Urokinase binding and catabolism by Hep G2 cells is plasminogen activator inhibitor-1 dependent, analogous to interactions of tissue-type plasminogen activator with these cells

• Published in: Thrombosis research 1995-08, Vol.79 (4), p.353-361
• Main Authors: Grimsley, Philip G., Normyle, John F., Brandt, Ruth A., Joulianos, Georgina, Chesterman, Colin N., Hogg, Philip J., Owensby, Dwain A.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal; Binding Sites; Binding, competitive; Cell Line; Endocytosis; Humans; Kinetics; Molecular Weight; Plasminogen Activator Inhibitor 1 - immunology; Plasminogen Activator Inhibitor 1 - metabolism; plasminogen activator inhibitor-1; Receptors, Cell Surface - metabolism; Receptors, Urokinase Plasminogen Activator; Tissue Plasminogen Activator - metabolism; Tumor Cells, Cultured; urokinase; Urokinase-Type Plasminogen Activator - chemistry; Urokinase-Type Plasminogen Activator - metabolism
• ISSN: 0049-3848; 1879-2472
• DOI: 10.1016/0049-3848(95)00123-9

The adherent human hepatoma cell line Hep G2 exhibits receptor mediated endocytosis and catabolism of tissue-type plasminogen activator•plasminogen activator inhibitor type-1 (t-PA•PAI-1) complexes formed when exogenous t-PA combines with endogenous PAI-1 in the extracellular matrix. To determine whether the other major PA, urokinase (u-PA), which also complexes with PAI-1, is metabolised via the same mechanism, 125I-labelled high (hmw) and low (lmw) molecular weight forms of u-PA were incubated with Hep G2 cells at 4°C for 2hr in the absence and presence of a 100-fold excess of unlabelled ligand in order to detect specific binding. Both hmw and lmw 125I-u-PA formed complexes with PAI-1 and these bound specifically and with high affinity (apparent K d 3.9 and 4.1 nM, with B max 78 × 10 3 and 83 × 10 3 binding sites/cell respectively). Binding by each form of radiolabelled u-PA was inhibited in a dose-dependent fashion by unlabelled t-PA, hmw-u-PA, lmw-u-PA, and by monoclonal anti-PAI-1 antibody. At 37°C, bound hmw and lmw 125I-u-PA•PAI-1 complexes were internalised and degraded rapidly. These findings indicate that the specificity of the previously described receptor which mediates PAI-1 dependent catabolism of t-PA by Hep G2 cells extends to complexes of u-PA with this inhibitor.