title: Characterization of the murine plasminogen/urokinase-type plasminogen-activator system.
abstract: The murine plasminogen/urokinase-type plasminogen-activator (u-PA) system was studied using purified proteins, plasma and endothelioma cells. Recombinant murine u-PA was obtained as a single-chain molecule of 45 kDa which was converted to two-chain u-PA with plasmin by cleavage of the Lys159-Ile160 peptide bond. Murine plasminogen, purified from plasma as a single-chain protein of 95 kDa, was resistant to quantitative activation with murine recombinant two-chain u-PA: only 15% activation within 1 h at 37 degrees C was obtained in mixtures of 1 microM plasminogen and 5 nM recombinant two-chain u-PA, whereas quantitative activation was observed in the autologous human system. Addition of 6-aminohexanoic acid to native murine plasminogen resulted in quantitative activation within 1 h. In murine plasma in vitro, plasminogen was also resistant to quantitative activation with u-PA (50% activation within 1 h at 37 degrees C with 50 nM recombinant two-chain u-PA, whereas in the human system nearly quantitative activation was obtained). Murine plasma clots submerged in murine plasma were resistant to lysis with u-PA; < or = 2% clot lysis in 2 h was obtained with 80 nM recombinant two-chain u-PA in the autologous murine system whereas 50% clot lysis in 2 h required only 15 nM recombinant two-chain u-PA in the autologous human system. Saturable binding of murine recombinant two-chain u-PA was observed to murine endothelioma cells that are genetically deficient in u-PA (u-PA-/- End cells). Binding was characterized by a Kd of 5.5 nM and 800000 binding sites/cell. However, u-PA-/- End cells did not significantly stimulate the activation rate of murine plasminogen by murine recombinant two-chain u-PA and did not enhance the plasmin-mediated conversion rate of murine recombinant single-chain u-PA to its two-chain derivative. Murine recombinant two-chain u-PA bound to murine endothelioma cells was quantitatively inhibited by murine plasminogen-activator inhibitor-1 (PAI-1). Thus, the interactions between murine plasminogen, u-PA and PAI-1 are qualitatively similar to those between their human counterparts. However, quantitative differences were observed both in the presence of cells and in plasma which may contribute to a reduced u-PA-mediated fibrinolytic activity in the murine systems.
authors.affiliation: Center for Molecular and Vascular Biology, University of Leuven, Belgium. roger.lijnen@med.kuleuven.ac.be
authors.forename: H R
authors.initials: HR
authors.lastname: Lijnen
authors.forename: B
authors.initials: B
authors.lastname: Van Hoef
authors.forename: D
authors.initials: D
authors.lastname: Collen
mesh_terms: D000818:Animals; D001769:Blood / Q000378:metabolism; D002021:Buffers; D004789:Enzyme Activation; D005342:Fibrinolysis; D006801:Humans; D051379:Mice; D010958:Plasminogen / Q000302:isolation & purification / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013045:Species Specificity; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D002021:Buffers; D017395:Plasminogen Activator Inhibitor 1; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; D010958:Plasminogen; D014568:Urokinase-Type Plasminogen Activator