Purification and Characterization of a Novel Inhibitor of Urokinase from Human Urine

Urokinase-related proteins in human urine occur mainly as a 1: 1 complex of urokinase with an inhibitor (Stump, D. C., Thienpont, M., and Collen, D. (1986) J. Biol. Chem. 261, 1267-1273). BALB/c mice were immunized with this urokinase-urokinase inhibitor complex and spleen cells fused with mouse myeloma cells, resulting in hybridomas producing monoclonal antibodies. Three antibodies reacting with the complex but not with urokinase were utilized to develop a sensitive (0.5 ng/ml) enzyme-linked immunosorbent assay for the urokinase inhibitor, which was used for monitoring its purification by chromatography on zinc chelateSepharose, concanavalin A-Sepharose, SP-Sephadex C-50, and Sephadex G-100. A homogeneous glycoprotein of apparent M , 50,000 was obtained with a yield of 40 pg/liter urine and a purification factor of 320. One mg of the purified protein inhibited 35,000 IU of urokinase within 30 min at 37 "C. This protein was immunologically related to both the purified urokinase-urokinase inhibitor complex and to the inhibitor portion dissociated from it by nucleophilic dissociation. It was immunologically distinct from all known protease inhibitors, including the endothelial cell-derived fast-acting inhibitor of tissue-type plasminogen activator, the placental inhibitor of urokinase and protease nexin. In electrophoresis the protein migrated with &mobility. Inhibition of urokinase occurred with a second order rate constant ( k ) of 8 x lo3 M" s-' in the absence and of 9 X lo4 M" s" in the presence of 50 IU of heparin/ml. The urokinase inhibitor was inactive towards single-chain urokinasetype plasminogen activator and plasmin, but it inhibited two-chain tissue-type plasminogen activator with a k below lo3 M" s" and thrombin with a k of 4 X lo4 M" s" in the absence and 2 X lo6 M" s" in the presence of heparin. The concentration of this urokinase inhibitor in plasma from normal subjects determined by immunoassay was 2 2 0.7 pg/ml (mean f S.D., n = 25). The protein purified from plasma by immunoabsorption had the same M,, amino acid composition, and immunoreactivity as the urinary protein. Furthermore,