Characterization of the murine plasminogen/urokinase‐type plasminogen‐activator system

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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‐Ile1 60 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°C was obtained in mixtures of 1 μM 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°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; ≤2% clot lysis in 2 h was obtained with 80nM 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.