Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1987 Jul 15;245(2):543–550. doi: 10.1042/bj2450543

Glycosaminoglycans on fibroblasts accelerate thrombin inhibition by protease nexin-1.

D H Farrell 1, D D Cunningham 1
PMCID: PMC1148156  PMID: 2959275

Abstract

Protease nexin-1 (PN-1) is a proteinase inhibitor that is secreted by human fibroblasts in culture. PN-1 inhibits certain regulatory serine proteinases by forming a covalent complex with the catalytic-site serine residue; the complex then binds to the cell surface and is internalized and degraded. The fibroblast surface was recently shown to accelerate the rate of complex-formation between PN-1 and thrombin. The present paper demonstrates that the accelerative activity is primarily due to cell-surface heparan sulphate, with a much smaller contribution from chondroitin sulphate. This conclusion is supported by the effects of purified glycosaminoglycans on the second-order rate constant for the inhibition of thrombin by PN-1. Also, treatment of 35SO4(2-)-labelled cells with heparitin sulphate lyase or chondroitin sulphate ABC lyase demonstrated two discrete pools of 35S-labelled glycosaminoglycans; subsequent treatment of plasma membranes with these glycosidases showed that heparitin sulphate lyase treatment abolished about 80% of the accelerative activity and chondroitin sulphate ABC lyase removed the remaining 20%. These results show that two components are responsible for the acceleration of PN-1-thrombin complex-formation by human fibroblasts. Although dermatan sulphate is also present on fibroblasts, it did not accelerate the inhibition of thrombin by PN-1.

Full text

PDF
546

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker J. B., Low D. A., Simmer R. L., Cunningham D. D. Protease-nexin: a cellular component that links thrombin and plasminogen activator and mediates their binding to cells. Cell. 1980 Aug;21(1):37–45. doi: 10.1016/0092-8674(80)90112-9. [DOI] [PubMed] [Google Scholar]
  2. Bergman B. L., Scott R. W., Bajpai A., Watts S., Baker J. B. Inhibition of tumor-cell-mediated extracellular matrix destruction by a fibroblast proteinase inhibitor, protease nexin I. Proc Natl Acad Sci U S A. 1986 Feb;83(4):996–1000. doi: 10.1073/pnas.83.4.996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chase T., Jr, Shaw E. Comparison of the esterase activities of trypsin, plasmin, and thrombin on guanidinobenzoate esters. Titration of the enzymes. Biochemistry. 1969 May;8(5):2212–2224. doi: 10.1021/bi00833a063. [DOI] [PubMed] [Google Scholar]
  4. Chen L. B., Buchanan J. M. Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc Natl Acad Sci U S A. 1975 Jan;72(1):131–135. doi: 10.1073/pnas.72.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DAVIS B. J. DISC ELECTROPHORESIS. II. METHOD AND APPLICATION TO HUMAN SERUM PROTEINS. Ann N Y Acad Sci. 1964 Dec 28;121:404–427. doi: 10.1111/j.1749-6632.1964.tb14213.x. [DOI] [PubMed] [Google Scholar]
  6. Danø K., Andreasen P. A., Grøndahl-Hansen J., Kristensen P., Nielsen L. S., Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res. 1985;44:139–266. doi: 10.1016/s0065-230x(08)60028-7. [DOI] [PubMed] [Google Scholar]
  7. Eaton D. L., Baker J. B. Evidence that a variety of cultured cells secrete protease nexin and produce a distinct cytoplasmic serine protease-binding factor. J Cell Physiol. 1983 Nov;117(2):175–182. doi: 10.1002/jcp.1041170207. [DOI] [PubMed] [Google Scholar]
  8. Farrell D. H., Cunningham D. D. Human fibroblasts accelerate the inhibition of thrombin by protease nexin. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6858–6862. doi: 10.1073/pnas.83.18.6858. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Farrell D. H., Van Nostrand W. E., Cunningham D. D. A simple two-step purification of protease nexin. Biochem J. 1986 Aug 1;237(3):907–912. doi: 10.1042/bj2370907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fenton J. W., 2nd, Fasco M. J., Stackrow A. B. Human thrombins. Production, evaluation, and properties of alpha-thrombin. J Biol Chem. 1977 Jun 10;252(11):3587–3598. [PubMed] [Google Scholar]
  11. Fraker P. J., Speck J. C., Jr Protein and cell membrane iodinations with a sparingly soluble chloroamide, 1,3,4,6-tetrachloro-3a,6a-diphrenylglycoluril. Biochem Biophys Res Commun. 1978 Feb 28;80(4):849–857. doi: 10.1016/0006-291x(78)91322-0. [DOI] [PubMed] [Google Scholar]
  12. Gill P. J., Adler J., Silbert C. K., Silbert J. E. Removal of glycosaminoglycans from cultures of human skin fibroblasts. Biochem J. 1981 Jan 15;194(1):299–307. doi: 10.1042/bj1940299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Guenther J., Nick H., Monard D. A glia-derived neurite-promoting factor with protease inhibitory activity. EMBO J. 1985 Aug;4(8):1963–1966. doi: 10.1002/j.1460-2075.1985.tb03878.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kjellén L., Oldberg A., Hök M. Cell-surface heparan sulfate. Mechanisms of proteoglycan-cell association. J Biol Chem. 1980 Nov 10;255(21):10407–10413. [PubMed] [Google Scholar]
  15. Kleinman H. K., Silbert J. E., Silbert C. K. Heparan sulfate of skin fibroblasts grown in culture. Connect Tissue Res. 1975;4(1):17–23. doi: 10.3109/03008207509152193. [DOI] [PubMed] [Google Scholar]
  16. Klintworth G. K., Smith C. F. Difference between the glycosaminoglycans synthetized by corneal and cutaneous fibroblasts in culture. Lab Invest. 1981 Jun;44(6):553–559. [PubMed] [Google Scholar]
  17. 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]
  18. Low D. A., Baker J. B., Koonce W. C., Cunningham D. D. Released protease-nexin regulates cellular binding, internalization, and degradation of serine proteases. Proc Natl Acad Sci U S A. 1981 Apr;78(4):2340–2344. doi: 10.1073/pnas.78.4.2340. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Low D. A., Scott R. W., Baker J. B., Cunningham D. D. Cells regulate their mitogenic response to thrombin through release of protease nexin. Nature. 1982 Jul 29;298(5873):476–478. doi: 10.1038/298476a0. [DOI] [PubMed] [Google Scholar]
  20. Marcum J. A., Fritze L., Galli S. J., Karp G., Rosenberg R. D. Microvascular heparin-like species with anticoagulant activity. Am J Physiol. 1983 Nov;245(5 Pt 1):H725–H733. doi: 10.1152/ajpheart.1983.245.5.H725. [DOI] [PubMed] [Google Scholar]
  21. Marcum J. A., Rosenberg R. D. Anticoagulantly active heparin-like molecules from vascular tissue. Biochemistry. 1984 Apr 10;23(8):1730–1737. doi: 10.1021/bi00303a023. [DOI] [PubMed] [Google Scholar]
  22. Marcum J. A., Rosenberg R. D. Heparinlike molecules with anticoagulant activity are synthesized by cultured endothelial cells. Biochem Biophys Res Commun. 1985 Jan 16;126(1):365–372. doi: 10.1016/0006-291x(85)90615-1. [DOI] [PubMed] [Google Scholar]
  23. ORNSTEIN L. DISC ELECTROPHORESIS. I. BACKGROUND AND THEORY. Ann N Y Acad Sci. 1964 Dec 28;121:321–349. doi: 10.1111/j.1749-6632.1964.tb14207.x. [DOI] [PubMed] [Google Scholar]
  24. Rosenberg R. D., Damus P. S. The purification and mechanism of action of human antithrombin-heparin cofactor. J Biol Chem. 1973 Sep 25;248(18):6490–6505. [PubMed] [Google Scholar]
  25. Scott R. W., Bergman B. L., Bajpai A., Hersh R. T., Rodriguez H., Jones B. N., Barreda C., Watts S., Baker J. B. Protease nexin. Properties and a modified purification procedure. J Biol Chem. 1985 Jun 10;260(11):7029–7034. [PubMed] [Google Scholar]
  26. Scott R. W., Eaton D. L., Duran N., Baker J. B. Regulation of extracellular plasminogen activator by human fibroblasts. The role of protease nexin. J Biol Chem. 1983 Apr 10;258(7):4397–4403. [PubMed] [Google Scholar]
  27. Sim R. B., Arlaud G. J., Colomb M. G. Kinetics of reaction of human C1-inhibitor with the human complement system proteases C1r and C1s. Biochim Biophys Acta. 1980 Apr 11;612(2):433–449. doi: 10.1016/0005-2744(80)90126-6. [DOI] [PubMed] [Google Scholar]
  28. Thom D., Powell A. J., Lloyd C. W., Rees D. A. Rapid isolation of plasma membranes in high yield from cultured fibroblasts. Biochem J. 1977 Nov 15;168(2):187–194. doi: 10.1042/bj1680187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tollefsen D. M., Blank M. K. Detection of a new heparin-dependent inhibitor of thrombin in human plasma. J Clin Invest. 1981 Sep;68(3):589–596. doi: 10.1172/JCI110292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tollefsen D. M., Pestka C. A., Monafo W. J. Activation of heparin cofactor II by dermatan sulfate. J Biol Chem. 1983 Jun 10;258(11):6713–6716. [PubMed] [Google Scholar]
  31. Wight T. N. Proteoglycans in pathological conditions: atherosclerosis. Fed Proc. 1985 Feb;44(2):381–385. [PubMed] [Google Scholar]
  32. Witt I., Kaiser C., Schrenk W. J., Wunderwald P. Amino acid composition and N-terminal sequence of antithrombin BM. Thromb Res. 1983 Dec 1;32(5):513–518. doi: 10.1016/0049-3848(83)90261-x. [DOI] [PubMed] [Google Scholar]
  33. Yamagata T., Saito H., Habuchi O., Suzuki S. Purification and properties of bacterial chondroitinases and chondrosulfatases. J Biol Chem. 1968 Apr 10;243(7):1523–1535. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES