Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1990 Jul 15;269(2):475–482. doi: 10.1042/bj2690475

Binding of tissue plasminogen activator to human aortic endothelial cells.

M A Sanzo 1, S C Howard 1, A J Wittwer 1, H M Cochrane 1
PMCID: PMC1131601  PMID: 2117440

Abstract

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.

Full text

PDF
475

Selected References

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

  1. Bachmann F., Kruithof I. E. Tissue plasminogen activator: chemical and physiological aspects. Semin Thromb Hemost. 1984 Jan;10(1):6–17. doi: 10.1055/s-2007-1004403. [DOI] [PubMed] [Google Scholar]
  2. Barnathan E. S., Kuo A., Van der Keyl H., McCrae K. R., Larsen G. R., Cines D. B. Tissue-type plasminogen activator binding to human endothelial cells. Evidence for two distinct binding sites. J Biol Chem. 1988 Jun 5;263(16):7792–7799. [PubMed] [Google Scholar]
  3. Beebe D. P., Aronson D. L. Turnover of human tissue plasminogen activator (tPA) in rabbits. Thromb Res. 1986 Sep 15;43(6):663–674. doi: 10.1016/0049-3848(86)90103-9. [DOI] [PubMed] [Google Scholar]
  4. Beebe D. P. Binding of tissue plasminogen activator to human umbilical vein endothelial cells. Thromb Res. 1987 Apr 15;46(2):241–254. doi: 10.1016/0049-3848(87)90286-6. [DOI] [PubMed] [Google Scholar]
  5. Blasi F., Vassalli J. D., Danø K. Urokinase-type plasminogen activator: proenzyme, receptor, and inhibitors. J Cell Biol. 1987 Apr;104(4):801–804. doi: 10.1083/jcb.104.4.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chen J. K., Hoshi H., McClure D. B., McKeehan W. L. Role of lipoproteins in growth of human adult arterial endothelial and smooth muscle cells in low lipoprotein-deficient serum. J Cell Physiol. 1986 Nov;129(2):207–214. doi: 10.1002/jcp.1041290212. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Dickson R. B., Willingham M. C., Pastan I. Binding and internalization of 125I-alpha 2-macroglobulin by cultured fibroblasts. J Biol Chem. 1981 Apr 10;256(7):3454–3459. [PubMed] [Google Scholar]
  9. Friesel R., Maciag T. Internalization and degradation of heparin binding growth factor-I by endothelial cells. Biochem Biophys Res Commun. 1988 Mar 30;151(3):957–964. doi: 10.1016/s0006-291x(88)80459-5. [DOI] [PubMed] [Google Scholar]
  10. Haigler H. T., Maxfield F. R., Willingham M. C., Pastan I. Dansylcadaverine inhibits internalization of 125I-epidermal growth factor in BALB 3T3 cells. J Biol Chem. 1980 Feb 25;255(4):1239–1241. [PubMed] [Google Scholar]
  11. 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]
  12. Howard E. W., Knauer D. J. Characterization of the receptor for protease nexin-I:protease complexes on human fibroblasts. J Cell Physiol. 1987 May;131(2):276–283. doi: 10.1002/jcp.1041310219. [DOI] [PubMed] [Google Scholar]
  13. Kirchheimer J. C., Remold H. G. Functional characteristics of receptor-bound urokinase on human monocytes: catalytic efficiency and susceptibility to inactivation by plasminogen activator inhibitors. Blood. 1989 Sep;74(4):1396–1402. [PubMed] [Google Scholar]
  14. 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]
  15. McKanna J. A., Haigler H. T., Cohen S. Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5689–5693. doi: 10.1073/pnas.76.11.5689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Munson P. J. LIGAND: a computerized analysis of ligand binding data. Methods Enzymol. 1983;92:543–576. doi: 10.1016/0076-6879(83)92044-x. [DOI] [PubMed] [Google Scholar]
  17. Nilsson S., Einarsson M., Ekvärn S., Häggroth L., Mattsson C. Turnover of tissue plasminogen activator in normal and hepatectomized rabbits. Thromb Res. 1985 Aug 15;39(4):511–521. doi: 10.1016/0049-3848(85)90174-4. [DOI] [PubMed] [Google Scholar]
  18. Owensby D. A., Sobel B. E., Schwartz A. L. Receptor-mediated endocytosis of tissue-type plasminogen activator by the human hepatoma cell line Hep G2. J Biol Chem. 1988 Aug 5;263(22):10587–10594. [PubMed] [Google Scholar]
  19. Parekh R. B., Dwek R. A., Rudd P. M., Thomas J. R., Rademacher T. W., Warren T., Wun T. C., Hebert B., Reitz B., Palmier M. N-glycosylation and in vitro enzymatic activity of human recombinant tissue plasminogen activator expressed in Chinese hamster ovary cells and a murine cell line. Biochemistry. 1989 Sep 19;28(19):7670–7679. doi: 10.1021/bi00445a023. [DOI] [PubMed] [Google Scholar]
  20. Parekh R. B., Dwek R. A., Thomas J. R., Opdenakker G., Rademacher T. W., Wittwer A. J., Howard S. C., Nelson R., Siegel N. R., Jennings M. G. Cell-type-specific and site-specific N-glycosylation of type I and type II human tissue plasminogen activator. Biochemistry. 1989 Sep 19;28(19):7644–7662. doi: 10.1021/bi00445a021. [DOI] [PubMed] [Google Scholar]
  21. Pohl G., Källström M., Bergsdorf N., Wallén P., Jörnvall H. Tissue plasminogen activator: peptide analyses confirm an indirectly derived amino acid sequence, identify the active site serine residue, establish glycosylation sites, and localize variant differences. Biochemistry. 1984 Jul 31;23(16):3701–3707. doi: 10.1021/bi00311a020. [DOI] [PubMed] [Google Scholar]
  22. Rijken D. C., Emeis J. J. Clearance of the heavy and light polypeptide chains of human tissue-type plasminogen activator in rats. Biochem J. 1986 Sep 15;238(3):643–646. doi: 10.1042/bj2380643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rijken D. C., Emeis J. J., Gerwig G. J. On the composition and function of the carbohydrate moiety of tissue-type plasminogen activator from human melanoma cells. Thromb Haemost. 1985 Dec 17;54(4):788–791. [PubMed] [Google Scholar]
  24. Rånby M., Bergsdorf N., Pohl G., Wallén P. Isolation of two variants of native one-chain tissue plasminogen activator. FEBS Lett. 1982 Sep 20;146(2):289–292. doi: 10.1016/0014-5793(82)80936-8. [DOI] [PubMed] [Google Scholar]
  25. Rånby M. Studies on the kinetics of plasminogen activation by tissue plasminogen activator. Biochim Biophys Acta. 1982 Jun 24;704(3):461–469. doi: 10.1016/0167-4838(82)90068-1. [DOI] [PubMed] [Google Scholar]
  26. Sakata Y., Okada M., Noro A., Matsuda M. Interaction of tissue-type plasminogen activator and plasminogen activator inhibitor 1 on the surface of endothelial cells. J Biol Chem. 1988 Feb 5;263(4):1960–1969. [PubMed] [Google Scholar]
  27. Simionescu N., Heltianu C., Antohe F., Simionescu M. Endothelial cell receptors for histamine. Ann N Y Acad Sci. 1982;401:132–149. doi: 10.1111/j.1749-6632.1982.tb25713.x. [DOI] [PubMed] [Google Scholar]
  28. Stoppelli M. P., Corti A., Soffientini A., Cassani G., Blasi F., Assoian R. K. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4939–4943. doi: 10.1073/pnas.82.15.4939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Verstraete M., Collen D. Thrombolytic therapy in the eighties. Blood. 1986 Jun;67(6):1529–1541. [PubMed] [Google Scholar]
  31. Wiley H. S., Cunningham D. D. The endocytotic rate constant. A cellular parameter for quantitating receptor-mediated endocytosis. J Biol Chem. 1982 Apr 25;257(8):4222–4229. [PubMed] [Google Scholar]
  32. Wiman B., Mellbring G., Rånby M. Plasminogen activator release during venous stasis and exercise as determined by a new specific assay. Clin Chim Acta. 1983 Jan 24;127(2):279–288. doi: 10.1016/s0009-8981(83)80012-6. [DOI] [PubMed] [Google Scholar]
  33. Wojta J., Hoover R. L., Daniel T. O. Vascular origin determines plasminogen activator expression in human endothelial cells. Renal endothelial cells produce large amounts of single chain urokinase type plasminogen activator. J Biol Chem. 1989 Feb 15;264(5):2846–2852. [PubMed] [Google Scholar]
  34. Wun T. C., Schleuning W. D., Reich E. Isolation and characterization of urokinase from human plasma. J Biol Chem. 1982 Mar 25;257(6):3276–3283. [PubMed] [Google Scholar]
  35. van Zonneveld A. J., Chang G. T., van den Berg J., Kooistra T., Verheijen J. H., Pannekoek H., Kluft C. Quantification of tissue-type plasminogen activator (t-PA) mRNA in human endothelial-cell cultures by hybridization with a t-PA cDNA probe. Biochem J. 1986 Apr 15;235(2):385–390. doi: 10.1042/bj2350385. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES