Mechanism of the enhanced intrinsic activity of single-chain urokinase-type plasminogen activator during ongoing fibrinolysis.
The activation of plasminogen at the surface of fibrin by single-chain urokinase-type plasminogen activator (scu-PA) was investigated using recombinant forms of a plasmin-resistant mutant of scu-PA, rscu-PA-Glu158, and an inactive catalytic site mutant of human plasminogen, rPg-Ala741. Conversion of cleavable 125I-labeled single-chain proteins to their two-chain forms, was quantitated by radioisotope counting of protein bands on reduced SDS-polyacrylamide gels. The efficiency of the activation (moles of plasmin generated per mol of plasminogen activator) of native Glu-plasminogen bound to degraded fibrin was comparable for scu-PA and its two-chain form (tcu-PA) and approximately 4-fold lower for rscu-PA-Glu158. The corresponding values with rPg-Ala741 were 4-fold or 9-fold lower for scu-PA or rscu-PA-Glu158, as compared to tcu-PA. In contrast, in solution in the absence of fibrin, the efficiency of scu-PA for activation of rPg-Ala741 was 100-fold lower than that of tcu-PA. Initial activation rates of rPg-Ala741 (32.7 fmol/well containing 50 microliters of solution) with 4 nM tcu-PA were comparable in solution and bound to degraded fibrin (v(o) = 1.01 and 1.16 fmol/min, respectively). In contrast, with 4 nM scu-PA the corresponding values when rPg-Ala741 was bound to degraded fibrin were 20-fold higher as compared to the soluble phase (v(o) = 0.23 and 0.012 fmol/min, respectively). Comparable results were obtained when using Glu- or Lys-forms of rPg-Ala741. Furthermore, in the presence of normal human plasma, activation of Glu-plasminogen bound to degraded fibrin was found to be about 2.5-fold more efficient with scu-PA than with tcu-PA. These findings indicate that the fibrin specificity of scu-PA does not require its conversion to tcu-PA, nor conversion of Glu- to Lys-plasminogen, but appears to be due to the additional binding of plasminogen to partially digested fibrin; scu-PA may thus represent a physiological functional form of u-PA in plasma.
Fleury
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Institut National de la Santé et de la Recherche Médicale U.143, Hôpital de Bicêtre, France.
Lijnen
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Anglés-Cano
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D000409:Alanine; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D004789:Enzyme Activation; D005337:Fibrin / Q000378:metabolism; D005342:Fibrinolysis; D005971:Glutamates; D018698:Glutamic Acid; D006801:Humans; D007700:Kinetics; D008239:Lysine; D016297:Mutagenesis, Site-Directed; D010958:Plasminogen / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000097:blood; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D012996:Solutions; D014568:Urokinase-Type Plasminogen Activator / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005971:Glutamates; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D012996:Solutions; D018698:Glutamic Acid; D012694:Serine; D005337:Fibrin; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator; D008239:Lysine; D000409:Alanine