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. 1986 Sep 1;103(3):1129–1133. doi: 10.1083/jcb.103.3.1129

Alpha-thrombin induces release of platelet-derived growth factor-like molecule(s) by cultured human endothelial cells

PMCID: PMC2114288  PMID: 3745265

Abstract

Cultured endothelial cells secrete a platelet-derived growth factor- like molecule (PDGFc). We examined the effects of purified human alpha- thrombin on the production of PDGFc in cultures of human umbilical vein endothelial cells (HUVE) using a specific radioreceptor assay for PDGF. Addition of physiologically relevant concentrations of alpha-thrombin (0.1 to 10 U/ml) induced a time- and dose-dependent increase in the release of PDGFc into the culture medium. Significant stimulation of PDGFc release was observed as early as 1.5 h after addition of alpha- thrombin (10 U/ml) with a 4.9 +/- 1.1 fold increase at 24 h (mean +/- SEM of nine experiments, P less than 0.01). alpha-Thrombin treatment of HUVE did not affect cell viability as assessed by trypan blue dye exclusion. The receptor binding of PDGFc secreted by HUVE in response to alpha-thrombin was inhibited by monospecific antibody to purified human PDGF indicating that the molecule(s) is closely related to PDGF. alpha-Thrombin inactivated with diisopropylfluorophosphate was without stimulatory effect. Lysis of HUVE by repeated cycles of freeze/thaw released minimal PDGFc (less than 0.3 ng per 10(6) cells) compared to levels of PDGFc released into supernatant medium in response to alpha- thrombin (greater than 5.0 ng per 10(6) cells after a 24-h incubation with 10 U/ml alpha-thrombin). Moreover, incubation of freeze/thaw lysates of HUVE with alpha-thrombin failed to release PDGFc. Over a 3-h time course, however, alpha-thrombin-induced secretion of PDGFc was not prevented by cycloheximide. We conclude that alpha-thrombin induces secretion of PDGFc from HUVE by a nonlytic mechanism requiring the serine esterase activity of the enzyme. Although this effect does not initially require de novo protein synthesis, it does require cell- mediated conversion of PDGFc from an inactive to an active form.

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Selected References

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  1. Awbrey B. J., Hoak J. C., Owen W. G. Binding of human thrombin to cultured human endothelial cells. J Biol Chem. 1979 May 25;254(10):4092–4095. [PubMed] [Google Scholar]
  2. Barrett T. B., Gajdusek C. M., Schwartz S. M., McDougall J. K., Benditt E. P. Expression of the sis gene by endothelial cells in culture and in vivo. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6772–6774. doi: 10.1073/pnas.81.21.6772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bowen-Pope D. F., Ross R. Methods for studying the platelet-derived growth factor receptor. Methods Enzymol. 1985;109:69–100. doi: 10.1016/0076-6879(85)09078-4. [DOI] [PubMed] [Google Scholar]
  4. Bowen-Pope D. F., Ross R. Platelet-derived growth factor. II. Specific binding to cultured cells. J Biol Chem. 1982 May 10;257(9):5161–5171. [PubMed] [Google Scholar]
  5. Bowen-Pope D. F., Vogel A., Ross R. Production of platelet-derived growth factor-like molecules and reduced expression of platelet-derived growth factor receptors accompany transformation by a wide spectrum of agents. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2396–2400. doi: 10.1073/pnas.81.8.2396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Camussi G., Aglietta M., Malavasi F., Tetta C., Piacibello W., Sanavio F., Bussolino F. The release of platelet-activating factor from human endothelial cells in culture. J Immunol. 1983 Nov;131(5):2397–2403. [PubMed] [Google Scholar]
  7. Collins T., Ginsburg D., Boss J. M., Orkin S. H., Pober J. S. Cultured human endothelial cells express platelet-derived growth factor B chain: cDNA cloning and structural analysis. Nature. 1985 Aug 22;316(6030):748–750. doi: 10.1038/316748a0. [DOI] [PubMed] [Google Scholar]
  8. DiCorleto P. E., Bowen-Pope D. F. Cultured endothelial cells produce a platelet-derived growth factor-like protein. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1919–1923. doi: 10.1073/pnas.80.7.1919. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Fox P. L., DiCorleto P. E. Regulation of production of a platelet-derived growth factor-like protein by cultured bovine aortic endothelial cells. J Cell Physiol. 1984 Nov;121(2):298–308. doi: 10.1002/jcp.1041210206. [DOI] [PubMed] [Google Scholar]
  11. Gajdusek C., DiCorleto P., Ross R., Schwartz S. M. An endothelial cell-derived growth factor. J Cell Biol. 1980 May;85(2):467–472. doi: 10.1083/jcb.85.2.467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Galdal K. S., Evensen S. A., Nilsen E. The effect of thrombin on fibronectin in cultured human endothelial cells. Thromb Res. 1985 Mar 1;37(5):583–593. doi: 10.1016/0049-3848(85)90091-x. [DOI] [PubMed] [Google Scholar]
  13. Galdal K. S., Lyberg T., Evensen S. A., Nilsen E., Prydz H. Thrombin induces thromboplastin synthesis in cultured vascular endothelial cells. Thromb Haemost. 1985 Aug 30;54(2):373–376. [PubMed] [Google Scholar]
  14. Gelehrter T. D., Sznycer-Laszuk R. Thrombin induction of plasminogen activator-inhibitor in cultured human endothelial cells. J Clin Invest. 1986 Jan;77(1):165–169. doi: 10.1172/JCI112271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jaye M., McConathy E., Drohan W., Tong B., Deuel T., Maciag T. Modulation of the sis gene transcript during endothelial cell differentiation in vitro. Science. 1985 May 17;228(4701):882–885. doi: 10.1126/science.3890179. [DOI] [PubMed] [Google Scholar]
  16. Laposata M., Dovnarsky D. K., Shin H. S. Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro. Blood. 1983 Sep;62(3):549–556. [PubMed] [Google Scholar]
  17. Levin E. G., Marzec U., Anderson J., Harker L. A. Thrombin stimulates tissue plasminogen activator release from cultured human endothelial cells. J Clin Invest. 1984 Dec;74(6):1988–1995. doi: 10.1172/JCI111620. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Levine J. D., Harlan J. M., Harker L. A., Joseph M. L., Counts R. B. Thrombin-mediated release of factor VIII antigen from human umbilical vein endothelial cells in culture. Blood. 1982 Aug;60(2):531–534. [PubMed] [Google Scholar]
  19. Loesberg C., Gonsalves M. D., Zandbergen J., Willems C., van Aken W. G., Stel H. V., Van Mourik J. A., De Groot P. G. The effect of calcium on the secretion of factor VIII-related antigen by cultured human endothelial cells. Biochim Biophys Acta. 1983 Sep 22;763(2):160–168. doi: 10.1016/0167-4889(83)90039-3. [DOI] [PubMed] [Google Scholar]
  20. Lollar P., Owen W. G. Evidence that the effects of thrombin on arachidonate metabolism in cultured human endothelial cells are not mediated by a high affinity receptor. J Biol Chem. 1980 Sep 10;255(17):8031–8034. [PubMed] [Google Scholar]
  21. Loskutoff D. J. Effect of thrombin on the fibrinolytic activity of cultured bovine endothelial cells. J Clin Invest. 1979 Jul;64(1):329–332. doi: 10.1172/JCI109457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Maciag T., Cerundolo J., Ilsley S., Kelley P. R., Forand R. An endothelial cell growth factor from bovine hypothalamus: identification and partial characterization. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5674–5678. doi: 10.1073/pnas.76.11.5674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Niman H. L., Houghten R. A., Bowen-Pope D. F. Detection of high molecular weight forms of platelet-derived growth factor by sequence-specific antisera. Science. 1984 Nov 9;226(4675):701–703. doi: 10.1126/science.6494905. [DOI] [PubMed] [Google Scholar]
  24. Pearson J. D., Gordon J. L. Vascular endothelial and smooth muscle cells in culture selectively release adenine nucleotides. Nature. 1979 Oct 4;281(5730):384–386. doi: 10.1038/281384a0. [DOI] [PubMed] [Google Scholar]
  25. Prescott S. M., Zimmerman G. A., McIntyre T. M. Human endothelial cells in culture produce platelet-activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) when stimulated with thrombin. Proc Natl Acad Sci U S A. 1984 Jun;81(11):3534–3538. doi: 10.1073/pnas.81.11.3534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Raines E. W., Ross R. Platelet-derived growth factor. I. High yield purification and evidence for multiple forms. J Biol Chem. 1982 May 10;257(9):5154–5160. [PubMed] [Google Scholar]
  27. Robbins K. C., Leal F., Pierce J. H., Aaronson S. A. The v-sis/PDGF-2 transforming gene product localizes to cell membranes but is not a secretory protein. EMBO J. 1985 Jul;4(7):1783–1792. doi: 10.1002/j.1460-2075.1985.tb03851.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Ross R. The pathogenesis of atherosclerosis--an update. N Engl J Med. 1986 Feb 20;314(8):488–500. doi: 10.1056/NEJM198602203140806. [DOI] [PubMed] [Google Scholar]
  29. Shimokado K., Raines E. W., Madtes D. K., Barrett T. B., Benditt E. P., Ross R. A significant part of macrophage-derived growth factor consists of at least two forms of PDGF. Cell. 1985 Nov;43(1):277–286. doi: 10.1016/0092-8674(85)90033-9. [DOI] [PubMed] [Google Scholar]
  30. Shuman M. A., Majerus P. W. The measurement of thrombin in clotting blood by radioimmunoassay. J Clin Invest. 1976 Nov;58(5):1249–1258. doi: 10.1172/JCI108579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Thornton S. C., Mueller S. N., Levine E. M. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science. 1983 Nov 11;222(4624):623–625. doi: 10.1126/science.6635659. [DOI] [PubMed] [Google Scholar]
  32. Wall R. T., Harker L. A., Quadracci L. J., Striker G. E. Factors influencing endothelial cell proliferation in vitro. J Cell Physiol. 1978 Aug;96(2):203–213. doi: 10.1002/jcp.1040960209. [DOI] [PubMed] [Google Scholar]
  33. Weksler B. B., Ley C. W., Jaffe E. A. Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A 23187. J Clin Invest. 1978 Nov;62(5):923–930. doi: 10.1172/JCI109220. [DOI] [PMC free article] [PubMed] [Google Scholar]

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