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. 1988 Nov;82(5):1769–1778. doi: 10.1172/JCI113790

Endothelial cell-mediated conversion of Glu-plasminogen to Lys-plasminogen. Further evidence for assembly of the fibrinolytic system on the endothelial cell surface.

K A Hajjar 1, R L Nachman 1
PMCID: PMC442747  PMID: 3141482

Abstract

Lysine-plasminogen (Lys-PLG), the plasmin-modified form of native glutamic acid-plasminogen (Glu-PLG), displays enhanced binding affinity for fibrin and also enhanced activation by urokinase and tissue plasminogen activator. We previously demonstrated high-affinity, specific, and functional binding of Glu-PLG as well as tissue plasminogen activator to cultured human umbilical vein endothelial cells (HUVEC). In the present study, we demonstrate binding of Lys-PLG to HUVEC, as well as conversion of Glu-PLG to Lys-PLG at the cell surface. Binding of Lys-PLG to HUVEC was saturable, reversible, epsilon-aminocaproic acid-sensitive, and involved two saturable sites with Kd's of 142 pM and 120 nM, respectively. Upon incubation with Glu-PLG, HUVEC, as well as endothelium in situ, partially converted the ligand to a Lys-PLG-like species. Conversion by HUVEC was blocked by diisopropyl-fluorophosphate, but not by other serine protease inhibitors, including alpha 2-plasmin inhibitor. Eluates of intact umbilical cord vessels contained Lys-PLG by immunoblot analysis. Lys-PLG was also identified immunohistochemically on the endothelial surface of vessels from a variety of normal and inflamed tissues. Thus, endothelial cells appear to actively modify circulating Glu-PLG, bind Lys-PLG to their surface, and thus enhance the fibrinolytic potential of the blood vessel wall.

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Selected References

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

  1. Bauer P. I., Machovich R., Büki K. G., Csonka E., Koch S. A., Horváth I. Interaction of plasmin with endothelial cells. Biochem J. 1984 Feb 15;218(1):119–124. doi: 10.1042/bj2180119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beisiegel U., Schneider W. J., Brown M. S., Goldstein J. L. Immunoblot analysis of low density lipoprotein receptors in fibroblasts from subjects with familial hypercholesterolemia. J Biol Chem. 1982 Nov 10;257(21):13150–13156. [PubMed] [Google Scholar]
  3. Booyse F. M., Osikowicz G., Feder S., Scheinbuks J. Isolation and characterization of a urokinase-type plasminogen activator (Mr = 54,000) from cultured human endothelial cells indistinguishable from urinary urokinase. J Biol Chem. 1984 Jun 10;259(11):7198–7205. [PubMed] [Google Scholar]
  4. Brown C. R., Hartree E. F. Effects of acrosin inhibitors on the soluble and membrane-bound forms of ram acrosin, and a reappraisal of the role of the enzyme in fertilization. Hoppe Seylers Z Physiol Chem. 1976 Jan;357(1):57–65. doi: 10.1515/bchm2.1976.357.1.57. [DOI] [PubMed] [Google Scholar]
  5. Burtin P., Chavanel G., Andre J. The plasmin system in human colonic tumors: an immunofluorescence study. Int J Cancer. 1985 Mar 15;35(3):307–314. doi: 10.1002/ijc.2910350305. [DOI] [PubMed] [Google Scholar]
  6. Burtin P., Chavanel G., André-Bougaran J., Gentile A. The plasmin system in human adenocarcinomas and their metastases. A comparative immunofluorescence study. Int J Cancer. 1987 Feb 15;39(2):170–178. doi: 10.1002/ijc.2910390208. [DOI] [PubMed] [Google Scholar]
  7. Castellino F. J. Biochemistry of human plasminogen. Semin Thromb Hemost. 1984 Jan;10(1):18–23. doi: 10.1055/s-2007-1004404. [DOI] [PubMed] [Google Scholar]
  8. Chapman H. A., Jr, Stone O. L., Vavrin Z. Degradation of fibrin and elastin by intact human alveolar macrophages in vitro. Characterization of a plasminogen activator and its role in matrix degradation. J Clin Invest. 1984 Mar;73(3):806–815. doi: 10.1172/JCI111275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chapman H. A., Jr, Vavrin Z., Hibbs J. B., Jr Macrophage fibrinolytic activity: identification of two pathways of plasmin formation by intact cells and of a plasminogen activator inhibitor. Cell. 1982 Mar;28(3):653–662. doi: 10.1016/0092-8674(82)90220-3. [DOI] [PubMed] [Google Scholar]
  10. Daniel T. O., Schneider W. J., Goldstein J. L., Brown M. S. Visualization of lipoprotein receptors by ligand blotting. J Biol Chem. 1983 Apr 10;258(7):4606–4611. [PubMed] [Google Scholar]
  11. Del Rosso M., Dini G., Fibbi G. Receptors for plasminogen activator, urokinase, in normal and Rous sarcoma virus-transformed mouse fibroblasts. Cancer Res. 1985 Feb;45(2):630–636. [PubMed] [Google Scholar]
  12. Del Rosso M., Pucci M., Fibbi G., Dini G. Interaction of urokinase with specific receptors abolishes the time of commitment to terminal differentiation of murine erythroleukaemia (Friend) cells. Br J Haematol. 1987 Jul;66(3):289–294. doi: 10.1111/j.1365-2141.1987.tb06912.x. [DOI] [PubMed] [Google Scholar]
  13. Fibbi G., Dini G., Pasquali F., Pucci M., Del Rosso M. The Mr 17500 region of the A chain of urokinase is required for interaction with a specific receptor in A431 cells. Biochim Biophys Acta. 1986 Mar 14;885(3):301–308. doi: 10.1016/0167-4889(86)90245-4. [DOI] [PubMed] [Google Scholar]
  14. Gross J. L., Krupp M. N., Rifkin D. B., Lane M. D. Down-regulation of epidermal growth factor receptor correlates with plasminogen activator activity in human A431 epidermoid carcinoma cells. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2276–2280. doi: 10.1073/pnas.80.8.2276. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hajjar K. A., Hamel N. M., Harpel P. C., Nachman R. L. Binding of tissue plasminogen activator to cultured human endothelial cells. J Clin Invest. 1987 Dec;80(6):1712–1719. doi: 10.1172/JCI113262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hajjar K. A., Harpel P. C., Jaffe E. A., Nachman R. L. Binding of plasminogen to cultured human endothelial cells. J Biol Chem. 1986 Sep 5;261(25):11656–11662. [PubMed] [Google Scholar]
  17. Holvoet P., Lijnen H. R., Collen D. A monoclonal antibody specific for Lys-plasminogen. Application to the study of the activation pathways of plasminogen in vivo. J Biol Chem. 1985 Oct 5;260(22):12106–12111. [PubMed] [Google Scholar]
  18. Hoylaerts M., Rijken D. C., Lijnen H. R., Collen D. Kinetics of the activation of plasminogen by human tissue plasminogen activator. Role of fibrin. J Biol Chem. 1982 Mar 25;257(6):2912–2919. [PubMed] [Google Scholar]
  19. Huarte J., Belin D., Bosco D., Sappino A. P., Vassalli J. D. Plasminogen activator and mouse spermatozoa: urokinase synthesis in the male genital tract and binding of the enzyme to the sperm cell surface. J Cell Biol. 1987 May;104(5):1281–1289. doi: 10.1083/jcb.104.5.1281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LaBombardi V. J., Shaw E., DiStefano J. F., Beck G., Brown F., Zucker S. Isolation and characterization of a trypsin-like serine proteinase from the membranes of Walker 256 carcino-sarcoma cells. Biochem J. 1983 Jun 1;211(3):695–700. doi: 10.1042/bj2110695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Levin E. G., Loskutoff D. J. Cultured bovine endothelial cells produce both urokinase and tissue-type plasminogen activators. J Cell Biol. 1982 Sep;94(3):631–636. doi: 10.1083/jcb.94.3.631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lijnen H. R., Collen D. Interaction of plasminogen activators and inhibitors with plasminogen and fibrin. Semin Thromb Hemost. 1982 Jan;8(1):2–10. doi: 10.1055/s-2007-1005038. [DOI] [PubMed] [Google Scholar]
  24. Loskutoff D. J., Edgington T. E. Synthesis of a fibrinolytic activator and inhibitor by endothelial cells. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3903–3907. doi: 10.1073/pnas.74.9.3903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lottenberg R., Broder C. C., Boyle M. D. Identification of a specific receptor for plasmin on a group A streptococcus. Infect Immun. 1987 Aug;55(8):1914–1918. doi: 10.1128/iai.55.8.1914-1918.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lucas M. A., Fretto L. J., McKee P. A. The binding of human plasminogen to fibrin and fibrinogen. J Biol Chem. 1983 Apr 10;258(7):4249–4256. [PubMed] [Google Scholar]
  27. Markus G., Evers J. L., Hobika G. H. Comparison of some properties of native (Glu) and modified (Lys) human plasminogen. J Biol Chem. 1978 Feb 10;253(3):733–739. [PubMed] [Google Scholar]
  28. Markus G., Priore R. L., Wissler F. C. The binding of tranexamic acid to native (Glu) and modified (Lys) human plasminogen and its effect on conformation. J Biol Chem. 1979 Feb 25;254(4):1211–1216. [PubMed] [Google Scholar]
  29. Martin B. M., Wasiewski W. W., Fenton J. W., 2nd, Detwiler T. C. Equilibrium binding of thrombin to platelets. Biochemistry. 1976 Nov 2;15(22):4886–4893. doi: 10.1021/bi00667a021. [DOI] [PubMed] [Google Scholar]
  30. Mellgren R. L. Calcium-dependent proteases: an enzyme system active at cellular membranes? FASEB J. 1987 Aug;1(2):110–115. doi: 10.1096/fasebj.1.2.2886390. [DOI] [PubMed] [Google Scholar]
  31. Miles L. A., Ginsberg M. H., White J. G., Plow E. F. Plasminogen interacts with human platelets through two distinct mechanisms. J Clin Invest. 1986 Jun;77(6):2001–2009. doi: 10.1172/JCI112529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Miles L. A., Plow E. F. Binding and activation of plasminogen on the platelet surface. J Biol Chem. 1985 Apr 10;260(7):4303–4311. [PubMed] [Google Scholar]
  33. Miles L. A., Plow E. F. Receptor mediated binding of the fibrinolytic components, plasminogen and urokinase, to peripheral blood cells. Thromb Haemost. 1987 Oct 28;58(3):936–942. [PubMed] [Google Scholar]
  34. Munson P. J., Rodbard D. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal Biochem. 1980 Sep 1;107(1):220–239. doi: 10.1016/0003-2697(80)90515-1. [DOI] [PubMed] [Google Scholar]
  35. Plow E. F., Freaney D. E., Plescia J., Miles L. A. The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type. J Cell Biol. 1986 Dec;103(6 Pt 1):2411–2420. doi: 10.1083/jcb.103.6.2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sjöholm I. Studies on the conformational changes of plasminogen induced during activation to plasmin and by 6-aminohexanoic acid. Eur J Biochem. 1973 Nov 15;39(2):471–479. doi: 10.1111/j.1432-1033.1973.tb03146.x. [DOI] [PubMed] [Google Scholar]
  37. Smaje L., Zweifach B. W., Intaglietta M. Micropressures and capillary filtration coefficients in single vessels of the cremaster muscle of the rat. Microvasc Res. 1970 Jan;2(1):96–110. doi: 10.1016/0026-2862(70)90055-5. [DOI] [PubMed] [Google Scholar]
  38. Stoppelli M. P., Tacchetti C., Cubellis M. V., Corti A., Hearing V. J., Cassani G., Appella E., Blasi F. Autocrine saturation of pro-urokinase receptors on human A431 cells. Cell. 1986 Jun 6;45(5):675–684. doi: 10.1016/0092-8674(86)90782-8. [DOI] [PubMed] [Google Scholar]
  39. Tanaka K., Nakamura T., Ichihara A. A unique trypsin-like protease associated with plasma membranes of rat liver. J Biol Chem. 1986 Feb 25;261(6):2610–2615. [PubMed] [Google Scholar]
  40. Turner B. M. Use of alkaline phosphatase-conjugated antibodies for detection of protein antigens on nitrocellulose filters. Methods Enzymol. 1986;121:848–855. doi: 10.1016/0076-6879(86)21081-2. [DOI] [PubMed] [Google Scholar]
  41. Vassalli J. D., Baccino D., Belin D. A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase. J Cell Biol. 1985 Jan;100(1):86–92. doi: 10.1083/jcb.100.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wiman B., Collen D. Molecular mechanism of physiological fibrinolysis. Nature. 1978 Apr 6;272(5653):549–550. doi: 10.1038/272549a0. [DOI] [PubMed] [Google Scholar]
  43. Wiman B., Wallén P. Activation of human plasminogen by an insoluble derivative of urokinase. Structural changes of plasminogen in the course of activation to plasmin and demonstration of a possible intermediate compound. Eur J Biochem. 1973 Jul 2;36(1):25–31. doi: 10.1111/j.1432-1033.1973.tb02880.x. [DOI] [PubMed] [Google Scholar]

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