| Synthesis and secretion of plasminogen activator inhibitor 1 by human endothelial cells in vitro. Effect of active site mutagenized tissue-type plasminogen activator. | |
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MedLine Citation:
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PMID: 1898737 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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The effects of recombinant tissue-type plasminogen activator (rt-PA) and of an inactive mutant of rt-PA, obtained by mutagenesis of the active site Ser478 to Ala (rt-PA-Ala478), on the synthesis and secretion of plasminogen activator inhibitor-1 (PAI-1) by human umbilical vein endothelial cells (HUVEC) in culture were studied. Under base-line conditions, PAI-1 antigen secretion was 4.3 +/- 1.0 micrograms (mean +/- S.D., n = 8) per 10(6) cells in 24 h. This PAI-1 had a low specific activity (6,000 +/- 1,600 units/mg) and Mr of 50,000, which was not altered by addition of rt-PA. In HUVEC cultured with 2 micrograms/ml rt-PA-Ala478, PAI-1 antigen secretion was 2.1 +/- 0.8 micrograms (n = 5) per 10(6) cells in 24 h with a specific activity of 120,000 +/- 42,000 units/mg and Mr of 50,000. Addition of rt-PA to this conditioned medium resulted in generation of three main components: 16% migrated as an Mr 106,000 rt-PA.PAI-1 complex, 16% as an Mr 81,000 degraded rt-PA.PAI-1 complex and the remainder as an Mr 45,000 degradation product of PAI-1. HUVEC cultured with 2 micrograms/ml rt-PA secreted 3.9 +/- 0.6 micrograms (n = 8) PAI-1 antigen per 10(6) cells within 24 h, of which 20-50% occurred as intact or degraded complexes with t-PA (Mr 106,000 and 81,000) and the rest as an inactive Mr 45,000 degradation product of PAI-1. PAI-1 mRNA levels, determined by Northern blot analysis and expressed relative to beta-actin mRNA levels, were very similar for HUVEC cultured in the absence or the presence of rt-PA or rt-PA-Ala478. It is concluded that PAI-1 is secreted by HUVEC in culture in fully active form which spontaneously inactivates. PAI-1 can be stabilized by addition of rt-PA-Ala478 to the culture medium, resulting in a 20-fold increase in specific activity. Interaction of rt-PA with active PAI-1 produces both t-PA.PAI-1 complex and an inactive degradation product of PAI-1. |
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Authors:
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K Bartha; P J Declerck; H Moreau; L Nelles; D Collen |
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Publication Detail:
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Type: In Vitro; Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: The Journal of biological chemistry Volume: 266 ISSN: 0021-9258 ISO Abbreviation: J. Biol. Chem. Publication Date: 1991 Jan |
Date Detail:
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Created Date: 1991-02-12 Completed Date: 1991-02-12 Revised Date: 2006-11-15 |
Medline Journal Info:
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Nlm Unique ID: 2985121R Medline TA: J Biol Chem Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 792-7 Citation Subset: IM |
Affiliation:
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Center for Thrombosis and Vascular Research, University of Leuven, Belgium. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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Antigens
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genetics Binding Sites Blotting, Northern Endothelium, Vascular / metabolism* Humans Mutation Plasminogen Inactivators / metabolism* RNA, Messenger / analysis Recombinant Proteins / genetics Tissue Plasminogen Activator / genetics* |
| Chemical | |
Reg. No./Substance:
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0/Antigens; 0/Plasminogen Inactivators; 0/RNA, Messenger; 0/Recombinant Proteins; EC 3.4.21.68/Tissue Plasminogen Activator |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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