,and binding to neutrophils

Platelet activation with GPIIb/IIIa binding to fibrinogen, aggregation and interaction with leukocytes constitute the principal mediator of thrombosis. Although the clinical benefits of GPIIb/IIIa antagonists have been documented, the relationship between their anti-platelet properties, platelet activation and binding to leukocytes is still debated. We investigated the effects of abciximab, tirofiban, roxifiban, and an anti-P-selectin blocking monoclonal antibody (Mab) on isolated human platelet aggregation using optical aggregometer, and on platelet P-selectin and GPIIb/IIIa expression, and platelet-neutrophil binding using flow cytometry. Thrombin at 0.025 U/ml induced maximal platelet aggregation (76.3 +/- 2.6%), P-selectin expression (88.5 +/- 4%), GPIIb/IIIa activation (PAC-1 binding, 86.2 +/- 8.9%) and platelet-neutrophil binding (58.0 +/- 6.4%). The GPIIb/IIIa antagonists inhibited in a concentration-dependent manner platelet aggregation (IC50 of 100 nM for abciximab and tirofiban and 50 nM for roxifiban) and PAC-1 binding, without any effect on P-selectin. None of these agents affected significantly platelet-neutrophil binding, whereas an anti-P-selectin Mab abolished this binding and amplified the effect of abciximab on platelet aggregation. These results indicate that the effects of these GPIIb/IIIa antagonists on platelet aggregation are not related to inhibition of platelet activation, as P-selectin levels and platelet-neutrophil binding remained unaffected, and highlight the participation of P-selectin with GPIIb/IIIa in platelet aggregation.     

Estimation of anti-platelet drugs on human platelet aggregation with a novel whole blood aggregometer by a screen filtration pressure method

1. The effects of anti-platelet drugs on human whole blood aggregation were evaluated using a novel whole blood aggregometer by a screen filtration pressure (SFP) method. 2. The SFP whole blood aggregometer was found to successfully detect whole blood aggregation induced by ADP, collagen and TRAP by measuring the SFP of blood samples. The platelet aggregation threshold index (PATI), the concentration of agonist required with an inducing pressure rate of 50%, varied time-dependently after collection of blood. High values for ADP and collagen were noted immediately after blood collection, suggesting low aggregation activity of platelets, and gradually increase thereafter. 3. Cilostazol (phosphodiesterase 3 inhibitor), dipyridamole, aspirin and tirofiban all inhibited whole blood aggregation in vitro. Inhibitory effects of cilostazol and dipyridamole, but not tirofiban, were markedly enhanced 6 or 7 fold by long pre-incubation (60 min), compared with short pre-incubation (2 min). Such enhancement was only observed with ADP- and not collagen-induced whole blood aggregation. A similar phenomenon was also observed for aggregation with platelet rich plasma (PRP). Cilostazol inhibition of ADP-induced platelet aggregation was more potent with PRP than whole blood (PATI(200)=3.80+/-0.95 microM for whole blood; 2.04+/-0.61 microM for PRP). Inhibitory effects of dipyridamole were attenuated in PRP without erythrocytes. 4. These results demonstrate that the SFP aggregometer can sensitively detect anti-platelet aggregatory effects of various kinds of drugs. So that it is a useful tool for evaluation of anti-platelet drugs.     

Beta-lactamases and outer membrane investigations in beta-lactam-resistant Comamonas acidovorans strains.

Imipenem-induced beta-lactamase (level of expression, specific activity and kinetic parameters (Vmax and Km) in response to nitrocefin) and outer membrane proteins (OMPs) (hydrophobicity, permeability and electrophoretic pattern) were characterized in, one beta-lactam sensitive (PAC-9), one resistant (PAC-1) and two resistant laboratory mutants (PAC-9M, PAC-9M2) of Comamonas acidovorans strains. Beta-lactamases from both mutant strains showed different Vmax values compared to the parental strains. Beta-lactam resistance was found to be associated in PAC-1 with inducible beta-lactamase production and OMP alteration by the appearance of a 102-KDa protein. Moreover, PAC-1 was less permeable to nitrocefin than PAC-9. These data indicate that C. acidovorans resistance to beta-lactam resulted from synergy between beta-lactamase and OMP alterations.     

4-甲氧基-1,3-苯二甲酸-4′-取代苯酚二酯类化合物的合成及其抗血小板聚集活性研究

目的 寻找新的高效低毒的血小板聚集抑制剂。方法 以 4-甲氧基-N,N′-二苯基-1,3-苯二甲酰胺为先导化合物,用 4-取代苯氧基代替苯氨基对先导化合物进行结构改造：以苯甲醚为原料,采用文献方法经 3 步反应制得重要中间体4-甲氧基-1,3-苯二甲酰氯;该中间体与 4-取代苯酚类化合物进行取代反应制得目标化合物。以吡考他胺和阿司匹林为阳性对照药物,采用 Born 比浊法对目标化合物进行体外抗血小板聚集活性初筛。结果与结论 包括先导化合物在内共制得 9 个化合物,其中 8 个未见文献报道,目标化合物的结构均经 IR、¹H-NMR 和 MS 谱确证。药理试验结果表明,化合物 PO3 的抗血小板聚集活性最高,优于吡考他胺和阿司匹林,化合物 PO4、PO5 和 PO7 的活性超过吡考他胺。     

Mesoionic oxatriazoles (MOTA): NO-donating characteristics and pharmacology.

Biological role of nitric oxide (NO), functioning of isoforms of NO synthetases (NOS) and pharmacology of principle NO-donors were reviewed. NO donating characteristics and pharmacology of 23 mesoionic oxatriazoles (MOTA) were compared with those of 5-morpholinosydnonimine (SIN-1), S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (NaNP) and glyceryl trinitrate (GTN). It is concluded that in vitro NO donating profile of MOTA hardly can be used as a predicting measure for their pharmacological activities either in vitro or in vivo. If anything, fast NO releasers seem to be stronger vasorelaxants than MOTA with slow NO releasing properties. Still, among representatives of this last category of MOTA one may find efficient antithrombotic and thrombolytic agents. For instance, MOTA 5-oxides were more potent thrombolytics than SIN-1, SNAP or NaNP. Also MOTA with potent anti-platelet action in vitro seem to be potent relaxants of tracheal strips. In summary, by manipulating the chemical structures of MOTA one may obtain relative selectivity towards vasorelaxant, anti-platelet, thrombolytic or tracheorelaxant properties. Thus different categories of MOTA might be designed with a hope of achieving hypotensive, antithrombotic, thrombolytic or anti-asthmatic drugs.     

4-甲(乙)氧基-1,3-苯二甲酰胺类化合物的合成及其体外抗血小板聚集活性

目的本文参照前期吡考他胺衍生物的构效关系,设计制得10个4-乙氧基-1,3-苯二甲酰胺类化合物(系列2);为了评价和比较其体外抗血小板聚集活性,同时进行了相应10个4-甲氧基-1,3-苯二甲酰胺类化合物(系列1)的合成,以期寻找新的抗血栓药物。方法以2,4-二甲基苯酚为起始原料,经Willianmson反应、氧化、氯代和胺解反应共制得10个未见文献报道的目标化合物(系列2),各化合物结构均经1H-NMR、IR和MS确证。采用Born比浊法对20个目标化合物进行了体外抗血小板聚集活性初筛。结果与结论在新制得的10个4-乙氧基系列化合物中,2b的活性最高,2a、2b、2d、2f的活性优于阳性对照药物吡考他胺。结果表明,新制得的4-乙氧基系列化合物与系列1各化合物相比,在抗血小板聚集方面同样具有研究价值。