Considerable attention has been focused recently on the therapeutic value of tissue plasminogen activator (t-PA) as a thrombolytic agent for the treatment of heart attacks and strokes. One possible problem with this therapy is the rapid reocclussion of the affected artery. Therefore, patients treated with t-PA are treated concomitantly with heparin. However, heparin cannot prevent coronary reocclussion effectively, since it promotes binding of thrombin to fibrin polymer by forming a ternary complex, thus lowering the inactivation of thrombin by anti-thrombin III. On the other hand, fucoidan [sulfated poly (L-fucopyranose)] from marine algae Fucus Vesiculosis, is reported to show in vitro and in vivo anticoagulant activity mediated by heparin co-factor II, which will inactivate thrombin, even on a fibrin surface. The enzyme response for lysis mediated by heparin co-factor II, which will inactivate thrombin, even on a fibrin surface. The enzyme responsible for lysis of fibrin is plasmin (Plm), which is generated from the inactive plasma precursor plasminogen (plg) by the action of specific activators. Our laboratory has reported that fucoidan enhanced the in vitro activation of Plg to Plm by t- pA or by urokinase (u-PA). This proposal is concerned with the mechanism of this enhancement and will consist of the following investigations. 1) Purification of fucoidan by DEAE or ECTEOLA cellulose, or QAE sepharose chromatography followed by degradation, using fucosidase or hydrochloric acid and isolation of the degradation products by gel filtration or ultra-filtration procedures. 2) The purified fucoidan or the degradation products will be characterized according to the molecular weight by using size exclusion chromatography, and 1H- nmr OR 13 C-NMR spectroscopy will be used to determine the location of the sulfate groups and the nature of the glycosidic bonds. 3) Fucoidan, which exhibits the optimum enhance of Plm generation, will be used for studying its interactions in the various in vitro systems during the activation enhancement of Plm generation, will be used for studying its interactions in the various in vitro systems during the activation of Glu- POLG or Lys-Plg, by t-PA or uPA, or during inhibition of the activation by plasminogen activator inhibitor (PAI-1). 4) The binding site of fucoidan, with Glu-Plg, t-PA, uPA, or PAI-1 will be investigated by affinity chromatography, using fucoidan-sepharose. 5) Conformation changes of Glu-Plg or Lys-Plg induced during the activation of Plg in the presence of fucoidan or 6-amino hexanoic acid (6-AHA), alone or in combination, will be examined by using circular dichroism spectroscopy. In each of the above studies the results of the fucoidan will be compared with sulfated glycosaminoglycans occurring in human tissues or heparinoids. The proposed study will give an insight into the mechanisms by which these compounds modify the normal processes and may eventually resulted in the development of a new generation of anticoagulants that may replace heparin as adjunct therapy with t-PA.
