Catabolism of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. Modulation by plasminogen activator inhibitor type 1.
Catalytic activity of tissue-type plasminogen activator (t-PA) in plasma is regulated in part by formation of complexes with specific inhibitors as well as by hepatic clearance. Potential interaction of these two regulatory mechanisms was examined in the human hepatoma cell line Hep G2. These cells secrete plasminogen activator inhibitor type-1 (PAI-1) and initiate catabolism of exogenous t-PA by receptor-mediated endocytosis. Specific binding of 125I-t-PA to cells at 4 degrees C results in dose-dependent formation of a 95-kDa species recognized by monospecific anti-PAI-1 and anti-t-PA antibodies and stable in the presence of low (0.2%) concentrations of sodium dodecyl sulfate (SDS). Specific binding of 125I-t-PA and formation of the 95-kDa SDS-stable species are inhibited in a concentration-dependent manner following preincubation of cells with anti-PAI-1 antibodies. High and low molecular weight forms of urokinase plasminogen activator (u-PA) capable of forming specific complexes with PAI-1 complete for 125I-t-PA binding sites. However, the proenzyme form of u-PA (scu-PA), incapable of forming complexes with PAI-1, does not compete for 125I-t-PA binding sites. The role of the serine protease active site of t-PA in mediating both interaction with PAI-1 and specific binding was examined using 125I-t-PA that had been functionally inactivated with D-phenylalanyl-L-propyl-L-arginyl-chloromethyl ketone (PPACK). 125I-t-PA-PPACK, despite a 6-fold lower affinity than active 125I-t-PA, exhibited specific binding to cells without detectable formation of SDS-stable complexes with PAI-1. Both surface-bound 125I-t-PA and 125I-t-PA-PPACK are internalized and degraded by cells at 37 degrees C. 125I-t-PA is internalized as a stable complex with PAI-1, whereas 125I-t-PA-PPACK is internalized with similar kinetics but without the presence of an SDS-stable complex. Thus, PAI-1 appears capable of modulating t-PA catabolism in the human hepatocyte.
Morton
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PA
Edward Malinckrodt Department of Pediatrics, Children's Hospital, St. Louis, Missouri.
Owensby
D A
DA
Sobel
B E
BE
Schwartz
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AL
D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites / Q000187:drug effects; D001667:Binding, Competitive; D006528:Carcinoma, Hepatocellular / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D004705:Endocytosis; D006023:Glycoproteins / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D008113:Liver Neoplasms; D046911:Macromolecular Substances; D008970:Molecular Weight; D015849:Plasminogen Inactivators; D011485:Protein Binding / Q000187:drug effects; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
D000911:Antibodies, Monoclonal; D006023:Glycoproteins; D046911:Macromolecular Substances; D015849:Plasminogen Inactivators; D012967:Sodium Dodecyl Sulfate; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator