NDLI: Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells.

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Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells.

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Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Lawrence, D. Erickson, L. A. Van Mourik, J. A. Loskutoff, D. J.
Abstract	Fibrin/agar films were prepared and used to detect plasminogen activators produced by cultured bovine aortic endothelial cells (fibrin autography). One preparation of fibrin underwent spontaneous lysis upon incubation at 37 degrees C. This lysis was prevented by antibodies to tissue-type plasminogen activator but not by antibodies to urokinase. Conditioned medium from the confluent endothelial cells was fractionated by polyacrylamide gel electrophoresis in the presence of NaDodSO4. The gels were analyzed on indicator films prepared with the spontaneously lysing fibrin (reverse fibrin autography). Unexpectedly, as the opaque fibrin film cleared, a distinct lysis-resistant zone appeared in the indicator gel at a region corresponding to Mr 55,000. Experiments were devised to determine whether the lysis-resistant zone in the indicator film reflected the presence of a cellular inhibitor in the polyacrylamide gel. The corresponding region was excised from a polyacrylamide gel, extracted with buffer, and tested directly for antifibrinolytic activity by the 125I-labeled fibrin plate method. Urokinase-mediated fibrinolytic activity was inhibited by the gel extract in a dose-dependent manner indicating the presence of such an inhibitor. Inhibitor activity was detected in Triton X-100 extracts of washed monolayers and in conditioned medium, where it accumulated with time. The endothelial cell inhibitor not only survived exposure to NaDodSO4 but also was active after incubation at pH 12 or treatment with 5% (vol/vol) 2-mercaptoethanol, 6 M urea, 4 M guanidine hydrochloride, or 1 M acetic acid. Considerable activity also remained after heating at 100 degrees C for 30 min. These results indicate that cultured bovine aortic endothelial cells synthesize and secrete a previously undetected, unusually stable fibrinolytic inhibitor of Mr 55,000. Reverse fibrin autography offers a convenient approach for studying such molecules.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	10
Volume Number	80
Language	English
Publisher	National Academy of Sciences
Publisher Date	1983-07-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Antifibrinolytic Agents Endothelium Enzymology Fibrinolysis Animals Aorta Cytology Cells, Cultured Culture Media Molecular Weight Urokinase-Type Plasminogen Activator Antagonists & Inhibitors Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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