Fragment E-2 from fibrin substantially enhances pro-urokinase-induced Glu-plasminogen activation. A kinetic study using the plasmin-resistant mutant pro-urokinase Ala-158-rpro-UK.
In a previous study, it was shown that fibrin fragment E-2 selectively promotes the activation of plasminogen by pro-urokinase (pro-UK) [Liu, J., & Gurewich, V. (1991) J. Clin. Invest. 88, 2012-2017]. In this study, the kinetics of this promotion by fragment E-2 was studied. Alanine-158-rpro-UK (A-pro-UK), a recombinant plasmin-resistant mutant, was used in order to avoid interference by UK generation during the reaction. In some experiments, pro-UK was substituted in order to validate the mutant as a surrogate. In the presence of a range of concentrations (0-20 microM) of fragment E-2, a linear promotion of the catalytic efficiency of A-pro-UK against native Glu-plasminogen was seen which was 245.5-fold at the highest concentration of fragment E-2 and 450-fold at the highest ratio of E-2/plasminogen used. The promotion was largely a function of an increase in kcat, since fragment E-2 induced a less than 10-fold reduction in KM (8.50-1.40 microM). In contrast to this ligand, epsilon-aminocaproic acid (EACA) induced a biphasic promotion of the activation of Glu-plasminogen which was only 18-fold at maximum. Fragment E-2 did not promote the activation of Lys-plasminogen, but the catalytic efficiency of A-pro-UK was 19.7-fold greater against the open Lys-form than against the closed Glu- form of plasminogen. Fragment E-2 had no effect on the amidolytic activity of A-pro-UK or pro-UK, suggesting that the promotion of their activities was indirect and related to a fragment E-2-induced conformational change in Glu-plasminogen.(ABSTRACT TRUNCATED AT 250 WORDS)
Liu
J N
JN
Vascular Research Laboratory, New England Deaconess Hospital, Harvard Medical School, Boston, MA 02215.
Gurewich
V
V
D000409:Alanine; D000595:Amino Acid Sequence; D004789:Enzyme Activation; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism*; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008433:Mathematics; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000378:metabolism*; D010960:Plasminogen Activators / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
D005338:Fibrin Fibrinogen Degradation Products; D011994:Recombinant Proteins; C072054:fibrin fragment E-2; D010958:Plasminogen; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine; D000409:Alanine; C067551:saruplase