Mechanism of enhancement by fucoidan and CNBr-fibrinogen digest of the activation of glu-plasminogen by tissue plasminogen activator.
The interactions of fucoidan with human glutamic type plasminogen (Glu-Plg), porcine pancreatic elastase digested plasminogen fractions and two chain tissue plasminogen activator t-PA) were investigated using fucoidan-Sepharose affinity chromatography. The results showed a high degree of affinity between fucoidan-Sepharose and Glu-Plg or PlgK(1-3) but not with PlgK4 or mini-Plg. Fucoidan-Sepharose also showed a high affinity for t-PA, which was largely reversed by 0.002 M 6-aminohexanoic acid (6-AH). The addition of fucoidan and CNBr-fibrinogen digest (CNBr-Fbg) gave the highest enhancement of the in vitro activation of Glu-Plg by t-PA in the presence of 0.002 M 6-AH. The results of affinity chromatography and enhancement studies suggested a template mechanism, since increasing the concentrations of any one of the two cofactors reversed the enhancement. Enzyme kinetic studies, using double reciprocal plots, showed that the addition of fucoidan-6-AH increased Kcat by 7-fold without affecting Km and addition of CNBr-Fbg lowered Km by 5-fold without significantly affecting Kcat while addition of the two cofactors lowered Km by 16-fold without significantly affecting Kcat. The enhancement by fucoidan-6-AH or by CNBr-Fbg of the in vitro activation of Glu-Plg by t-PA was reversed by plasminogen activator inhibitor 1 (PAI-1). Fucoidan-Sepharose affinity chromatography revealed that the binding of PAI-1 with fucoidan may be responsible for the reversal of the enhancement by fucoidan-6-AH.
Muneer
E
E
Department of Chemistry, Prairie View A&M University, Texas 77446, USA.
Bell
J
J
Doctor
V M
VM
D002846:Chromatography, Affinity; D003488:Cyanogen Bromide / Q000494:pharmacology*; D005340:Fibrinogen / Q000494:pharmacology*; D006801:Humans; D010958:Plasminogen / Q000378:metabolism; D011134:Polysaccharides / Q000494:pharmacology*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D011134:Polysaccharides; D005340:Fibrinogen; D010958:Plasminogen; C007789:fucoidan; D010959:Tissue Plasminogen Activator; D003488:Cyanogen Bromide