抗CD59对CVF激活人血小板的影响

目的:研究补体的激活对人血小板的作用及其抗CD59对该作用的影响。方法:应用眼镜蛇毒因子(CVF)制成补体活化血小板的模型,通过测定健康成年男性血小板的聚集及释放曲线,观察抗CD59应用前后补体对血小板变形、聚集及释放功能的影响。结果:CVF能诱导健康成年男性的血小板显著、持久地变形和释放,但不能诱导血小板聚集。当CVF浓度在12.5-50.0g/L范围内,血小板的最大变形幅度与CVF浓度的对数呈线性相关(r=0.970,P<0.01,n=36)。抗CD59能增强健康成年男性血小板的最大变形幅度,并与剂量相关,其血小板最大变形幅度的最大增加值为空白对照的1.36倍(P<0.01)。同时抗CD59也促进补体诱导的血小板ATP分泌增多。结论:活化补体后能诱导健康成年男性血小板发生变形、释放,但不诱导血小板的聚集,且抗CD59能增加补体诱导的血小板功能改变。     

ERK5对体外血小板活化及在体血栓的影响

目的:探讨细胞外信号调节激酶5(ERK5)对体外血小板聚集及在体血栓的影响及机制。方法:采用Western blot对人血小板中ERK5的表达及其在血小板活化后的磷酸化水平进行检测;采用血小板聚集仪检测ERK5特异性抑制剂XMD8-92对血小板聚集及致密颗粒释放的影响;采用Fe Cl3颈动脉血栓模型检测ERK5对在体血栓的影响;采用Western blot检测XMD8-92对蛋白激酶B(Akt)和人第10号染色体缺失的磷酸酶及张力蛋白同源蛋白(PTEN)磷酸化的影响。结果:人血小板中存在ERK5的稳定表达,其磷酸化水平在血小板活化后显著升高(P0.05)。XMD8-92可抑制多种血小板激活剂引起的血小板聚集和致密颗粒释放(P0.05)。Western blot结果表明,XMD8-92可通过下调PTEN Ser370位点磷酸化而增强PTEN的活性,从而抑制Akt的磷酸化,这种抑制效果也通过血小板特异PTEN基因敲除小鼠得到了验证。在体血栓研究表明,XMD8-92经尾静脉给药,可显著延长小鼠第一次颈动脉血栓的形成时间。结论:ERK5可通过影响PTEN的磷酸化调节Akt的活化,进而影响到体外血小板的聚集和在体血栓的形成。     

绞股蓝对血小板聚集及微循环的影响

本文观察了100例人体内服绞股蓝水煎液后血小板聚集功能及微循环的变化,发现绞股蓝水煎液内服对血小板聚集功能有明显的抑制作用(P<0.001)。对100例高粘滞血症患者的球结膜微循环的血流障碍有明显的改善(P<0.001)。其机理是绞股蓝降低血栓素A_2/前列环素的比值,从而抑制血小板聚集使血液粘度下降,微循环血流障碍得以改善。同时用西洋参水煎液内服作对比观察,发现西洋参在抑制血小板聚集功能方面与绞股蓝有类似作用。     

离体人类血小板与Ⅰ型骨胶原、四硫酸软骨素及六硫酸软骨素表面的相互作用

作者研究了离体人类血小板与Ⅰ型骨胶原六硫酸软骨素(CH-6-S)、四硫酸软骨素(CH-4-S)H-6-S/骨胶原层。以及骨胶原-CH-6-S的复合物表面之间的相互作用,把聚苯乙烯和硅烷玻璃作为对照。将血小板数目、血小板因子Ⅳ释放量以及血小板在不同表面的聚集能力与对照相比较,血小板数目和血小板因子Ⅳ释放量的数据表明:CH-6-S、CH-4-S与对照表面之间无差异。然而当将骨胶原、CH-6-S/骨胶原层以及骨胶原与CH-6-S的复合物与对照比较时发现血小板目及血小板因子Ⅳ释放量有显著差异。纯Ⅰ型骨胶原表面对血小板     

高脂血清对内皮细胞前列环素,血栓素和脂质过氧化物的影响

实验比较了高脂兔血清(HRS)和正常兔血清(RS)对培养的小牛主动脉内皮细胞(EC)的PGI_2和TXA_2代谢的影响及其与脂质过氧化物(Lpo)形成和超氧化物歧化酶(SOD)活性的关系。结果表明:HRS对EC合成PGI_2的影响有一个时间和浓度相关的反应过程。用HRS孵育生长融合状的EC、在早期阶段可刺激PGI_2的产生,以后则呈现为持续下降,同时其抗血小板聚集能力也降低,而Lpo含量增高,SOD活性下降。统计处理发现,HRS浓度与Lpo含量呈正相关关系,而HRS和Lpo两者与PGI_2均呈负相关。经HRS孵育的EC其TXA_2产量也较对照组低。结果提示HRS及其代谢过程产生的高浓度Lpo引起的EC PGI_2产量由上升至下降的变化过程可能是EC对损伤反应的表现,最终表现为PGI_2合成障碍,抗血小板聚集能力减弱。     

通用血小板的研究

目的研究有效修饰血小板表面HLA的方法。方法采用浓度梯度法用mPEG修饰血小板表面HLA。通过微量淋巴细胞毒试验检测修饰效果,并对血小板功能进行检测。修饰后血小板输注到中国白兔体内检测血小板计数。结果mPEG可有效修饰血小板表面HLA,黏附与聚集能力分别由修饰前的(30.2±5.59)%和(1.43±0.74)%升至修饰后的(45.5±2.72)%,(3.86±1.21)%,Ca2 释放无显著差异。中国白兔输注修饰血小板24h时血小板升高指数及血小板回收率分别为69.93±11.20和(52.06±10.92)%。结论mPEG修饰可有效阻断血小板表面HLA与其相应抗体的结合,修饰后血小板黏附及聚集功能有所增强,Ca2 释放功能不受影响,中国白兔输注修饰后血小板计数增高。     

H_2S对ATP诱导小胶质细胞活化的影响

目的:研究硫化氢(hydrogen sulfide,H_2S)对三磷酸腺苷(adenosine triphosphate,ATP)诱导大鼠小胶质细胞炎性因子释放的调节,并探讨小胶质细胞条件培养基对神经元样细胞凋亡的影响。方法:选定的大鼠小胶质细胞随机分4组:(1)正常对照组:常规培养;(2)ATP组:细胞接种24 h后用ATP处理;(3)Na HS(sodium hydrosulfide,H_2S供体)+ATP组:ATP处理前用Na HS预孵育30 min,且Na HS始终存在于反应体系中;(4)KN-62(异喹啉衍生物,嘌呤受体拮抗剂)+ATP组:ATP处理前用KN-62预孵育30 min。用ELISA检测各组细胞上清液中TNF-α、IL-6的变化,用Western Blot检测各组丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)P38和JNK表达水平。用人神经母细胞瘤细胞(SH-SY5Y)研究条件培养基对神经元的影响,其方法与上述大鼠小胶质细胞处理相同。用倒置显微镜观察SH-SY5Y细胞形态变化及凋亡情况,用MTT法检测各组细胞活力。结果:当ATP浓度3 mmol/L时,大鼠小胶质细胞释放TNF-α和IL-6水平增加(P0.05),同时上调大鼠小胶质细胞p-P38、p-JNK蛋白表达水平(P0.05)。当ATP浓度5 mmol/L时,小胶质细胞活化后的条件培养基可以使SH-SY5Y细胞形态发生改变,其细胞活力降低(P0.05)。ATP引起的上述变化可以通过Na HS预处理而逆转(P0.05),其作用与P2X7R阻断剂KN-62相类似。结论:H_2S可下调被ATP诱导的p-P38和pJNK MAPK蛋白表达,减少TNF-α和IL-6等炎性因子的释放,从而对神经元产生保护作用。     

血小板聚集功能和尿11-脱氢血栓素B_2水平测定对评价脑梗死患者阿司匹林治疗作用的价值

目的:探讨血小板聚集功能和尿11-脱氢血栓素B2(11-DTB2)检测对评价一次及反复脑梗死患者服用阿司匹林治疗作用的价值。方法:42例反复脑梗死(≥2次)患者和50例发生一次脑梗死患者服用阿司匹林(100mg/天)至少7天后,用二磷酸腺苷(ADP)和花生四烯酸(AA)作诱导剂,测定两组血小板最大聚集率,并测定其11-DTB2的含量。结果:AA作诱导剂时,反复脑梗死患者与一次脑梗死患者比较,其血小板最大聚集率、阿司匹林半抵抗发生率明显较高(P<0.01);尿11-DTB2也是前者高于后者,差异有显著性统计学意义(P<0.01)。结论:脑梗死患者服用阿司匹林时检测血小板聚集功能和尿11-DTB2水平,可以观察阿司匹林抑制血小板功能的程度及临床疗效。     

血小板膜的粘附蛋白受体

粘附蛋白包括纤维蛋白原(Fg)、血管性假血友病因子(vWF)、纤维连接蛋白(Fn)和血小板反应素(TSP),分别参与血小板粘附的聚集。血小板被激活时,粘附蛋白从α颗粒释放,与膜表面相应受体结合。血小板膜蛋白有100多种,主要为糖蛋白(GP),构成受体。GPⅡ_b-Ⅲ_a占血小板膜蛋白的主要部分,在血小板激活过程中起着各种粘附蛋白受体的作用。但GPⅠ_b是vWF的受体,参与血小板与内皮下粘附。     

脑梗塞患者血小板形态学与微循环的研究

本文对血小板超微结构、球结膜微循环与脑梗塞之间的关系进行了观察,进一步探讨血小板在脑梗塞发病中的动态改变及球结膜微循环的变化。结果显示:正常血小板呈园盘形或椭园形,中间为密度很高约2-3个致密体,并有多个溶酶体颗粒,以及较小的密度很高的许多糖元颗粒等。而患者组血小板形态、线粒体、致密斑颗粒、糖元颗粒与对照组比较均有显著变化或减少(P<0.001)。球结膜微循环的改变亦更明显,微血管血流慢、血管迂曲、形态不规则、局部扩张、细胞聚集等(P<0.01)。脑梗塞患者组血小板超微结构及球结膜微循环的改变与疾病程度成同步变化倾向。故脑梗塞患者除治疗缺血外,还应解除微循环障碍。     

Mechanisms of platelet response to monosodium urate crystals.

The mechanisms of urate-crystal-induced release of platelet constituents has been studied morphologically and biochemically. Urate crystals provoked an early energy-dependent release of the dense-body constituents serotonin, ADP, and ATP from washed platelets. Concurrently, platelet ultrastructure showed evidence of shape change, contractile wave, and aggregation. These are typical morphologic concomitants of platelet secretion. By 30 minutes' incubation, urate-induced platelet lysis occurred, as shown by loss of the cytoplasmic enzyme lactic dehydrogenase (LDH) and ultrastructurally by disruption of platelet membrane integrity. Cytochalasin B inhibited the urate-crystal-induced shape change, aggregation, and disruption of cell membranes. Platelet degranulation was not inhibited and the initial component of serotonin release was not affected. Cytochalasin B also abrogated crystal-induced LDH loss. Thus, the initial crystal-induced serotonin release does not depend on platelet lysis. It is concluded that urate-crystal--induced release of serotonin, ATP, and ADP represents an example of platelet secretion.     

Rat platelet aggregation by ATP. Aggregometrical and ultrastructural comparison with aggregations induced by ADP and collagen.

This paper describes the aggregation of rat platelets by adenosine triphosphate (ATP). The aggregometry of ATP-induced aggregation and the ultrastructure of ATP-aggregated platelets were compared and contrasted with those of adenosine diphosphate (ADP)-treated and collagen-treated samples. Human platelets were also studied alongside with rat specimens. Several lines of evidence indicate that the ATP-induced aggregation of rat platelet-rich plasma (PRP) is not a result of contaminating ADP in the ATP preparation. ATP did not cause aggregation of human platelets; it inhibited ADP- and collagen-induced human platelet aggregation. ATP pretreated with a creatine phosphate/creatine phosphokinase system caused similar rat platelet aggregation as did ATP not treated with this system. The aggregometry of ATP-induced aggregation of rat PRP was similar to that of collagen-induced aggregation but markedly different from that of ADP-induced aggregation. However, the nature of ATP-induced aggregation was similar to that induced by ADP. Both ATP- and ADP-induced rat platelet aggregations were not affected by adenosine, adenosine monophosphate, or acetylsalicylic acid. The ultrastructure of ATP-aggregated platelets was similar to that of ADP-aggregated ones. It appears that either platelets of rats possess specific ATP receptors or the rat plasma contains a material, lacking or insufficiently present in human plasma, that converts ATP to ADP in a fashion similar to the release of ADP from platelet storage granules.     

Platelet aggregation induced by latex particles. I. Effects of size, surface potential and hydrophobicity of particles

Latex particles induced platelet aggregation associated with the release of ATP from the platelets. The smaller the diameter of particles having the same surface structure, the greater numbers or greater total surface area of particles were required for both reactions. The higher hydrophobic and higher negatively charged particles, having a diameter of about 0.3 micron, induced platelet aggregation most easily. Hydrophilic particles without high negative surface potential activated platelets only a little. Particle-induced platelet aggregation is not only caused by colloidal electronic force and hydrophobic interaction between platelets and latex particles but also by factors concerning cell activation.     

Shiga toxin 2 and lipopolysaccharide induce human microvascular endothelial cells to release chemokines and factors that stimulate platelet function

Shiga toxins (Stxs) produced by Shigella dysenteriae type 1 and enterohemorrhagic Escherichia coli are the most common cause of hemolytic-uremic syndrome (HUS). It is well established that vascular endothelial cells, mainly those located in the renal microvasculature, are targets for Stxs. The aim of the present research was to evaluate whether E. coli-derived Shiga toxin 2 (Stx2) incubated with human microvascular endothelial cells (HMEC-1) induces release of chemokines and other factors that might stimulate platelet function. HMEC-1 were exposed for 24 h in vitro to Stx2, lipopolysaccharide (LPS), or the Stx2-LPS combination, and chemokine production was assessed by immunoassay. More interleukin-8 was released than stromal cell-derived factor 1alpha (SDF-1alpha) or SDF-1beta and RANTES. The Stx2-LPS combination potentiated chemokine release, but Stx2 alone caused more release of SDF-1alpha at 24 h than LPS or Stx2-LPS did. In the presence of low ADP levels, HMEC-1 supernatants activated platelet function assessed by classical aggregometry, single-particle counting, granule secretion, P-selectin exposure, and the formation of platelet-monocyte aggregates. Supernatants from HMEC-1 exposed only to Stx2 exhibited enhanced exposure of platelet P-selectin and platelet-THP-1 cell interactions. Blockade of platelet cyclooxygenase by indomethacin prevented functional activation. The chemokine RANTES enhanced platelet aggregation induced by SDF-1alpha, macrophage-derived chemokine, or thymus and activation-regulated chemokine in the presence of very low ADP levels. These data support the hypothesis that microvascular endothelial cells exposed to E. coli O157:H7-derived Stx2 and LPS release chemokines and other factors, which when combined with low levels of primary agonists, such as ADP, cause platelet activation and promote the renal thrombosis associated with HUS.     

Diagnostic usefulness of a lumi-aggregometer adenosine triphosphate release assay for the assessment of platelet function disorders

Platelet dense granule release assays are recommended for diagnosing platelet function disorders and are commonly performed by Lumi-Aggregometer (Chrono-Log, Havertown, PA) assays of adenosine triphosphate (ATP) release. We conducted a prospective cohort study of people tested for ATP release defects to assess bleeding symptoms. Reduced release, with 1 or more agonists, was more common among patients with bleeding disorders than among healthy control subjects (P < .001). The respective likelihood (odds ratio [95% confidence interval]) of a bleeding disorder or an inherited platelet function disorder were high when release was reduced with 1 or more agonists (17 [6-46]; 128 [30-545]), even if aggregation was normal (12 [4-34]; 105 [20-565]). ATP release had high specificity and moderate sensitivity for inherited platelet function disorders, with most abnormalities detected by the combination of 6 μmol/L epinephrine, 5.0 μg/mL collagen, and 1 μmol/L U46619. Platelet ATP release assays are useful for evaluating common bleeding disorders, regardless of aggregation findings.     

The effect of the platelet count on the aggregation response and adenosine triphosphate release in an impedance lumi-aggregometer

Platelet aggregation studies are usually performed in either electrical impedance or optical systems and the release reaction assessed by quantitating adenosine triphosphate (ATP) from luminesence produced by the firefly luciferin-luciferase system. In evaluating the results of such studies, attention is paid to a variety of parameters such as the slope of the aggregation response and the maximum aggregation expressed as percent light transmission in optical and as Ohms in electrical impedance systems. Although threshold platelet counts are frequently cited below which the performance of these studies is technically difficult, the influence of the platelet count within the normal range on the results of such studies has not been prospectively addressed. This study examines the relationship between the aggregation response, ATP release and the platelet count in a lumi-impedance system. It is clear that the platelet count influences the results in this system and requires consideration in the interpretation if an erroneous conclusion is to be avoided.     

Age-related changes in human platelet function in vitro

Platelet aggregation and ATP release were simultaneously measured in platelet-rich plasma samples obtained from humans of various age. In subjects aged over 59 years, an increase was found in platelet sensitivity to ADP and collagen as well as elevated aggregation amplitudes 5 min after induction with low concentrations of the aggregation agents. At 1 mumol/l ADP, old persons had a higher incidence of a secondary aggregation wave. Platelets from old subjects also released more ATP in response to collagen stimulation. Since no age-related changes are commonly found in the platelet count, the observed increase in platelet sensitivity to aggregation agents cannot be regarded as a mere compensatory reaction. An attempt is made to relate altered platelet function to peculiarities of lipid metabolism associated with ageing.     

Polymorphonuclear leucocytes release a factor(s) that induces platelet aggregation and ATP release after interaction with insoluble and surface-fixed immune complexes.

We have found that human polymorphonuclear leucocytes (PMN) can be stimulated by large aggregates (heat-aggregated IgG, chemically polymerized IgG, heavily aggregated human immune complexes) and by surface-bound immune complexes (IC) to release enzymes (lysozyme, beta glucuronidase) and a factor(s) able to induce platelet aggregation and ATP release from the platelets. Surface-bound IC were most effective in stimulating the release of this factor(s). We used several substrates for their preparation: plastic-adsorbed antigen. Sepharose-coupled antigen and polymerized antigen. The platelet-aggregating factor(s) released by IC-stimulated PMN and zymosan-stimulated PMN were compared for their susceptibility to inhibition by indomethacin. Both induced a first phase of platelet aggregation that was resistant to indomethacin, but the second phase of aggregation and the release of platelet ATP were inhibited to a variable degree, more pronounced in the case of the factor(s) released after PMN-IC interaction. The lack of inhibition of the early phases of aggregation induced by our factor(s) when platelets were simultaneously exposed to indomethacin suggests that the classical, phospholipid PAF is released under these experimental conditions. Although, further experiments will be necessary to fully characterize the factor(s) involved, our observations suggest a complex interrelationship between human PMN and platelet activation, which may play an important role in the sequence of events that mediate the tissue deposition of IC and appearance of inflammatory changes.     

Effects of simvastatin/ezetimibe on microparticles, endothelial progenitor cells and platelet aggregation in subjects with coronary heart disease under antiplatelet therapy

It is not known whether the addition of ezetimibe to statins adds cardiovascular protection beyond the expected changes in lipid levels. Subjects with coronary heart disease were treated with four consecutive 1-week courses of therapy (T) and evaluations. The courses were: T1, 100 mg aspirin alone; T2, 100 mg aspirin and 40 mg simvastatin/10 mg ezetimibe; T3, 40 mg simvastatin/10 mg ezetimibe, and 75 mg clopidogrel (300 mg initial loading dose); T4, 75 mg clopidogrel alone. Platelet aggregation was examined in whole blood. Endothelial microparticles (CD51), platelet microparticles (CD42/CD31), and endothelial progenitor cells (CD34/CD133; CDKDR/CD133, or CD34/KDR) were quantified by flow cytometry. Endothelial function was examined by flow-mediated dilation. Comparisons between therapies revealed differences in lipids (T2 and T3<T1 and T4 for total cholesterol, LDL-C, and triglycerides; P<0.002 for all), as well as for endothelial function (T2>T1 and T4, P=0.001). Decreased platelet aggregation was observed after aspirin (arachidonic acid, T1<T3 and T4, P=0.034) and clopidogrel (adenosine, T3 and T4<T1 and T2, P<0.0001) therapy. Simvastatin/ezetimibe diphosphate did not change platelet aggregation, the amount of circulating endothelial and platelet microparticles, or endothelial progenitor cells. Cardiovascular protection following therapy with simvastatin/ezetimibe seems restricted to lipid changes and improvement of endothelial function not affecting the release of microparticles, mobilization of endothelial progenitor cells or decreased platelet aggregation.