Substrate inhibition of fibrin-dependent plasminogen activation by tissue-type plasminogen activator.
Tissue plasminogen activator (t-PA) is a poor activator of plasminogen, but its catalytic efficiency is greatly enhanced in the presence of fibrin or certain fibrin derivatives. These two very different rates of plasminogen activation provide a basis for the phenomenon of substrate inhibition, which was the subject of the present study. The reaction mixture contained Glu-plasminogen (0-4.0 microM), +/- D-dimer (0.06-1.0 microM), t-PA (0.05-50.0 nM), and S2251 (1.5 mM). Inhibition of the reaction at higher plasminogen concentrations was seen at lower concentrations of t-PA (< 1.0 nM) and D-dimer (< 0.5 microM). The maximum rate of plasminogen activation occurred at 0.2 microM Glu-plasminogen but then decreased progressively above this concentration. At a physiological concentration of plasminogen (2.0 microM), activation was about half that at 0.2 microM. This phenomenon was shown not to be related to Glu-plasminogen aggregation or to a non-competitive inhibiting contaminant. Instead, the non-linear Lineweaver-Burk curve in the presence of D-dimer was consistent with substrate inhibition. Increasing the concentration of D-dimer (1.0 microM) and/or t-PA (50 nM) overcame the inhibition and reestablished linearity in the Lineweaver-Burk plots. However, at the higher concentrations of t-PA, the catalytic efficiency was reduced by 50-120-fold, suggesting that t-PA may be less efficient at pharmacological than at physiological concentrations. In conclusion, substrate inhibition in t-PA-induced plasminogen activation is reported for the first time. This phenomenon was confined to low concentrations of t-PA and D-dimer in the presence of physiological concentrations of plasminogen. The observation suggests that small fibrin clots (hemostatic plugs) may be more resistant to t-PA-induced lysis than an occlusive thrombus.
Vascular Research Laboratory, Deaconess Hospital, Harvard Medical School, Boston, Massachusetts 02215.
J
J
Liu
V
V
Gurewich
D004789:Enzyme Activation; D005246:Feedback; D005337:Fibrin / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008954:Models, Biological; D010446:Peptide Fragments / Q000494:pharmacology; D010958:Plasminogen / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D005337:Fibrin; D010958:Plasminogen; D010959:Tissue Plasminogen Activator