Effects of intact fibrin and partially plasmin-degraded fibrin on kinetic properties of one-chain tissue-type plasminogen activator.
A comparative kinetic analysis of the enzymatic activities of one-chain and two-chain tissue-type plasminogen activator (t-PA) demonstrates that two-chain t-PA catalyzes the hydrolysis of the peptide substrate D-Val-Leu-Arg-pNA about 4-fold more effectively than one-chain t-PA. The difference is accounted for almost entirely by a corresponding difference is the kcat values of the enzymes, whereas the Km values are similar. The amidolytic activity of two-chain t-PA is not enhanced by intact or partially plasmin-degraded fibrin. In contrast, the activity of one-chain t-PA is stimulated up to 3.7-fold by intact fibrin and up to 4.7-fold by plasmin-degraded fibrin (fibrin X-fragment). The stimulatory effects are realized via increases in the kcat values. It appears thus that in the presence of fibrin the intrinsically inferior catalytic properties of one-chain t-PA become similar to the properties of two-chain t-PA. The dependency of the activity of one-chain t-PA on the concentration of fibrin monomer is consistent with a single association site of both proteins and an association constant of Kass = 6.25 x 10(6) l/mol. Stimulation of one-chain t-PA by plasmin-degraded fibrin is more complex and appears to involve two different binding sites with association constants of Kass = 0.67 x 10(9) l/mol and Kass = 3.85 x 10(6) l/mol, respectively. The stimulatory effects of fibrin and partially plasmin-degraded fibrin on one-chain t-PA are suppressed by epsilon-aminocaproic acid and by a monoclonal antibody directed against the lysine binding site of t-PA. The latter findings support the notion that fibrin activation of one-chain t-PA is mediated by the lysine binding site on kringel domains of the enzyme.
Fischer
B E
BE
Research Center of Biotechnology, Academy of Sciences of the G.D.R., Berlin.
Will
H
H
D000595:Amino Acid Sequence; D005337:Fibrin / Q000494:pharmacology*; D005340:Fibrinogen / Q000378:metabolism; D005341:Fibrinolysin / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008545:Melanoma / Q000201:enzymology; D008969:Molecular Sequence Data; D009842:Oligopeptides; D010446:Peptide Fragments / Q000494:pharmacology; D013379:Substrate Specificity; D010959:Tissue Plasminogen Activator / Q000302:isolation & purification / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000201:enzymology
D009842:Oligopeptides; D010446:Peptide Fragments; D005337:Fibrin; D005340:Fibrinogen; D010959:Tissue Plasminogen Activator; D005341:Fibrinolysin