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

肝素是一种糖胺聚糖，广泛用于预防 血栓栓塞以及与组织纤溶酶原激活剂结合 （t-PA），以防止持续的血栓形成或最初 成功的血栓溶解。 由于肝素促进凝血酶与 通过形成三元复合物的纤维蛋白聚合物， 冠状动脉再闭塞有限。 因此，寻找一个理想 使血栓溶解剂的功效最大化的抗凝剂， 最大限度地减少不良副作用仍然在继续。 酶 负责纤维蛋白溶解的是纤溶酶， 非活性血浆前体纤溶酶原（Plg）通过特异性 活化剂。 我们实验室的研究比较了岩藻依聚糖[硫酸化 聚（L-吡喃岩藻糖））与6-氨基己酸（6-AHA）或CNBr裂解 纤维蛋白原（CNBr-Fbg）单独或组合增强活化 谷氨酸纤溶酶原（Glu-Plg）或赖氨酸纤溶酶原（Lys-Plg）的两个 链组织纤溶酶原激活剂（t-PA）或LMwt尿激酶或通过 链激酶 褐藻糖胶可增强Glu-Plg或Lys- Plg和高度的协同作用之间观察到6-AMA和 而CNBr-Fbg的增强作用不受褐藻糖胶的影响 并被6-AHA逆转。 在较高浓度下单独的岩藻依聚糖是 有效增强尿激酶对Glu-Plg的激活， 岩藻依聚糖和6-AMA的组合显示出加和效应， Glu-Plg的激活。 这项建议涉及以下方面： 1)岩藻依聚糖硫酸酯的分离纯化， 通过使用尺寸排阻对分子量进行表征 通过色谱和3C-NMR光谱来确定化合物的位置。 硫酸基团和糖苷键的性质。 2)的 岩藻依聚糖硫酸酯在不同体外系统中活化过程中的相互作用 Glu-Plg或Lys-Plg通过t-PA或尿激酶或链激酶。 的 岩藻依聚糖硫酸酯激活或抑制的动力学和机制 这些系统将通过测量纤溶酶（Plm）的速率来研究。 使用显色底物形成。 SDS-PAGE将用于确认 PIg激活过程中Plm的产生。 所述结合区域 将通过研究岩藻依聚糖与t-PA的相互作用， 与两条t-PA链分离的功能活性A和B链 在温和还原和烷基化之后。 Lys-Plg将被降解， 弹性蛋白酶消化和岩藻依聚糖与Kringle 1-3的结合。Kringle 4和Val 442-Plg馏分也将进行研究。 3)动力学 AT-III-凝血酶和HC-II-凝血酶相互作用的分析将 在添加或不添加岩藻依聚糖的情况下进行研究。 的每一个中 上述研究结果将与硫酸化岩藻依聚糖硫酸酯进行比较， 存在于人体组织或类肝素中的糖胺聚糖。 的 建议研究岩藻依聚糖和其他硫酸盐的相互作用， 多糖与蛋白酶，和纤维蛋白溶解的激活剂， 凝固将使我们深入了解 这些属性之间的关系和机制，他们 修改这些程序。

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.