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

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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.