FUCOIDAN/PROTEASE--ACTIVATORS AND INHIBITORS OF COAGULATION AND FIBRINOLYSIS

肝素是一种糖胺多聚糖，广泛用于预防 血栓栓塞症及与组织型纤溶酶原激活剂联合应用 (t-PA)预防持续血栓形成或首次闭塞后再闭塞 溶栓成功。由于肝素促进凝血酶与 纤维蛋白聚合物通过形成三元络合物，其预防 冠状动脉再闭塞是有限的。因此，对理想的追求 使溶栓剂的效果最大化的抗凝剂 将不良副作用降至最低的做法仍在继续。这种酶 负责分解纤维蛋白的是纤溶酶，它是由 特异性纤溶酶原激活的血浆前体型纤溶酶原失活 激活剂。我们实验室的研究比较了岩藻糖胶[硫酸盐] 6-氨基己酸(6-AHA)或溴化氰基裂解的聚(L-岩藻糖) 纤维蛋白原(CNBR-FBG)单独或联合应用增强活化作用 谷氨酸纤溶酶原(Glu-PLG)或赖氨酸纤溶酶原(Lys-PLG) 链式组织型纤溶酶原激活剂(t-PA)或LMwt尿激活剂或 链激酶素。褐藻糖胶增强Glu-PLG或Lys-PA的t-PA活性 6-AMA和6-AMA之间存在高度的协同作用 褐藻糖胶对CNBrFBG的增强作用不受褐藻糖胶的影响 并被6-AHA逆转。单独的岩藻糖胶在较高浓度下 有效增强尿激酶激活Glu-PLG的作用 岩藻糖胶与6-AMA联用具有相加效应。 激活Glu-PLG。这项提议涉及以下几个方面： 1)用DEAE和ECTEOLA纤维素柱层析纯化岩藻糖胶， 分子量的大小排除法表征 用层析和3c-核磁共振波谱确定 硫酸盐基团和糖苷键的性质。2) 岩藻糖胶在不同体外体系中的相互作用 T-PA或尿激酶或链激酶法测定Glu-PLG或Lys-PLG。这个 褐藻糖胶的激活或抑制作用机理及动力学 这些系统将通过测量纤溶酶(PLM)的比率来进行研究 使用显色底物形成。将使用SDS-PAGE确认 猪激活过程中PLM的产生。结合区 将通过研究其与t-PA的相互作用来定位岩藻糖胶 从两个t-PA链分离出来的功能活性A和B链 经过温和还原和烷基化反应。Lys-PLG将被降解 弹性酶消化和岩藻糖胶与kringle 1-3的结合。克林格尔 4和Val442-PLG组分也将被调查。3)动力学 将分析AT-III-凝血酶和HC-II-凝血酶的相互作用 研究了添加或不添加岩藻糖胶的情况。在每一个 以上研究将岩藻糖胶的结果与硫酸盐化的结果进行比较 存在于人体组织或类肝素中的糖胺聚糖。这个 岩藻糖胶与其他硫酸盐类化合物相互作用的研究 含有蛋白酶和纤溶激活剂的多糖和 凝固术将使我们对结构-功能有一个深入的了解 这些特性之间的关系以及它们所通过的机制 修改这些流程。

Heparin, a glycosaminoglycan is widely used for the prevention of thromboembolism and also in conjunction with tissue plasminogen activator (t-PA) to prevent ongoing thrombosis or reocclusion after initially successful thrombolysis. Since heparin promotes binding of thrombin to fibrin polymer by forming a ternary complex, its efficacy in preventing coronary reocclusion is limited. Therefore, the search for an ideal anticoagulant that maximizes efficacy of the thrombolytic agent and minimizes undesirable side effects still continues. The enzyme responsible for the lysis of fibrin is plasmin which is generated from the inactive plasma precursor plasminogen (Plg) by the action of specific activators. Studies in our laboratory compared fucoidan [sulfated poly(L-Fucopyranose)) with 6-aminohexanoic acid (6-AHA) or CNBr cleaved fibrinogen (CNBr-Fbg) alone or in combination in enhancing the activation of glutamic plasminogen (Glu-Plg) or lysine plasminogen (Lys-Plg) by two chain tissue plasminogen activator (t-PA) or LMwt urokinase or by streptokinase. Fucoidan enhanced the t-PA activation of Glu-Plg or Lys- Plg and a high degree of synergism was observed between 6-AMA and fucoidan while the enhancement by CNBr-Fbg was not influenced by fucoidan and was reversed by 6-AHA. Fucoidan alone at higher concentrations was effective in enhancing the activation of Glu-Plg by urokinase while the combination of fucoidan and 6-AMA showed additive effect in enhancing the activation of Glu-Plg. This proposal is concerned with the following: 1) Purification of fucoidan by DEAE and ECTEOLA cellulose chromatography, characterization as to the molecular weight by using size exclusion chromatography and 3C-NMR spectroscopy to determine the location of the sulfate groups and the nature of the glycosidic bonds. 2) The interaction of fucoidan in various in vitro systems during the activation of Glu-Plg or Lys-Plg by t-PA or urokinase or streptokinase. The kinetics and the mechanism of activation or inhibition by fucoidan in these systems will be investigated by measuring the rate of plasmin (Plm) formation using chromogenic substrate. SDS-PAGE will be used to confirm the generation of Plm during the activation of PIg. The binding region of fucoidan with t-PA will be localized by studying its interaction with functionally active A and B chains separated from the two chain t-PA after mild reduction and alkylation. Lys-Plg will be degraded by elastase digestion and the binding of fucoidan with kringle 1-3. Kringle 4 and Val442-Plg fractions will also be investigated. 3) Kinetic analyses of AT-III-thrombin and HC-II-thrombin interactions will be investigated with or without the addition of fucoidan. In each of the above studies the results of fucoidan will be compared with sulfated glycosaminoglycans occurring in human tissues or heparinoids. The proposed study of the interactions of fucoidan and other sulfated polysaccharides with proteases, and activators of fibrinolysis and coagulation will give an insight into the structure-function relationships between these properties and the mechanisms by which they modify these processes.