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. 1987 Apr;84(7):1886–1890. doi: 10.1073/pnas.84.7.1886

Isolation of the tissue factor inhibitor produced by HepG2 hepatoma cells.

G J Broze Jr, J P Miletich
PMCID: PMC304546  PMID: 3031657

Abstract

Progressive inhibition of tissue factor activity occurs upon its addition to human plasma (serum). This process requires the presence of factor VII(a), facto-X(a), Ca2+, and another component in plasma that we have called the tissue factor inhibitor (TFI). A TFI secreted by HepG2 cells (human hepatoma cell line) was isolated from serum-free conditioned medium in a four-step procedure including CdCl2 precipitation, diisopropylphosphoryl-factor Xa affinity chromatography, Sephadex G-75 superfine gel filtration, and Mono Q ion-exchange chromatography. The purified TFI contained a predominant band at Mr 38,000 on NaDodSO4/polyacrylamide gel electrophoresis that comigrates with inhibitory activity. Like the activity present in plasma, this TFI requires the presence of factor VII(a), factor X(a), and Ca2+ to express inhibitory activity. Its specific activity (assuming an extinction coefficient of 10 at 280 nM, for a 1-cm path length through a 1% solution) was 9800 units/mg of protein, where 1 unit of TFI activity was defined as that present in 1 ml of normal pooled serum.

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Selected References

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  1. Bajaj S. P., Rapaport S. I., Prodanos C. A simplified procedure for purification of human prothrombin, factor IX and factor X. Prep Biochem. 1981;11(4):397–412. doi: 10.1080/00327488108065531. [DOI] [PubMed] [Google Scholar]
  2. Bevilacqua M. P., Pober J. S., Majeau G. R., Cotran R. S., Gimbrone M. A., Jr Interleukin 1 (IL-1) induces biosynthesis and cell surface expression of procoagulant activity in human vascular endothelial cells. J Exp Med. 1984 Aug 1;160(2):618–623. doi: 10.1084/jem.160.2.618. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bevilacqua M. P., Pober J. S., Majeau G. R., Fiers W., Cotran R. S., Gimbrone M. A., Jr Recombinant tumor necrosis factor induces procoagulant activity in cultured human vascular endothelium: characterization and comparison with the actions of interleukin 1. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4533–4537. doi: 10.1073/pnas.83.12.4533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Broze G. J., Jr, Hickman S., Miletich J. P. Monoclonal anti-human factor VII antibodies. Detection in plasma of a second protein antigenically and genetically related to factor VII. J Clin Invest. 1985 Sep;76(3):937–946. doi: 10.1172/JCI112093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Broze G. J., Jr, Majerus P. W. Purification and properties of human coagulation factor VII. J Biol Chem. 1980 Feb 25;255(4):1242–1247. [PubMed] [Google Scholar]
  6. Broze G. J., Jr, Miletich J. P. Characterization of the inhibition of tissue factor in serum. Blood. 1987 Jan;69(1):150–155. [PubMed] [Google Scholar]
  7. Carson S. D. Plasma high density lipoproteins inhibit the activation of coagulation factor X by factor VIIa and tissue factor. FEBS Lett. 1981 Sep 14;132(1):37–40. doi: 10.1016/0014-5793(81)80422-x. [DOI] [PubMed] [Google Scholar]
  8. Hubbard A. R., Jennings C. A. Inhibition of tissue thromboplastin-mediated blood coagulation. Thromb Res. 1986 May 15;42(4):489–498. doi: 10.1016/0049-3848(86)90212-4. [DOI] [PubMed] [Google Scholar]
  9. LANCHANTIN G. F., WARE A. G. Identification of a thromboplastin inhibitor in serum and in plasma. J Clin Invest. 1953 Apr;32(4):381–389. doi: 10.1172/JCI102749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. Lerner R. G., Goldstein R., Cummings G., Lange K. Stimulation of human leukocyte thromboplastic activity by endotoxin. Proc Soc Exp Biol Med. 1971 Oct;138(1):145–148. doi: 10.3181/00379727-138-35848. [DOI] [PubMed] [Google Scholar]
  12. Maynard J. R., Dreyer B. E., Stemerman M. B., Pitlick F. A. Tissue-factor coagulant activity of cultured human endothelial and smooth muscle cells and fibroblasts. Blood. 1977 Sep;50(3):387–396. [PubMed] [Google Scholar]
  13. Maynard J. R., Heckman C. A., Pitlick F. A., Nemerson Y. Association of tissue factor activity with the surface of cultured cells. J Clin Invest. 1975 Apr;55(4):814–824. doi: 10.1172/JCI107992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Muhlfelder T. W., Niemetz J., Kreutzer D., Beebe D., Ward P. A., Rosenfeld S. I. C5 chemotactic fragment induces leukocyte production of tissue factor activity: a link between complement and coagulation. J Clin Invest. 1979 Jan;63(1):147–150. doi: 10.1172/JCI109269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Peterson C. B., Blackburn M. N. Isolation and characterization of an antithrombin III variant with reduced carbohydrate content and enhanced heparin binding. J Biol Chem. 1985 Jan 10;260(1):610–615. [PubMed] [Google Scholar]
  16. Rothberger H., Zimmerman T. S., Spiegelberg H. L., Vaughan J. H. Leukocyte procoagulant activity: enhancement of production in vitro by IgG and antigen-antibody complexes. J Clin Invest. 1977 Mar;59(3):549–557. doi: 10.1172/JCI108670. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Sanders N. L., Bajaj S. P., Zivelin A., Rapaport S. I. Inhibition of tissue factor/factor VIIa activity in plasma requires factor X and an additional plasma component. Blood. 1985 Jul;66(1):204–212. [PubMed] [Google Scholar]
  18. Silverberg S. A., Nemerson Y., Zur M. Kinetics of the activation of bovine coagulation factor X by components of the extrinsic pathway. Kinetic behavior of two-chain factor VII in the presence and absence of tissue factor. J Biol Chem. 1977 Dec 10;252(23):8481–8488. [PubMed] [Google Scholar]
  19. Zur M., Nemerson Y. Kinetics of factor IX activation via the extrinsic pathway. Dependence of Km on tissue factor. J Biol Chem. 1980 Jun 25;255(12):5703–5707. [PubMed] [Google Scholar]

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