猪凝血酶的亲和层析纯化及其酶原的激活研究

采用柠檬酸钡作吸附剂、ＥＤＴＡ溶解的方法从猪血浆提取凝血酶原，建立了凝血酶原激活的简单方法。并以环氧氯丙烷活化的Ｓｅｐｈａｒｏｓｅ４Ｂ为载体，采用１－乙基－３－（３－二甲氨丙基）碳二亚胺进行肝素的固定化，将固定化肝素用于猪凝血酶的亲和层析纯化。结果表明，通过一步亲和层析将猪凝血酶粗品提纯了８．２倍，获得了比活超过１１００Ｕ／ｍｇ的凝血酶，收率为６０％。     

槲皮素单硫酸酯和5’-氯-5’-脱氧腺苷单用与合用对凝血酶诱导兔血小板聚集的影响

目的：研究槲皮素单硫酸酯和5’-氨-5’-脱氧腺苷合用对凝血酶诱导兔血小板聚集的影响。方法：用比浊法检测血小板聚集。结果：槲皮素单硫酸酯和5’-氨-5’-脱氧腺苷对凝血酶诱导的兔血小板聚集都有抑制作用，两者合用作用增加。结论：槲皮素单硫酸酯与5’-氨-5’-脱氧腺苷具有协同抗血小板聚集的作用。     

Research about Preparation of Porcine Thrombin

A rapid, simple, and reproducible method for the preparation of porcine thrombin was investigated.Porcine prothrombin was isolated by carrying out isoeiectric precipitation. Porcine thrombin was prepared from pro-thrombin which was activated by the prothrombinase complex containing Factor Xa, Factor V, Ca^2 and phospholipids.Factor Xa and Factor V were isolated by DEAE-cellulose chromatography respectively. They were both identified bySDS-PAGE. Yields and specific activity of porcine thrombin were 107μg per milliliter plasma and 4 U per milligram protein.     

解毒化瘀方对凝血酶合并缺氧诱导PC12细胞损伤ERK1/2信号通路表达的影响

目的 探讨急性缺血性中风（AIS）瘀毒病机的分子机制及解毒化瘀方对凝血酶合并缺氧损伤的保护作用。方法 （1）用凝血酶合并缺氧损伤PC12细胞,模拟急性缺血性中风的体外细胞模型。（2）将细胞分为空白组,模型组,解毒化瘀方含药血浆组和PD98059组（MEK抑制剂组）,应用荧光定量实时RT-PCR方法检测MEK1、PAR-1及ERK1/2的mRNA表达,应用免疫荧光法检测ERK1/2、P-ERK1/2的蛋白表达。结果 PCR结果提示：模型组MEK1、PAR-1及ERK1/2的mRNA表达与空白对照组相比显著升高（P<0.01）,解毒化瘀方组和PD98059组MEK1、PAR-1及ERK1/2的mRNA表达较模型组显著降低（P<0.01）,免疫荧光结果提示：解毒化瘀方组和PD98059组ERK1/2、P-ERK1/2的蛋白表达较模型组显著降低（P<0.01）。结论 解毒化瘀方以凝血酶为作用的分子靶点,能够显著降低ERK1/2信号通路在缺血性中风体外细胞模型中的表达。     

Influence of Fucoidans on Hemostatic System

Three structurally different fucoidans from the brown seaweeds Saccharina latissima (SL), Fucus vesiculosus (FV), and Cladosiphon okamuranus (CO), two chemically modified fucoidans with a higher degree of sulfation (SL-S, CO-S), and a synthetic totally sulfated octasaccharide (OS), related to fucoidans, were assessed on anticoagulant and antithrombotic activities in different in vitro experiments. The effects were shown to depend on the structural features of the compounds tested. Native fucoidan SL with a degree of sulfation (DS) of 1.3 was found to be the most active sample, fucoidan FV (DS 0.9) demonstrated moderate activity, while the polysaccharide CO (DS 0.4) was inactive in all performed experiments, even at high concentrations. Additional introduction of sulfate groups into fucoidan SL slightly decreased the anticoagulant effect of SL-S, while sulfation of CO, giving rise to the preparation CO-S, increased the activity dramatically. The high level of anticoagulant activity of polysaccharides SL, SL-S, and CO-S was explained by their ability to form ternary complexes with ATIII-Xa and ATIII-IIa, as well as to bind directly to thrombin. Synthetic per-O-sulfated octasaccharide OS showed moderate anticoagulant effect, determined mainly by the interaction of OS with the factor Xa in the presence of ATIII. Comparable tendencies were observed in the antithrombotic properties of the compounds tested.     

聚乙二醇对猪凝血酶的化学修饰及其效果分析

利用亲和层析法,从粗酶中纯化得到猪凝血酶.采用活化的单甲氧基聚乙二醇对凝血酶进行了共价修饰,以此修饰酶为研究对象,进行了凝血酶在修饰前后的主要酶学性质的比较研究.结果表明,凝血酶经mPEGl修饰后,Km有所增加,pH和盐离子浓度的稳定性、热稳定性以及抗胰蛋白酶的水解能力均有所增强.     

猪凝血酶的分离纯化及部分性质研究

经等电点沉淀,DEAE离子交换纤维素柱层析和Sephadex G-100 分子筛层析从猪血浆中纯化得到猪凝血酶,酶比活力为2 000 U/m g,纯化倍数为100,酶活力回收为56% ,酶反应的最适pH 和温度分别为7.0 和37℃,SephadexG-100层析测得酶分子量为36 000。猪凝血酶的稳定性较差,40℃保温27 h, 酶活力下降84% 。高浓度的盐对酶稳定性有较大的影响,在2 m ol/LNaCl下放置3 h,酶活力下降50% 。     

Evaluation of Potential Thrombin Inhibitors from the White Mangrove (Laguncularia racemosa (L.) C.F. Gaertn.)

The aim of this work was to verify the effects of methanol (MeOH) and hydroalcoholic (HA) extracts and their respective partition phases obtained from white mangrove (Laguncularia racemosa (L.) C.F. Gaertn.) leaves on human thrombin activity. Among the extracts and phases tested, only the ethyl acetate and butanolic partitions significantly inhibited human thrombin activity and the coagulation of plasma in the presence of this enzyme. Chromatographic analyses of the thrombin samples incubated with these phases revealed that different compounds were able to interact with thrombin. The butanolic phase of the MeOH extract had the most potent inhibitory effects, reducing enzymatic activity and thrombin-induced plasma coagulation. Two glycosylated flavonoids in this partition were identified as the most potent inhibitors of human thrombin activity, namely quercetin-3-O-arabinoside (QAra) and quercetin-3-O-rhamnoside (Qn). Chromatographic analyses of thrombin samples incubated with these flavonoids demonstrated the chemical modification of this enzyme, suggesting that the MeOH extract contained other compounds that both induced structural changes in thrombin and diminished its activity. In this article, we show that despite the near absence of the medical use of mangrove compounds, this plant contains natural compounds with potential therapeutic applications.