Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the α-phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

尖吻蝮蛇毒凝血酶样酶(TLE)的酶联免疫吸附分析(ELISA)

本文以辣根过氧化物酶(HRP)为标记酶,聚苯乙烯微量血凝板为固相载体,运用夹心酶联免疫吸附(ELISA)技术研究了部分纯化的尖吻蝮蛇毒凝血酶样酶(TLE)的微量检测,并用四参数方程模拟了TLE的对数剂量反应曲线。     

蛇毒溶栓素对动物凝血功能的影响

本文报道蛇毒溶栓素(ATE)对家犬、猕猴体内外凝血作用的影响.体外实验表明:ATE具有类凝血酶样作用,能使去钙血浆和纤维蛋白原直接凝固,它不激活Ⅻ因子,产生是非交联的纤维蛋白,并且有纤溶酶样作用,能直接溶解纤维蛋白,产生抗凝作用.体内实验表明:ATE显著延长全血凝固时间、凝血酶时间和凝血酶原时间,注射ATE后血浆中纤维蛋白原降低,优球蛋白溶解时间缩短,三P试验阳性,血小板减少.体内外实验表明,ATE具有去纤、纤溶、抗血小板功能和抗凝效应.     

尖吻蝮蛇毒凝血酶样酶的长期毒性及连续给药后药理效应的研究

本文报道尖吻蝮蛇毒凝血酶样酶对犬及家兔的亚急性毒性及生化药理效应。用药量(μg/kg·d)犬分别为200、65,家兔分别为540、180,连续静脉注射28天。用药后14天及28天,WBC,DC,Hb 含量、肝、臂功能均无明显异差。血浆纤维蛋白原含量明显降低,凝血酶样酶时间及凝血酶时间明显延长,血小板聚集功能明显抑制,高剂量组个别犬血浆尿素氮显著上升(停药2天后降低),实验结束时体重均增加。用药后10天起,个别犬于每天注药后出现短时反应,至3周后消失。病理解剖检查眼肉及镜检未见重要脏器发生病变。本资料提示凝血酶样酶是一低毒高效应制剂,用药2周后对血小板聚集的抑制作用有耐受现象,因此连续用药时间不宜过长。     

^125Ⅰ眼镜蛇毒及抗眼镜蛇毒血清在小白鼠体内分布的比较观察

应用氯胺T法对尖吻蝮蛇毒的促凝组份进行~125I标记,以放射性参与量(脏器与血液放射比)的比值作为促凝组份在组织中分布的依据,观察其在小白鼠体内的分布状态.结果表明,一次性快速iv凝组份后2小时,以肾分布最高,肺、脾和肝的放射性参比量也较高,尿中含量很高,提示其主要由肾脏排泄.     

Haemostasis during transvesical prostatic adenomectomy

Summary Blood loss, plasma fibrinolytic activity, prothrombin, P.T.T., and fibrinogen plasma levels were measured in 30 patients subjected to transvesical adenomectomy. These parameters were evaluated during 7 consecutive days after the operation. Ten patients received the antifibrinolytic substance (AMCHA), ten patients received Bothrops Jararaca venom extract, and ten patients served as controls. The results show that the coagulating fraction of Bothrops Jararaca snake venom reduces intraoperative bleeding without influencing the haemostatic balance. Postoperative haematuria could be reduced by antifibrinolytic agents.     

Mechanism of action and determination of the best substrate for a thrombin-like enzyme from Lachesis muta muta venom by regression analysis of the kinetic parameters determined with peptidyl p-nitroanilide substrates.

The kinetic behavior of a thrombin-like enzyme from Lachesis muta muta venom has been studied with 13 tripeptidyl p-nitroanilide substrates. Eight substrates were unprotected at the N terminus and were used for the regression analysis of the experimentally determined kinetic parameters 1/Km, kcat and kcat/Km. The individual contribution of each amino acid side chain to the kinetic parameters was calculated. The amino acid sequence of the ideal substrate (D-Pro-Leu-Arg-pNA) was determined from a regression analysis for each kinetic parameter. This result was confirmed experimentally. The structural analysis of the tripeptides showed that the binding to the S3 sub-site had a small effect on Km. The binding of L-Leu to the S2 sub-site increased kcat without changing the value of Km. The analysis of the kinetic parameters revealed that, in the binding of L-Leu to the S2 sub-site, the enzyme bound the transition state configuration of the substrate/product transformation more tightly than that of the substrate.     

液相色谱分离纯化五步蛇毒凝血酶样酶

目的：改进分离工艺，找到液相色谱分离纯化五步蛇毒中凝血酶样酶的最佳方案。方法：五步蛇毒依次经SuperdexG75凝胶过滤、DEAE．SephroseF．F阴离子交换和SuperdexG30凝胶过滤，通过改变洗脱液pH值、流速、Tris浓度、盐浓度及上样量来考察对分离纯化五步蛇毒中凝血酶样酶的影响。结果：Superdex75凝胶过滤色谱分离纯化受洗脱液流速、Tris浓度、盐浓度、pH值、上样量的影响；DEAE—SephroseF．FN离子交换色谱受盐浓度的影响：SuperdexG30凝胶过滤色谱受上样量的影响。三步分离纯化后获得一种五步蛇毒凝血酶样酶，分子量约23kD。结论：依次经SuperdexG75、DEAE—SephroseF．F和SuperdexG30凝胶，通过改变洗脱液流速、Tris浓度、盐浓度、pH值、上样量等能得到最佳分离条件．最终可获得五步蛇毒凝血酶样酶的纯品。     

蝮蛇蛇毒类凝血酶的研究

为研制蝮蛇抗栓酶的换代产品，开发单一组分的类凝血酶制剂。采用离子交换和亲和层析等现代纯化方法，从东北白眉蝮蛇蛇毒中分离出了单一组分的类凝血酶。半成品经高效液相色谱凝胶柱测定，纯度大于90％，最高可达97.94％，分子量38200D，SDS－PAGE电泳测定为一条带，分子量为42100D，活性为1137BU／mg，比活性为1705BU／mg·pro，不含出血毒和神经毒，冻干成品制剂全部符合注射用品标准。     

Neurotoxicological effects of a thrombin-like enzyme isolated from Crotalus durissus terrificus venom (preliminary study).

A thrombin-like enzyme, purified from the venom of Crotalus durissus terrificus by gel filtration and affinity chromatography, showed a single protein band in Sodium dodecyl sulfate-polyacrilamide gel electrophoresis (SDS-PAGE) with a molecular weight of about 33kDa. Clear cellular morphological changes, deep ganglioside level modifications in some brain areas and behavioral alterations in pup rats injected with this protein were detected. Ganglioside composition, one of the chemical markers of brain maturation, was altered specially in the hypothalamus, hippocampus and prefrontal cortex. The most reliable behavioral effects were a delayed, maturation of the righting reflex, posture and motor response after treatment. These effects were consistent with the histological changes revealed in the cerebellum and prefrontal cortex of treated neonate rats, areas related to motor activities.