Detection and partial characterization of a specific plasminogen activator inhibitor in human chondrocyte cultures.
Serum-free culture medium collected from primary monolayer cultures of human articular chondrocytes was found to inhibit human urokinase [EC 3.4.21.31] activity. Although chondrocyte culture medium contained a small amount of endothelial-type plasminogen activator inhibitor which could be demonstrated by reverse fibrin autography, most of the urokinase inhibitory activity of chondrocyte culture medium was shown to be due to a different molecule from endothelial-type inhibitor, since it did not react with a specific antibody to this type of inhibitor. The dominant urokinase inhibitor in chondrocyte culture medium was partially purified by concanavalin A-Sepharose affinity chromatography. The partially purified inhibitor inhibited high-Mr urokinase more effectively than low-Mr urokinase, but no obvious inhibition was detected against tissue-type plasminogen activator, plasmin, trypsin, and thrombin. The inhibitor had an apparent Mr of 43,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis, and it was unstable to sodium dodecyl sulfate, acid, and heat treatments. Inhibition of urokinase by the inhibitor was accompanied with the formation of a sodium dodecyl sulfate-stable high-Mr complex between them. Inhibition and complex formation required the active site of urokinase. The partially purified inhibitor was thought to be immunologically different from the known classes of plasminogen activator inhibitors, including endothelial-type inhibitor, macrophage/monocyte inhibitor, and protease nexin, since it did not react with specific antibodies to these inhibitors.
Yamada
H
H
Department of Medicine and Clinical Science, John Curtin School of Medical Research, Australian National University, Canberra.
Stephens
R W
RW
Nakagawa
T
T
McNicol
D
D
D000906:Antibodies; D002358:Cartilage, Articular / Q000032:analysis*; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D005342:Fibrinolysis / Q000187:drug effects; D006023:Glycoproteins / Q000276:immunology / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors*; D015849:Plasminogen Inactivators; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors
D000906:Antibodies; D006023:Glycoproteins; D015849:Plasminogen Inactivators; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator