Plasminogen activation with single-chain urokinase-type plasminogen activator (scu-PA). Studies with active site mutagenized plasminogen (Ser740----Ala) and plasmin-resistant scu-PA (Lys158----Glu).
The mechanism of the activation of plasminogen by single-chain urokinase-type plasminogen activator (single-chain u-PA, scu-PA) was studied using rscu-PA-Glu158, a recombinant plasmin-resistant mutant of human scu-PA obtained by site-specific mutagenesis of Lys158 to Glu, and rPlg-Ala740, a recombinant human plasminogen in which the catalytic site is destroyed by mutagenesis of the active-site Ser740 to Ala. Conversion of 125I-labeled single-chain plasminogen to two-chain plasmin was quantitated on reduced sodium dodecyl sulfate-gel electrophoresis combined with autoradiography and radioisotope counting of gels bands. The efficiencies of both rscu-PA-Glu158 and rscu-PA for the activation of rPlg-Ala740 and of natural plasminogen were comparable and were 250-500-fold lower than that of recombinant two-chain u-PA (rtcu-PA) for rscu-PA-Glu158 and 100-200-fold lower for rscu-PA. Pretreatment of rscu-PA-Glu158 or rscu-PA with excess alpha 2-antiplasmin, which efficiently neutralizes all contaminating rtcu-PA, did not significantly reduce the catalytic efficiency of these single-chain moieties, indicating that they have a low but significant intrinsic plasminogen activating potential. The low intrinsic catalytic efficiency of rscu-PA for the conversion of plasminogen to plasmin may be sufficient to generate trace amounts of plasmin, which may regulate plasminogen activation by converting poorly active rscu-PA to very active rtcu-PA.
Lijnen
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HR
Center for Thrombosis and Vascular Research, University of Leuven, Belgium.
Van Hoef
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B
Nelles
L
L
Collen
D
D
D000409:Alanine; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005341:Fibrinolysin / Q000378:metabolism*; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008239:Lysine; D009154:Mutation; D010958:Plasminogen / Q000235:genetics / Q000378:metabolism*; D010960:Plasminogen Activators / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D004792:Enzyme Precursors; D005971:Glutamates; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012694:Serine; D010958:Plasminogen; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine; D000409:Alanine