The effect of polymerised fibrin on the catalytic activities of one-chain tissue-type plasminogen activator as revealed by an analogue resistant to plasmin cleavage.
A one-chain recombinant tissue-type plasminogen activator (EC 2.4.31.-) (tPA) analogue was constructed in which Arg-275 of the activation site was changed to Gly by site-directed mutagenesis. This analogue, tPA-Gly275, was very resistant to plasmin (EC 2.4.21.5) cleavage. It has been used to gain information about the activity of the uncleaved one-chain tPA form, also when plasmin is generated as a result of a plasminogen activation reaction. The amidolytic activity of tPA-Gly275 with less than Glu-Gly-Arg-pNA was investigated and compared to that of one-chain and two-chain wild-type recombinant tPA. A small but significant intrinsic amidolytic activity was observed with the analogue as well as the wild-type one-chain tPA form. However, it was much lower than that of two-chain tPA. Polymerised fibrin enhanced the amidolytic activity of both one-chain tPA forms but not of two-chain tPA. Measurements of the plasminogen activation kinetics in the absence of fibrin revealed that tPA-Gly275 possessed a significant intrinsic activity. However, it was 30-fold lower than that of two-chain tPA. Addition of polymerised fibrin profoundly enhanced the plasminogen activation rate of both tPA-Gly275 and wild-type one- and two-chain tPA to approximately the same maximal level. The results were interpreted to mean that fibrin binding can induce an activated state of the intact tPA one-chain form.
D001704:Biopolymers; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004347:Drug Interactions; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000494:pharmacology*; D005341:Fibrinolysin / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000031:analogs & derivatives / Q000302:isolation & purification / Q000378:metabolism*
D001704:Biopolymers; D010446:Peptide Fragments; D012967:Sodium Dodecyl Sulfate; D005337:Fibrin; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin