凝血酶在自发性脑出血后脑水肿形成中的作用

脑出血后脑水肿形成是导致继发性神经损害的一个重要因素。近年来，有关脑出血后脑水肿产生机制的研究表明，凝血时释放的凝血酶可能是引起脑出血后脑水肿的重要物质之一，凝血酶在脑出血后脑水肿形成、炎症反应等方面起着重要作用。本文对近年来凝血酶在自发性脑出血后脑水肿形成中的作用研究进展作一综述。     

凝血酶对脑出血后脑损伤的作用机制

凝血酶在对脑出血后脑组织的损伤中起着重要作用。本文通过介绍凝血酶的化学结构及其受体的分布，从脑水肿，炎症反应，脑缺血损伤，神经元损伤四个方面，对凝血酶在脑出血后脑损伤的作用机制进行综述。最后对小剂量凝血酶 预处理在脑组织中的保护作用进行了展望。     

脑出血凝血酶的神经毒性机制及其防治策略

本文介绍了凝血酶的结构、其在中枢神经系统产生和代谢特征、在脑出血（ICH）后神经损伤中的重要作用及可能机制。提出抗凝血酶毒性治疗的可能策略：内源性凝血酶抑制剂、外源性凝血酶抑制剂、单克隆和多克隆抗体阻断凝血酶受体（PARs）及阻断PARs下游的信号转导。随着凝血酶在ICH后作用研究的深入，抗凝血酶治疗可能是防治出血性脑损伤最有希望的措施之一。     

凝血酶对大鼠脑出血血肿周围组织的毒性损伤研究

目的 :研究脑出血血液凝固过程中产生凝血酶的神经毒性作用。方法 :90只大鼠分成 :对照组、脑出血组、脑出血水蛭素干预组、凝血酶组和凝血酶 +水蛭素干预组。术后 48h观察大鼠神经功能缺损评分 (NDS) ,检测血肿周边组织髓过氧化物酶 (MPO)的活性、脑水肿含量及TUNEL阳性细胞数量。结果 :局部注入重组水蛭素显著减轻了脑出血大鼠神经功能缺损 ,降低局部MPO活性、减少脑水分含量及TUNEL阳性细胞数量 ;脑内直接注入凝血酶后局部脑水分含量升高 ,MPO活性升高 ,并可检测到大量TUNEL阳性细胞。结论 :凝血酶与脑出血后的脑水肿、白细胞浸润和神经细胞DNA损伤有关     

凝血酶与脑白质损伤的研究进展

脑出血是脑卒中的一种亚型,占脑卒中15%~20%,具有极高的致残率和病死率。近年来,研究发现凝血酶可导致脑出血后继发性脑损伤,引起神经细胞坏死、凋亡,血脑屏障破坏、脑水肿及神经炎症反应。凝血酶是一种毒性因子,在脑出血继发性脑损伤中扮演重要角色。凝血酶不仅可损害大脑灰质,损伤局部脑功能,而且能引起大脑白质病变,导致认知障碍、痴呆等。凝血酶引起的大脑灰质损伤一直是业内研究的热点,而凝血酶对大脑白质的损伤鲜见研究报道。文中就凝血酶与大脑白质损伤的研究做一综述。     

局部亚低温对脑出血后水肿影响的实验研究

目的探讨局部亚低温对大鼠脑出血后水肿形成的影响及其可能机制。方法雄性Wistar大鼠230只随机分为:对照组;脑出血组;脑出血加局部亚低温组;凝血酶加局部亚低温组。应用Evans-Blue测定血脑屏障(BBB)通透性,应用干湿重法测定脑水含量。结果与对照组相比,大鼠注血后6h开始出现脑组织水含量和BBB通透性的增加,在72h达到高峰,然后逐渐消退。不同时程局部亚低温均可以显著降低脑出血后72h时脑组织水含量及BBB通透性(P<0.01),其中给以4h局部亚低温时,降低最明显。注射凝血酶6h后,脑组织水含量及BBB通透性显著增高(P<0.01),于24~48h达高峰,然后逐渐下降。凝血酶 局部亚低温组在各个时间点与凝血酶组相比,脑组织水含量及BBB通透性明显降低(P<0.01)。结论局部亚低温可能是通过抑制凝血酶的毒性作用来减轻脑出血后水肿的形成及血脑屏障的破坏。     

脑出血后血脑屏障损害研究进展

脑水肿是脑出血的严重并发症之一,其中血脑屏障损害导致的血管源性脑水肿是脑出血 后脑水肿的主要类型。凝血酶、炎症因子、基质金属蛋白酶、补体、血红蛋白降解产物、水通道蛋白4 等以及这些因子间的相互作用都可能在脑出血后血脑屏障损害病理过程中发挥重要作用,本文对此 做一综述。     

脑出血后凝血酶的神经毒性研究进展

脑出血(intracerebral hemorrhage,ICH)是神经科常见的急、危、重症之一,有较高的病死率和致残率。脑出血后除血肿扩大导致的机械性损伤外,还有脑水肿、细胞凋亡、炎症反应、脑血管痉挛等继发性损伤[1]。研究表明凝血酶是导致脑出血后继发性损伤的重要因素。本文就凝血酶及其神经毒性、抗凝治疗展望做一综述。     

大鼠脑出血后脑组织蛋白酶连接素-1、凝血酶和蛋白酶激活受体-1表达变化的实验研究

目的 研究大鼠实验性脑出血后脑组织内蛋白酶连接素-1(PN-1)、凝血酶及蛋白酶激活受体-1(PAR-1)的表达及变化规律. 方法采用自体血注入法制备大鼠脑出血模型,Westernblot方法检测假手术组及脑出m模型组不同时程(3 h、6 h、10 h、12 h、24 h、48 h、120 h)PN-1、凝血酶和PAR-1蛋白的表达水平. 结果假手术组可以检测到少量PN-1、凝血酶和PAR-1蛋白的表达;PN-1于脑出血后3 h开始增加,此后持续上升,10 h达高峰,后逐渐回降;凝血酶于脑出血后12h显著增加,48 h达高峰;PAR-1于脑出血后3 h即显著增加,48 h达高峰,120 h仍处于较高水平.脑出血后各时间点PN-1、凝血酶和PAR-1蛋白的表达量与假手术组比较,差异均具有统计学意义(P<0.05).PN-1与PAR-1在脑出血后10h时呈负相关(r=-0.900,P<0.05),PN-1与凝血酶在脑出血后12h、120h时呈负相关(r=-0.900,P<0.05:r=-0.895,P<0.05). 结论脑出血后增多的凝血酶通过不断激活PAR-1从而介导了脑出血后的神经损伤过程,与此同时凝血酶抑制剂PN-1也大量表达,一定程度上抑制凝血酶过表达所引起的毒性效应:脑出血后三种蛋白的表达增加可能与脑出血后神经损伤的发病机制有关.     

大鼠脑出血后脑组织蛋白酶连接素-1、凝血酶和蛋白酶激活受体-1表达变化的实验研究

目的 研究大鼠实验性脑出血后脑组织内蛋白酶连接素-1(PN-1)、凝血酶及蛋白酶激活受体-1(PAR-1)的表达及变化规律. 方法采用自体血注入法制备大鼠脑出血模型,Westernblot方法检测假手术组及脑出m模型组不同时程(3 h、6 h、10 h、12 h、24 h、48 h、120 h)PN-1、凝血酶和PAR-1蛋白的表达水平. 结果假手术组可以检测到少量PN-1、凝血酶和PAR-1蛋白的表达;PN-1于脑出血后3 h开始增加,此后持续上升,10 h达高峰,后逐渐回降;凝血酶于脑出血后12h显著增加,48 h达高峰;PAR-1于脑出血后3 h即显著增加,48 h达高峰,120 h仍处于较高水平.脑出血后各时间点PN-1、凝血酶和PAR-1蛋白的表达量与假手术组比较,差异均具有统计学意义(P<0.05).PN-1与PAR-1在脑出血后10h时呈负相关(r=-0.900,P<0.05),PN-1与凝血酶在脑出血后12h、120h时呈负相关(r=-0.900,P<0.05:r=-0.895,P<0.05). 结论脑出血后增多的凝血酶通过不断激活PAR-1从而介导了脑出血后的神经损伤过程,与此同时凝血酶抑制剂PN-1也大量表达,一定程度上抑制凝血酶过表达所引起的毒性效应:脑出血后三种蛋白的表达增加可能与脑出血后神经损伤的发病机制有关.     

Effect of Different Types of Thrombin Inhibitors on Thrombin/Thrombomodulin Modulated Activation of Protein C In Vitro

The objectives of this study were to investigate whether the affinity of thrombin for small-molecule, active site-directed thrombin inhibitors and substrates is affected by the presence of thrombomodulin (TM), and to what extent thrombin inhibitors inhibit TM-bound thrombin. Inhibition of human -thrombin was studied in the presence and absence of solubilised rabbit lung TM in a buffer containing CaCl₂. TM inhibited thrombin-induced proteolysis of human fibrinogen with a dissociation constant (K_D) of 4 nmol/l. With at least 16-fold molar excess of TM over thrombin the affinity of thrombin both for the small thrombin substrates (S-2366 and S-2238) and the reversible, active site-directed thrombin inhibitors (inogatran and melagatran) increased twofold. In contrast, the ability of hirudin to inhibit thrombin was reduced by TM, since hirudin competes with TM in binding to thrombin. The effect of thrombin inhibitors on protein C activation by thrombin bound to human kidney cells transfected with cDNA for human TM was also studied. The mean binding capacity of the transfected cells was approximately 320,000 quantified by flow cytometry with antibodies against TM. Hirudin, inogatran and melagatran inhibited the activation of protein C by thrombin complexed with cell-bound TM in a dose-dependent manner, with mean IC₅₀ values±S.D. of 4.4±0.8, 20.0±1.1 and 6.4±0.2 nmol/l, respectively. Antithrombin inhibited protein C activation with an IC₅₀ value of 290±10 nmol/l, which was enhanced fourfold (IC₅₀ 60 nmol/l) by the addition of heparin 0.5 U/ml. Heparin alone, up to a concentration of 1 U/ml, had no effect on the activation of protein C. Small direct thrombin inhibitors thus inhibited both free and TM-bound thrombin and therefore also inhibited the activation of protein C. Whether this will influence their clinical efficacy or safety versus heparin and warfarin, which also inhibit protein activation, respectively, lowers the concentration of protein C, remains to be studied in clinical trials.     

组织型谷氨酰胺转移酶抑制剂对凝血酶脑组织毒性的影响

目的通过动物在体实验了解组织型谷氨酰胺转移酶(tissue transglutaminase,tTG)抑制剂(monodansyl-cadaverine,MDC)对凝血酶(thrombin,Tm)脑组织毒性的影响。方法第1组动物用4种剂量的Tm(0、5、10、15U溶于5μl生理盐水)通过立体定向仪注射入大鼠纹状体,第2组动物在各剂量Tm中同时加入MDC(5mM)加以干预作为相应对照,动物存活24h后取注射部位石蜡包埋切片,做常规HE染色和凋亡特异性TUNEL染色及照相。观察两种染色的切片细胞变化并对各组TUNEL特异染色的凋亡细胞进行计数,以了解在大鼠体内MDC抑制tTG对Tm诱导凋亡作用的影响。结果Tm处理脑组织的胞体发生肿胀,细胞核发生浓缩固缩现象,核仁消失,核膜完整性破坏,且浓度越高异常形态的细胞越多。而在对照的Tm MDC处理组织切片的异常细胞减少。TUNEL凋亡细胞特异性染色计数,5、10和15UTm凋亡细胞数分别为(13.78±3.31)、(25.56±4.16)和(35.00±5.96)个;而同时加入MDC3种不同浓度的凋亡细胞数分别为(8.56±2.51)、(16.89±2.03)和(28.33±5.83)个。结论MDC可通过抑制tTG减少Tm引起的细胞凋亡。     

Effects of sodium and lithium salts on the conformation of human alpha-thrombin

Chemical modification studies have demonstrated that the ultra-violet difference spectrum of alpha-thrombin produced in the presence of sodium is due primarily to changes in the environment of tyrosine residues. This is based on the observation that the spectrum could be abolished by treatment of alpha-thrombin with tetranitromethane but not with dimethyl-(2-hydroxy-5-nitrobenzyl) sulfonium bromide. Although lithium produces similar (UV) difference spectrum, circular dichroism studies indicate that sodium and lithium induce different conformational transitions. alpha-Thrombin tends to assume a more ordered structure in the presence of sodium whereas lithium has the reverse effect. This inverse behavior is consistent with the effects of these cations on the autolysis rate and thermal stability of the activities of alpha-thrombin.     

Hirudin insensitive thrombin-stimulated platelet release

It has been previously proposed that platelet stimulation may involve two platelet-thrombin complexes: an initial platelet-thrombin complex (P-T) which is converted to an activated platelet-thrombin complex (P*-T). By using the release of radioactive serotonin as a measure of thrombin stimulation, we have demonstrated that under appropriate conditions, a hirudin sensitive and a hirudin insensitive complex can be differentiated. At short platelet-thrombin preincubation times (0-2 minutes) at 4 degrees C, in a buffer containing 18.7 mM phosphate, added hirudin almost completely inhibited the release of radioactive serotonin obtained upon subsequent warming to 37 degrees C (only the hirudin sensitive complex exists). If platelets were preincubated with thrombin for longer periods of time (30 minutes), hirudin became less effective in inhibiting the release obtained upon subsequent warming to 37 degrees C. The same results were obtained whether or not the platelets were washed after incubation at 4 degrees C and before warming to 37 degrees C. We postulate that this change in hirudin sensitivity may reflect a slow conversion of the first platelet-thrombin complex (P-T) to an activated platelet-thrombin complex (P*-T) which can undergo release upon warming. This transition appears to be much faster in acetatetris buffer since at short platelet-thrombin incubation times at 4 degrees C, added hirudin had little or no effect on the release obtained upon warming to 37 degrees C. The difference in the ability of hirudin to inhibit thrombin-induced release in the two buffers was shown to depend upon the presence of phosphate and on variations in ionic strength, and not due to a change in the inhibition constant (Ki) for hirudin.     

A thrombin assay based upon the release of fibrinopeptide A from fibrinogen: definition of a new thrombin unit

An assay for thrombin is presented wherein thrombin-catalyzed hydrolysis at Arg-A alpha-16 to release fibrinopeptide A (FPA) from fibrinogen is measured using high-performance liquid chromatography (HPLC). In this assay one thrombin unit (TU) is defined as that amount of thrombin that will release half of the FPA in one min from one ml of a solution of greater than 90% clottable normal human fibrinogen (less than or equal to 0.35 microM) at 37 degrees C, pH 7.4, /2 0.15. One TU is equivalent to approximately 0.1 NIH unit of thrombin and approximately 1 pmol of pure human thrombin. At 37 degrees C, pH 7.4, and plasma levels of fibrinogen of 3 mg/ml, one TU will catalyze the release of 3.6 nmol FPA min-1. Variability in fibrinogen samples which produce dramatic differences in clotting time assays with the same sample of thrombin, produce little or no variation in the catalytic assay for TU. The assay for TU obviates the need for maintenance of a thrombin reference standard.