蝮蛇蛇毒中提取的一种具有激肽原酶活性的蛋白水解酶

目的:从蛇毒中获得大量高纯度的具有激肽原酶活性的蛋白水解酶.方法:采用离子交换和亲和层析技术进行纯化.结果:从江浙蝮蛇毒中提取出了一种高纯度的具有激肽原酶活性的蛋白水解酶,不需活化即可水解激肽原,释放激肽,并具有精氨酸酯酶活性.粗毒经提纯,比活可达800 U/mg以上,远远高于哺乳动物来源的激肽原酶,纯度可达95%以上.对其性质考察,Mr为38 000,N-末端的15个氨基酸序列分析表明,该酶与蛇毒丝氨酸蛋白酶及胰蛋白酶——激肽释放酶同源.结论:这种方法适合于工业化生产.     

逆流免疫电泳法检测蛇毒

蛇毒是一种潜在的新战剂。建立一种快速、灵敏度高的分析方法具有一定的现实意义。用逆流免疫电泳法对白眉蝮蛇毒、黑眉蝮蛇毒、五步蛇毒进行了半定量分析,检定范围前两种在0.048-1.5μg,后者在0.024-1.5μg。做区分鉴别试验,在0.15-1.5μg范围内,能区分蝮蛇毒、五步蛇毒,竹叶青蛇毒和烙铁头蛇毒,但不能将黑、白眉腹蛇毒区分开。     

蝮蛇毒蛋白C激活物对家兔实验性DIC的干预作用

目的:观察蝮蛇毒蛋白C激活物(PCA)对家兔实验性弥散性血管内凝血(DIC)的干预作用并探讨其作用机理。方法:健康青紫蓝家兔24只随机分为:正常对照组、DIC组、PCA干预组(n=8),采用耳缘静脉注射兔脑粉浸液建立DIC模型,检测各组的凝血酶原时间(PT)、活化部分凝血活酶时间(APTT)、凝血酶时间(TT)、纤维蛋白原(Fbg)含量和血小板计数(PLT)。结果:与正常对照组相比,DIC组PT、APTT延长的百分率、TT缩短的百分率、Fbg、PLT显著升高(P0.01);PCA干预组PT、APTT延长的百分率,TT缩短的百分率与Fbg、PLT降低的百分率均明显降低(P0.01)。结论:蝮蛇毒PCA对家兔实验性DIC有一定的防治作用,其机制可能与PCA抗凝、抑制血小板聚集有关。     

蛇毒F_(6.3.2)激活蛋白C的可能机制初探

本实验研究蛇毒蛋白C激活组份F6.3.2对蛋白C的激活作用。以BaCl2吸附血浆和纯化蛋白C为作用底物,采用发色底物法测定蛇毒组份F6.3.2对蛋白C的特异性激活作用,同时采用SDS-PAGE.测定该组份对蛋白C的激活机制。结果发现F6.3.2没有直接作用发色底物使之生色的能力。它对BaCl2吸附血浆不表现生色反应能力(与对照组相比P>0.05),而对纯化蛋白C则表现出很强的生色反应能力(与对照组Ⅰ、对照组Ⅱ相比P<0.01).SDS-PAGE结果表明,F6.3.2与纯化蛋白C作用后的混合物,由两条链组成,一条链分子量为19KDa,与纯化蛋白C的轻链相同,另一条链分子量为59.5 KDa。为纯化蛋白C重链与F6.3.2的分子量之和,表明F6.3.2已结合到纯化蛋白C的重链上。因此.我们认为蛇毒蛋白C激活组份F6.3.2对蛋白C有特异性激活作用,它激活蛋白C的可能机制是通过结合于蛋白C的重链,形成F6.3.2-蛋白C复合物,引起蛋白C变构,从而将蛋白C激活为活化蛋白C。     

血栓调节蛋白的研究进展

血栓调节蛋白(TM)作为内皮细胞表面一种具有抗凝活性的糖蛋白,不仅是重要的抗凝辅助因子,也是反映血管内皮细胞损伤的分子标志,通过与凝血酶结合,激活蛋白C而发挥抗凝作用.TM还可通过激活血管中Pro-PcPB而发挥纤溶抑制作用.组织中TM mRA的表达及血浆中TM浓度变化也可作为肿瘤预后的指标和动脉硬化,RDS等病情变化的敏感指标.     

白眉蝮蛇毒激肽原酶对糖尿病模型大鼠血液学变化的影响

目的:从白眉蝮蛇毒中提取激肽原酶,研究白眉蝮蛇毒激肽原酶对糖尿病大鼠血液学变化的影响.方法:用疏水层析技术从白眉蝮蛇毒中提取出的一种高纯度的激肽原酶,腹腔注射链脲佐菌素(STZ)制造大鼠糖尿病模型,将糖尿病大鼠随机分为三组,蛇毒激肽原酶组静脉注射白眉蝮蛇毒激肽原酶,阳性对照组不予处理,消渴丸组灌胃给予消渴丸研粉,另取正常大鼠作为阴性对照组.检测血糖、血小板聚集率、全血黏度.结果:蛇毒激肽原酶降糖作用不明显,但可显著降低实验大鼠模型的血小板聚集率及全血黏度,在改善全血黏度方面优于消渴丸.结论:蛇毒激肽原酶可通过对血液流变的改善作用达到防治糖尿病血管病变的作用.     

血浆蛋白C、蛋白S抗凝体系的研究进展

蛋白C是一种维生素K依赖性糖蛋白,它为血浆丝氨酸蛋白酶酶原;凝血酶能将蛋白C转化为活化蛋白C(APC);在一种叫做蛋白S(PS)的APC协同因子存在时,APC发挥抗凝活性;蛋白C和蛋白S缺陷或缺乏可引起动、静脉血栓形成.     

围术期患者右旋糖酐40急性扩容前后凝血、抗凝、纤溶功能的变化

目的:观察围术期患者使用右旋糖酐40溶液急性扩容后凝血、抗凝和纤溶功能指标的变化。方法:选择ASAⅠ~Ⅱ级全麻下行腹部手术患者40例,麻醉开始后即刻输注复方右旋糖酐40溶液1000ml,持续输注2h。于输注前后采集患者静脉血监测血小板计数(PLT)、活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)、纤维蛋白原(Fib)、纤溶酶原(PLG)、凝血因子VIII活性(FVIII:C)、抗凝血酶活性(AT:A)、活化蛋白C(APC)等凝血、抗凝及纤溶指标的变化。结果:应用右旋糖酐40后,APTT、PT延长,FVIII:C降低,差异有显著性意义(P<0.05~0.01);AT:A、APC和PLG明显降低(P<0.05~0.01)结论:在围术期使用1000ml右旋糖酐40急性扩容对患者的凝血、抗凝和纤溶指标有明显影响,临床应用时需防范其引起出血和血栓形成的危险。     

复方丹参注射液抗脂多糖诱导的兔弥漫性血管内凝血

目的: 研究复方丹参注射液对脂多糖(LPS)诱导的兔弥漫性血管内凝血(DIC)的作用。方法: 用LPS诱导兔DIC模型。凝固法测定纤维蛋白原含量,全自动凝血分析仪检测活化部分凝血活酶时间(APTT)、凝血酶原时间(PT)和纤维蛋白原含量;全自动血细胞分析仪进行血小板计数;全自动血浆分析仪测定谷丙转氨酶(ALT)以及血尿素氮(BUN);发色底物法测定蛋白C及抗凝血酶Ⅲ的活性。观察复方丹参注射液对LPS诱导的兔DIC的拮抗作用。结果: 兔耳缘静脉持续滴注LPS,观察到:APTT和PT显著延长;血小板计数和纤维蛋白原含量明显减少;ALT和BUN显著升高;蛋白C和抗凝血酶Ⅲ的活性明显降低。给予复方丹参注射液后,APTT和PT的延长明显缩短;血小板计数和纤维蛋白原的含量均明显恢复;ALT和BUN显著下降;蛋白C及抗凝血酶Ⅲ的活性显著改善。结论: 复方丹参注射液对LPS诱导的兔DIC有良好的拮抗作用。     

蝮蛇抗栓酶的不良反应

<正> 蝮蛇杭栓酶(下称svate)是从蛇毒中分离出的一种具有精氨酸酶活性的蛋白酶制剂。具有抗凝、溶栓、去纤、减少血小板聚集、扩血管、改善微循环等作用。临床广泛用于治疗脑血栓、冠心病、闭塞性血管性疾病、肾病综合征、高脂血症等多种疾病。疗效肯定,副作用较少。最近,有关副作用也有报告。为引起重视,现综述如下: 一、过敏反应:Svate是一种蛋白酶制剂,故可     

Role of protein C in endotoxin-induced release of plasminogen activator inhibitor from endothelial cell

To elucidate the role of protein C (PC) in the release of plasminogen activator inhibitor (PAI) from endothelial cells, the effect of PC and activated protein C (APC) on plasma levels of PAI in endotoxin (ET)-treated rats was examined. When activated by snake venom, human PC significantly prolonged the activated partial thromboplastin time (APTT) of both human and rat plasma samples. Addition of APC also prolonged the APTT of both human and rat plasma samples. PAI activity in plasma from septicemic patients and ET-treated rats was neutralized by APC. A small dose of ET (0.1 microgram/kg) gradually increased plasma PAI activity, which became maximum 3h after ET-treatment. APC administered prior to ET-treatment, PC decreased PAI activity, however, no such inhibition was seen when administered after ET-treatment. A significant negative correlation between PC concentrations and PAI activities was observed in plasma from septicemic patients. These findings indicated that activation of PC on endothelial surface plays a regulatory role in releasing PAI and that endotoxin might inhibit the surface activation of PC.     

Trimeresurus venom inhibition of anti-HPA-1a and anti-HPA-1b antibody binding to human platelets

A solid-phase red cell adherence assay was used to demonstrate the specific inhibitory effect of seven species of Trimeresurus snake venom on the binding of HPA-1a- and HPA-1b-specific platelet antibodies. Trimeresurus venom did not inhibit the binding of HLA-, HPA-3a-, HPA-3b-, HPA-4a-, HPA-5a-, and HPA-5b-specific platelet antibodies. Venom from other genera of snakes, including representatives from Agkistrodon, Ancistrodon, Bitis, Bothrops, Bungarus, Causus, Crotalus, Dendroaspis, Ecis, Micrurus, Naja, Notechis, Ophiophagus, Pseudechis, Sepedon (Hemachatus), and Vipera, all failed to specifically inhibit anti-HPA-1a and HPA-1b binding. These results may indicate that the component in Trimeresurus snake venom previously reported to bind to the platelet GPIIb-IIIa complex, inhibiting fibrinogen binding, binds close to the HPA-1a and HPA-1b epitopes.     

Monoclonal antibodies against Vipera lebetina venom nerve growth factor cross-react with other snake venom nerve growth factors

Nerve growth factor (NGF) was isolated from the venom of Vipera lebetina and was purified to homogeneity as judged by SDS gel electrophoresis. The biologically active NGF was used to immunize BALB/c mouse, and the spleen cells from immunized mouse were fused with mouse PAI myeloma cells. Forty-seven hybrid cell lines, secreting monoclonal antobodies to V. lebetina NGF, were isolated and nine of them purified from ascitic fluids. The isolated antibodies define two partially overlapping epitopes of the V. lebetina NGF which are not involved in the biological activity of the molecule. Both epitopes are also present on the β-NGF from the mouse salivary gland and on the NGFs from the following snake venoms: V. lebetina, V. ursini, V. berus berus, Echis carinatus, Bungarus caeruleus, Agkistrodon halys, Naja naja oxiana. Naja naja atra and Naja naja, but not on the bovine seminal plasma NGF. The mol. wts of the NGFs in these snake venoms were determined by Western immunoblot with monoclonal antibodies. The mol. wts of the NGFs from V. ursini (37,000), E. carinatus (36,000, 44,000) and A. halys (29,000) were determined for the first time.     

Study of chromogenic substrate on protein C activity assay--in patients treated with warfarin

Protein C is an anticoagulant protein that circulates in blood as a zymogen of a serine protease. Recently the exogenous Protein C activator has been obtained from the venom of Agkistrodon Contortrix Contortrix. We also reported a functional assay method which activates Protein C with PROTAC and then measure the amidolytic activity with chromogenic substrate (S-2366) by COBAS FARA. At present, S-2366 (Glu-Pro-Arg-pNA), CBS 65-25 (Lys-Pro-Arg-pNA) and SPECTROZYME PCa (Lys-Pro-Arg-pNA) are used as the chromogenic substrate for activated Protein C. We studied which substrate is more suitable for amidolytic activity assay for Protein C by PROTAC in 99 patients under long-term Warfarin therapy and 29 as normal subjects. Our results indicate that SPECTROZYME PCa and CBS 65-25 are unsuitable for Protein C activity assay with PROTAC. Because change of absorbance was detected in Protein C deficient plasma and we couldn't gain good correlation between amidolytic activity of activated Protein C measured with these chromogenic substrates and antigenicity of Protein C by EIA. On the other hand, S-2366 is the most specific for activated Protein C and we also obtained good correlation against Protein C antigen.     

Hypersensitivity to airborne spitting cobra snake venom.

BACKGROUND: Although the cytolytic, neurotoxic, and hemolytic actions of snake venoms are well known, the ability of airborne inhaled snake venom of the spitting cobra to induce asthma in snake handlers has not been reported. OBJECTIVE: To report the allergenicity of inhaled snake venom in a snake handler who developed increasing hypersensitivity to airborne venom, produced by spitting cobras during public demonstrations. METHODS: Serum samples were obtained from 2 handlers (our study patient and another snake handler who reported developing wheezing when handling spitting cobras), and desiccated venom was obtained from 9 species to which the handlers were exposed. Serum from an asymptomatic and nonatopic snake handler exposed to the same snake species was used as a control. Phosphate-buffered saline extracts were prepared from the desiccated venom, proteins in the venom extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting was performed. Inhibition enzyme-linked immunosorbent assays (ELISAs) were performed to demonstrate cross-reactivity. RESULTS: The study patient had never been previously bitten by a cobra. Wheezing occurred rapidly on inhalational exposure and was reversed by inhalation of salbutamol. The patient had developed IgE antibodies to 9 different snake venoms on Western immunoblots, with major IgE binding proteins of 59 to 63 kDa and 8 to 15 kDa. The cross-reactive nature of the IgE epitopes in the venoms in the different species was also confirmed by 50% inhibition of IgE binding in an ELISA by preincubation with unrelated species. Life-threatening sensitivity of the patient was sustained after a long period of avoidance. CONCLUSIONS: We propose that aerosolized snake venom be considered a new potential source of allergens that may result in anaphylaxis on subsequent exposure. Further studies of the development of specific IgE sensitization following snakebites and the risks of such sensitization should be conducted on snake handlers, particularly those who demonstrate the spitting species.     

蛇毒PCA改善冠脉微血栓大鼠血液流变学的机制研究

目的: 探讨皖南产蝮蛇毒(AHV)蛋白C激活物(PCA)改善冠脉微血栓(CAM)大鼠血液流变学的作用机制。方法: Sprague-Dawley大鼠50只,随机分为假手术组、CAM组、低、中、高剂量PCA 干预组,每组10只。通过从主动脉根部直接向左心室注入月桂酸钠1.0 mg/kg(浓度10 g/L)以建立大鼠冠脉微血栓模型,采用血栓弹力仪系统(TEG)描计血栓弹力图,测定各组血浆中内皮素-1(ET-1)、P-选择素(P-selectin)、肌酸激酶同工酶(CK-MB)、乳酸脱氢酶(LDH)和天冬氨酸氨基转移酶(AST)的水平;Western blotting检测心肌组织中P-selectin和肿瘤坏死因子α(TNF-α)蛋白表达;光镜观察心肌组织学改变。结果: 与模型组比较,PCA大剂量干预组凝血时间和血凝块形成时间延长,Alpha角度、最大幅度和血凝指数值减小(P<0.05),血浆CK-MB、LDH、AST、ET-1和P-selectin的水平降低(P<0.05),心肌组织P-selectin和TNF-α的蛋白表达下降(P<0.05)。病理观察显示中、高剂量PCA干预组大鼠冠脉未形成微血栓。结论: 蝮蛇毒PCA组分可以限制冠脉微血栓的形成,降低血浆ET-1和P-selectin的含量,抑制心肌组织P-selectin和TNF-α表达,改善微血栓后血液流变学变化,有效保护心肌细胞。     

Immunogenicity of venoms from four common snakes in the South of Vietnam and development of ELISA kit for venom detection

The antigenicity and antigenic relationship between venoms of four common snakes in the South of Vietnam-Trimeresurus popeorum, Calloselasma rhodostoma, Naja naja and Ophiophagus hannah-were studied. Most of venom components expressed antigenicity and produced high titre antivenoms. The venoms share common components and antivenoms cross-reacted along them. Furthermore, cross-reactions were observed among non-common antigens, indicating that they share common epitopes. Hence, using single component as immunogen for species diagnosis of snakebites can reduce cross-reaction, perhaps may not be totally specific. A three-step affinity purification protocol was set up for preparation of species-specific antivenom antibodies. The steps involved affinity chromatography of IgG from hyper-immunized rabbit sera with protein A columns, immuno-affinity chromatography of monovalent antivenom antibodies with respective homologous venom columns, and immuno-absorption of cross-species reacting antibody molecules with heterologous venom columns. The antibodies were then used for construction of an enzyme-linked immunosorbent assay (ELISA) test kit. The kit can differentiate among the four common snake venoms in various types of samples with the detection limit of 0.2-1.6 ng/ml, depending on the type of samples and species of the snake. The efficacy of this kit for snake venom detection was successfully demonstrated in experimental envenomation in rats. Preliminary evaluation with 140 samples taken from 88 human snakebite victims in Vietnam showed that the kit could detect venom in human samples and would be a very useful tool for fast identification of snakebites in clinics.     

Screening for fibrinolytic activity in eight Viperid venoms

Snake venoms contain direct-acting fibrinolytic metalloproteinases (MMP) that could have important applications in medicine. Fibrinolytic enzymes isolated from venom can induce in vitro clot lysis by directly acting on a fibrin clot. The most ideal fibrinolytic enzyme would have high affinity for clots, dissolve clots directly without causing hemorrhage, and would not be neutralized in vivo by endogenous metalloproteinase inhibitors. The purpose of this study was to compare DEAE/HPLC venom profiles from Viperid snakes and identify fractions that contain fibrinolytic activity with no hemorrhagic activity and are not neutralized by animal sera. The sera selected were from four (Virginia opossum, Gray woodrat, Mexican ground squirrel, and Hispid cottonrat) animals known to neutralize hemorrhagic activity in snake venoms. Nineteen fractions from the Viperid venoms had fibrinolytic activity. Agkistrodon venom fractions contained the highest specific fibrinolytic activities. A. piscivorus leucostoma fraction 4 contained a high specific fibrinolytic activity, no hemorrhagic activity, and the fibrinolytic activity was not neutralized by the proteinase inhibitors of the four animal sera. A. contortrix laticinctus fraction 1 also had a high specific fibrinolytic activity and no hemorrhagic activity. However, the fibrinolytic activity was neutralized by Didelphis virginiana (Virginia opossum) serum.     

Rapid determination of protein C activity

The diagnosis of Protein C (PC) congenital deficiency is of first importance because it leads, more frequently than Antithrombin III deficiency, to serious thromboembolic accidents in young patients, even when PC levels are slightly decreased (40 p. cent up to 60 p. cent). In order to measure PC activity, the authors developed a new method using the activator from Agkistrodon C. Contortrix snake venom and the synthetic chromogenic substrate CBS 65-25. Results obtained on plasma from normal individuals, congenital and acquired deficiencies, are comparable on the one hand, to those found with an ELISA method, and on the other hand with the clotting method, except for patients under anticoagulant therapy. This new rapid and sensitive method can be performed manually and is easily adapted on instruments used in clinical chemistry laboratories. This method is potentially available for routine use.     

Toxicity of venoms from snakes of medical importance in México

The characterization of the toxic activities of snake venoms is necessary to understand the physiopathology of the envenomation and to test the potency of the antivenoms used to treat this pathology. Because of the lack of data on the toxic activities of venoms from Mexican snakes of medical importance, we studied the venoms from Bothrops asper, Athropoides nummifrr, Agkistrodon billineatus, Crotalus durissus durissus, Crotalus basiliscus, Crotalus scutulatus, Crotalus atrox and Micrurus nigrocinctus. The studies performed were: SDS-PAOE, determination of lethal potency, hemorrhagic, necrotizing, coagulation on plasma and fibrinogen, phospholipasic and fibri(noge)nolytic activities. In addition we studied the neutralizing capacity of the toxic activities of an antivenom currently used for the treatment of snakebites in Mexico. The venom from viperids showed important hemorrhagic, necrotizing, coagulative on plasma, prothrombinic, fibrinogenolytic and phospholipase activities. The venoms with the highest lethal potency were those of Micrurus nigrocinctus and Crotalus scutulatus; however, the viperine venom that globally displayed the most potent toxic activities was from Bothrops asper. All the venoms showed toxic activities of similar range to those described for other American venomous snakes. The activity on plasma or fibrinogen varied widely among the different venoms but all displayed capacity to act on the coagulation system. The antivenom tested not only neutralized the lethality B. asper venom but also its other toxic activities.