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. 1987 Mar 1;104(3):697–704. doi: 10.1083/jcb.104.3.697

Perturbation of human endothelial cells by thrombin or PMA changes the reactivity of their extracellular matrix towards platelets

PMCID: PMC2114527  PMID: 3818796

Abstract

In this study we have examined the influence of perturbation of endothelial cells on the amounts of fibronectin and von Willebrand factor in their extracellular matrix and the consequences of a changed composition of the matrix on platelet adhesion. For this purpose, we have used an in vitro perfusion system with which we can investigate the interactions of platelets in flowing blood with cultured endothelial cells and their extracellular matrix (Sakariassen, K. S., P. A. M. M. Aarts, P. G. de Groot, W. P. M. Houdgk, and J. J. Sixma, 1983, J. Lab. Clin Med. 102:522-535). Treatment of endothelial cells with 0.1-1.0 U/ml thrombin for 2 h increased the reactivity of the extracellular matrix, isolated after the thrombin treatment, towards platelets by approximately 50%. The increased reactivity did not depend on de novo protein synthesis but was inhibited by 3-deazaadenosine, an inhibitor of phospholipid methylation, which also inhibits the stimulus- induced instantaneous release of von Willebrand factor from endothelial cells. However, no changes in the amounts of von Willebrand factor and fibronectin in the matrix were detected. Thrombin may change the organization of the matrix proteins, not the composition. When endothelial cells were perturbed with the phorbol ester PMA or thrombin for 3 d, the adhesion of platelets to the extracellular matrix of treated cells was strongly impaired. This impairment coincided with a decrease in the amounts of von Willebrand factor and fibronectin present in the matrix. These results indicate that, after perturbation, endothelial cells regulate the composition of their matrix, and that this regulation has consequences for the adhesion of platelets.

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Selected References

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  1. Booyse F. M., Quarfoot A. J., Feder S. Culture-produced subendothelium. I. Platelet interaction and properties. Haemostasis. 1982;11(1):49–61. doi: 10.1159/000214640. [DOI] [PubMed] [Google Scholar]
  2. Chazov E. I., Alexeev A. V., Antonov A. S., Koteliansky V. E., Leytin V. L., Lyubimova E. V., Repin V. S., Sviridov D. D., Torchilin V. P., Smirnov V. N. Endothelial cell culture on fibrillar collagen: model to study platelet adhesion and liposome targeting to intercellular collagen matrix. Proc Natl Acad Sci U S A. 1981 Sep;78(9):5603–5607. doi: 10.1073/pnas.78.9.5603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Engvall E., Ruoslahti E., Miller E. J. Affinity of fibronectin to collagens of different genetic types and to fibrinogen. J Exp Med. 1978 Jun 1;147(6):1584–1595. doi: 10.1084/jem.147.6.1584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Foidart J. M., Bere E. W., Jr, Yaar M., Rennard S. I., Gullino M., Martin G. R., Katz S. I. Distribution and immunoelectron microscopic localization of laminin, a noncollagenous basement membrane glycoprotein. Lab Invest. 1980 Mar;42(3):336–342. [PubMed] [Google Scholar]
  5. Gamse G., Fromme H. G., Kresse H. Metabolism of sulfated glycosaminoglycans in cultured endothelial cells and smooth muscle cells from bovine aorta. Biochim Biophys Acta. 1978 Dec 18;544(3):514–528. doi: 10.1016/0304-4165(78)90326-4. [DOI] [PubMed] [Google Scholar]
  6. Guisasola J. A., Cockburn C. G., Hardisty R. M. Plasmin digestion of factor VIII: characterization of the breakdown products with respect to antigenicity and von Willebrand activity. Thromb Haemost. 1978 Oct 31;40(2):302–315. [PubMed] [Google Scholar]
  7. Hormia M., Lehto V. P., Virtanen I. Intracellular localization of factor VIII-related antigen and fibronectin in cultured human endothelial cells: evidence for divergent routes of intracellular translocation. Eur J Cell Biol. 1984 Mar;33(2):217–228. [PubMed] [Google Scholar]
  8. Houdijk W. P., de Groot P. G., Nievelstein P. F., Sakariassen K. S., Sixma J. J. Subendothelial proteins and platelet adhesion. von Willebrand factor and fibronectin, not thrombospondin, are involved in platelet adhesion to extracellular matrix of human vascular endothelial cells. Arteriosclerosis. 1986 Jan-Feb;6(1):24–33. doi: 10.1161/01.atv.6.1.24. [DOI] [PubMed] [Google Scholar]
  9. Ill C. R., Engvall E., Ruoslahti E. Adhesion of platelets to laminin in the absence of activation. J Cell Biol. 1984 Dec;99(6):2140–2145. doi: 10.1083/jcb.99.6.2140. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jaffe E. A., Hoyer L. W., Nachman R. L. Synthesis of von Willebrand factor by cultured human endothelial cells. Proc Natl Acad Sci U S A. 1974 May;71(5):1906–1909. doi: 10.1073/pnas.71.5.1906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jaffe E. A., Mosher D. F. Synthesis of fibronectin by cultured human endothelial cells. J Exp Med. 1978 Jun 1;147(6):1779–1791. doi: 10.1084/jem.147.6.1779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jaffe E. A., Nachman R. L., Becker C. G., Minick C. R. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest. 1973 Nov;52(11):2745–2756. doi: 10.1172/JCI107470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Klebe R. J., Bentley K. L., Sasser P. J., Schoen R. C. Elution of fibronectin from collagen with chaotrophic agents. Exp Cell Res. 1980 Nov;130(1):111–117. doi: 10.1016/0014-4827(80)90047-6. [DOI] [PubMed] [Google Scholar]
  14. Laug W. E., Weinblatt M. E., Jones P. A. Endothelial cells degrade extracellular matrix proteins produced in vitro. Thromb Haemost. 1985 Aug 30;54(2):498–502. [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. Lyberg T., Galdal K. S., Evensen S. A., Prydz H. Cellular cooperation in endothelial cell thromboplastin synthesis. Br J Haematol. 1983 Jan;53(1):85–95. doi: 10.1111/j.1365-2141.1983.tb01989.x. [DOI] [PubMed] [Google Scholar]
  18. Madri J. A., Dreyer B., Pitlick F. A., Furthmayr H. The collagenous components of the subendothelium. Correlation of structure and function. Lab Invest. 1980 Oct;43(4):303–315. [PubMed] [Google Scholar]
  19. McPherson J., Sage H., Bornstein P. Isolation and characterization of a glycoprotein secreted by aortic endothelial cells in culture. Apparent identity with platelet thrombospondin. J Biol Chem. 1981 Nov 10;256(21):11330–11336. [PubMed] [Google Scholar]
  20. Mosher D. F., Vaheri A. Thrombin stimulates the production and release of a major surface-associated glycoprotein (fibronectin) in cultures of human fibroblasts. Exp Cell Res. 1978 Mar 15;112(2):323–334. doi: 10.1016/0014-4827(78)90215-x. [DOI] [PubMed] [Google Scholar]
  21. Mumby S. M., Raugi G. J., Bornstein P. Interactions of thrombospondin with extracellular matrix proteins: selective binding to type V collagen. J Cell Biol. 1984 Feb;98(2):646–652. doi: 10.1083/jcb.98.2.646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nawroth P. P., Stern D. M., Kaplan K. L., Nossel H. L. Prostacyclin production by perturbed bovine aortic endothelial cells in culture. Blood. 1984 Oct;64(4):801–806. [PubMed] [Google Scholar]
  23. Reinders J. H., de Groot P. G., Dawes J., Hunter N. R., van Heugten H. A., Zandbergen J., Gonsalves M. D., van Mourik J. A. Comparison of secretion and subcellular localization of von Willebrand protein with that of thrombospondin and fibronectin in cultured human vascular endothelial cells. Biochim Biophys Acta. 1985 Mar 21;844(3):306–313. doi: 10.1016/0167-4889(85)90131-4. [DOI] [PubMed] [Google Scholar]
  24. Ross R., Glomset J. A. The pathogenesis of atherosclerosis (second of two parts). N Engl J Med. 1976 Aug 19;295(8):420–425. doi: 10.1056/NEJM197608192950805. [DOI] [PubMed] [Google Scholar]
  25. Sage H., Balian G., Vogel A. M., Bornstein P. Type VIII collagen. Synthesis by normal and malignant cells in culture. Lab Invest. 1984 Feb;50(2):219–231. [PubMed] [Google Scholar]
  26. Sage H., Crouch E., Bornstein P. Collagen synthesis by bovine aortic endothelial cells in culture. Biochemistry. 1979 Nov 27;18(24):5433–5442. doi: 10.1021/bi00591a028. [DOI] [PubMed] [Google Scholar]
  27. Sakariassen K. S., Aarts P. A., de Groot P. G., Houdijk W. P., Sixma J. J. A perfusion chamber developed to investigate platelet interaction in flowing blood with human vessel wall cells, their extracellular matrix, and purified components. J Lab Clin Med. 1983 Oct;102(4):522–535. [PubMed] [Google Scholar]
  28. Sakariassen K. S., Banga J. D., de Groot P. G., Sixma J. J. Comparison of platelet interaction with subendothelium of human renal and umbilical arteries and the extracellular matrix produced by human venous endothelial cells. Thromb Haemost. 1984 Aug 31;52(1):60–65. [PubMed] [Google Scholar]
  29. Sakariassen K. S., Bolhuis P. A., Sixma J. J. Human blood platelet adhesion to artery subendothelium is mediated by factor VIII-Von Willebrand factor bound to the subendothelium. Nature. 1979 Jun 14;279(5714):636–638. doi: 10.1038/279636a0. [DOI] [PubMed] [Google Scholar]
  30. Sakariassen K. S., Bolhuis P. A., Sixma J. J. Platelet adherence to subendothelium of human arteries in pulsatile and steady flow. 1980 Aug 15-Sep 1Thromb Res. 19(4-5):547–559. doi: 10.1016/0049-3848(80)90027-4. [DOI] [PubMed] [Google Scholar]
  31. Sixma J. J., Sakariassen K. S., Stel H. V., Houdijk W. P., In der Maur D. W., Hamer R. J., de Groot P. G., van Mourik J. A. Functional domains on von Willebrand factor. Recognition of discrete tryptic fragments by monoclonal antibodies that inhibit interaction of von Willebrand factor with platelets and with collagen. J Clin Invest. 1984 Sep;74(3):736–744. doi: 10.1172/JCI111489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sixma J. J., Wester J. The hemostatic plug. Semin Hematol. 1977 Jul;14(3):265–299. [PubMed] [Google Scholar]
  33. Stel H. V., Sakariassen K. S., de Groot P. G., van Mourik J. A., Sixma J. J. Von Willebrand factor in the vessel wall mediates platelet adherence. Blood. 1985 Jan;65(1):85–90. [PubMed] [Google Scholar]
  34. Sussman I. I., Rand J. H. Subendothelial deposition of von Willebrand's factor requires the presence of endothelial cells. J Lab Clin Med. 1982 Oct;100(4):526–532. [PubMed] [Google Scholar]
  35. Vlodavsky I., Eldor A., HyAm E., Atzom R., Fuks Z. Platelet interaction with the extracellular matrix produced by cultured endothelial cells: a model to study the thrombogenicity of isolated subendothelial basal lamina. Thromb Res. 1982 Oct 15;28(2):179–191. doi: 10.1016/0049-3848(82)90260-2. [DOI] [PubMed] [Google Scholar]
  36. Wagner D. D., Olmsted J. B., Marder V. J. Immunolocalization of von Willebrand protein in Weibel-Palade bodies of human endothelial cells. J Cell Biol. 1982 Oct;95(1):355–360. doi: 10.1083/jcb.95.1.355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wagner D. D., Urban-Pickering M., Marder V. J. Von Willebrand protein binds to extracellular matrices independently of collagen. Proc Natl Acad Sci U S A. 1984 Jan;81(2):471–475. doi: 10.1073/pnas.81.2.471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Weiss H. J., Turitto V. T., Baumgartner H. R. Effect of shear rate on platelet interaction with subendothelium in citrated and native blood. I. Shear rate--dependent decrease of adhesion in von Willebrand's disease and the Bernard-Soulier syndrome. J Lab Clin Med. 1978 Nov;92(5):750–764. [PubMed] [Google Scholar]
  39. Willems C., Astaldi G. C., De Groot P. G., Janssen M. C., Gonsalvez M. D., Zeijlemaker W. P., Van Mourik J. A., Van Aken W. G. Media conditioned by cultured human vascular endothelial cells inhibit the growth of vascular smooth muscle cells. Exp Cell Res. 1982 May;139(1):191–197. doi: 10.1016/0014-4827(82)90332-9. [DOI] [PubMed] [Google Scholar]
  40. de Groot P. G., Gonsalves M. D., Loesberg C., van Buul-Wortelboer M. F., van Aken W. G., van Mourik J. A. Thrombin-induced release of von Willebrand factor from endothelial cells is mediated by phospholipid methylation. Prostacyclin synthesis is independent of phospholipid methylation. J Biol Chem. 1984 Nov 10;259(21):13329–13333. [PubMed] [Google Scholar]
  41. van Mourik J. A., Mochtar I. A. Purification of human antihemophilic factor (factor VIII) by gel chromatography. Biochim Biophys Acta. 1970 Dec 22;221(3):677–679. doi: 10.1016/0005-2795(70)90247-3. [DOI] [PubMed] [Google Scholar]

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