Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1991 Jan 2;112(2):335–344. doi: 10.1083/jcb.112.2.335

An Arg-Gly-Asp sequence within thrombin promotes endothelial cell adhesion

PMCID: PMC2288825  PMID: 1988465

Abstract

Thrombin, in addition to its central role in hemostasis, possesses diverse cellular bioregulatory functions implicated in wound healing, inflammation, and atherosclerosis. In the present study we demonstrate that thrombin molecules modified either at the procoagulant or catalytic sites induce endothelial cell (EC) adhesion, spreading, and cytoskeletal reorganization. The most potent adhesive thrombin analogue (NO2-alpha-thrombin) was obtained by nitration of tyrosine residues. The cell adhesion promoting activity of NO2-alpha-thrombin was blocked upon the formation of thrombin-antithrombin III (ATIII) complexes and by antiprothrombin antibodies, but was unaffected by hirudin. Arg-Gly- Asp-containing peptides, fully inhibited EC adhesion to NO2-alpha- thrombin, while synthetic peptides corresponding to thrombin "Loop B" mitogenic site and the thrombin-derived chemotactic fragment "CB67- 129", were uneffective. Immunofluorescence studies indicated that EC adhesion to NO2-alpha-thrombin was followed by cell spreading, actin microfilament assembly, and formation of focal contacts. By the use of specific antibodies, the vitronectin (vn) receptor (alpha v beta 3) was found to be localized in clusters upon cell adhesion to NO2-alpha- thrombin. An anti alpha v beta 3 antibody blocked EC adhesion and spreading while antifibronectin (fn) receptor (alpha 5 beta 1) antibodies were uneffective. While native thrombin exhibited a very low cell attachment activity, thrombin that was incubated at 37 degrees C before coating of plastic surfaces induced EC attachment and spreading. We propose that under certain conditions the naturally hindered RGD domain within thrombin is exposed for interaction with alpha v beta 3 on EC. This in turn promotes cell adhesion, spreading, and reorganization of cytoskeletal elements, which may altogether contribute to repair mechanisms in the disturbed vessel wall. This study defines a new biological role of thrombin and characterizes a new recognition mechanism on EC for this molecule.

Full Text

The Full Text of this article is available as a PDF (2.8 MB).

Selected References

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

  1. Bagdy D., Barabas E., Gráf L., Petersen T. E., Magnusson S. Hirudin. Methods Enzymol. 1976;45:669–678. doi: 10.1016/s0076-6879(76)45057-7. [DOI] [PubMed] [Google Scholar]
  2. Bar-Shavit R., Benezra M., Eldor A., Hy-Am E., Fenton J. W., 2nd, Wilner G. D., Vlodavsky I. Thrombin immobilized to extracellular matrix is a potent mitogen for vascular smooth muscle cells: nonenzymatic mode of action. Cell Regul. 1990 May;1(6):453–463. doi: 10.1091/mbc.1.6.453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bar-Shavit R., Eldor A., Vlodavsky I. Binding of thrombin to subendothelial extracellular matrix. Protection and expression of functional properties. J Clin Invest. 1989 Oct;84(4):1096–1104. doi: 10.1172/JCI114272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bar-Shavit R., Kahn A. J., Mann K. G., Wilner G. D. Identification of a thrombin sequence with growth factor activity on macrophages. Proc Natl Acad Sci U S A. 1986 Feb;83(4):976–980. doi: 10.1073/pnas.83.4.976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bar-Shavit R., Kahn A., Wilner G. D., Fenton J. W., 2nd Monocyte chemotaxis: stimulation by specific exosite region in thrombin. Science. 1983 May 13;220(4598):728–731. doi: 10.1126/science.6836310. [DOI] [PubMed] [Google Scholar]
  6. Barbieri B., Balconi G., Dejana E., Donati M. B. Evidence that vascular endothelial cells can induce the retraction of fibrin clots. Proc Soc Exp Biol Med. 1981 Nov;168(2):204–207. doi: 10.3181/00379727-168-41260. [DOI] [PubMed] [Google Scholar]
  7. Bennett J. S., Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest. 1979 Nov;64(5):1393–1401. doi: 10.1172/JCI109597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bode W., Mayr I., Baumann U., Huber R., Stone S. R., Hofsteenge J. The refined 1.9 A crystal structure of human alpha-thrombin: interaction with D-Phe-Pro-Arg chloromethylketone and significance of the Tyr-Pro-Pro-Trp insertion segment. EMBO J. 1989 Nov;8(11):3467–3475. doi: 10.1002/j.1460-2075.1989.tb08511.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Carney D. H., Cunningham D. D. Cell surface action of thrombin is sufficient to initiate division of chick cells. Cell. 1978 Aug;14(4):811–823. doi: 10.1016/0092-8674(78)90337-9. [DOI] [PubMed] [Google Scholar]
  10. Charo I. F., Bekeart L. S., Phillips D. R. Platelet glycoprotein IIb-IIIa-like proteins mediate endothelial cell attachment to adhesive proteins and the extracellular matrix. J Biol Chem. 1987 Jul 25;262(21):9935–9938. [PubMed] [Google Scholar]
  11. Cheresh D. A. Human endothelial cells synthesize and express an Arg-Gly-Asp-directed adhesion receptor involved in attachment to fibrinogen and von Willebrand factor. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6471–6475. doi: 10.1073/pnas.84.18.6471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Conforti G., Zanetti A., Colella S., Abbadini M., Marchisio P. C., Pytela R., Giancotti F., Tarone G., Languino L. R., Dejana E. Interaction of fibronectin with cultured human endothelial cells: characterization of the specific receptor. Blood. 1989 May 1;73(6):1576–1585. [PubMed] [Google Scholar]
  13. Dejana E., Bertocchi F., Bortolami M. C., Regonesi A., Tonta A., Breviario F., Giavazzi R. Interleukin 1 promotes tumor cell adhesion to cultured human endothelial cells. J Clin Invest. 1988 Oct;82(4):1466–1470. doi: 10.1172/JCI113753. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dejana E., Colella S., Conforti G., Abbadini M., Gaboli M., Marchisio P. C. Fibronectin and vitronectin regulate the organization of their respective Arg-Gly-Asp adhesion receptors in cultured human endothelial cells. J Cell Biol. 1988 Sep;107(3):1215–1223. doi: 10.1083/jcb.107.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dejana E., Colella S., Languino L. R., Balconi G., Corbascio G. C., Marchisio P. C. Fibrinogen induces adhesion, spreading, and microfilament organization of human endothelial cells in vitro. J Cell Biol. 1987 May;104(5):1403–1411. doi: 10.1083/jcb.104.5.1403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dejana E., Lampugnani M. G., Giorgi M., Gaboli M., Federici A. B., Ruggeri Z. M., Marchisio P. C. Von Willebrand factor promotes endothelial cell adhesion via an Arg-Gly-Asp-dependent mechanism. J Cell Biol. 1989 Jul;109(1):367–375. doi: 10.1083/jcb.109.1.367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dejana E., Lampugnani M. G., Giorgi M., Gaboli M., Marchisio P. C. Fibrinogen induces endothelial cell adhesion and spreading via the release of endogenous matrix proteins and the recruitment of more than one integrin receptor. Blood. 1990 Apr 1;75(7):1509–1517. [PubMed] [Google Scholar]
  18. Dejana E., Languino L. R., Colella S., Corbascio G. C., Plow E., Ginsberg M., Marchisio P. C. The localization of a platelet GpIIb-IIIa-related protein in endothelial cell adhesion structures. Blood. 1988 Mar;71(3):566–572. [PubMed] [Google Scholar]
  19. Engvall E., Ruoslahti E. Binding of soluble form of fibroblast surface protein, fibronectin, to collagen. Int J Cancer. 1977 Jul 15;20(1):1–5. doi: 10.1002/ijc.2910200102. [DOI] [PubMed] [Google Scholar]
  20. Fenton J. W., 2nd, Bing D. H. Thrombin active-site regions. Semin Thromb Hemost. 1986 Jul;12(3):200–208. doi: 10.1055/s-2007-1003551. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Fitzgerald L. A., Charo I. F., Phillips D. R. Human and bovine endothelial cells synthesize membrane proteins similar to human platelet glycoproteins IIb and IIIa. J Biol Chem. 1985 Sep 15;260(20):10893–10896. [PubMed] [Google Scholar]
  23. Fridman R., Alon Y., Doljanski F., Fuks Z., Vlodavsky I. Cell interaction with the extracellular matrices produced by endothelial cells and fibroblasts. Exp Cell Res. 1985 Jun;158(2):461–476. doi: 10.1016/0014-4827(85)90469-0. [DOI] [PubMed] [Google Scholar]
  24. Furie B., Bing D. H., Feldmann R. J., Robison D. J., Burnier J. P., Furie B. C. Computer-generated models of blood coagulation factor Xa, factor IXa, and thrombin based upon structural homology with other serine proteases. J Biol Chem. 1982 Apr 10;257(7):3875–3882. [PubMed] [Google Scholar]
  25. Glenn K. C., Carney D. H., Fenton J. W., 2nd, Cunningham D. D. Thrombin active site regions required for fibroblast receptor binding and initiation of cell division. J Biol Chem. 1980 Jul 25;255(14):6609–6616. [PubMed] [Google Scholar]
  26. Goldfarb R. H., Liotta L. A. Thrombin cleavage of extracellular matrix proteins. Ann N Y Acad Sci. 1986;485:288–292. doi: 10.1111/j.1749-6632.1986.tb34590.x. [DOI] [PubMed] [Google Scholar]
  27. Goldman R., Bar-Shavit Z. Dual effect of normal and stimulated macrophages and their conditioned media on target cell proliferation. J Natl Cancer Inst. 1979 Oct;63(4):1009–1016. [PubMed] [Google Scholar]
  28. Gospodarowicz D., Moran J., Braun D., Birdwell C. Clonal growth of bovine vascular endothelial cells: fibroblast growth factor as a survival agent. Proc Natl Acad Sci U S A. 1976 Nov;73(11):4120–4124. doi: 10.1073/pnas.73.11.4120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. MCLEAN J. R., MAXWELL R. E., HERTLER D. FIBRINOGEN AND ADENOSINE DIPHOSPHATE-INDUCED AGGREGATION OF PLATELETS. Nature. 1964 May 9;202:605–606. doi: 10.1038/202605a0. [DOI] [PubMed] [Google Scholar]
  30. Marguerie G. A., Plow E. F., Edgington T. S. Human platelets possess an inducible and saturable receptor specific for fibrinogen. J Biol Chem. 1979 Jun 25;254(12):5357–5363. [PubMed] [Google Scholar]
  31. Perdue J. F., Lubenskyi W., Kivity E., Sonder S. A., Fenton J. W., 2nd Protease mitogenic response of chick embryo fibroblasts and receptor binding/processing of human alpha-thrombin. J Biol Chem. 1981 Mar 25;256(6):2767–2776. [PubMed] [Google Scholar]
  32. Plow E. F., Loftus J. C., Levin E. G., Fair D. S., Dixon D., Forsyth J., Ginsberg M. H. Immunologic relationship between platelet membrane glycoprotein GPIIb/IIIa and cell surface molecules expressed by a variety of cells. Proc Natl Acad Sci U S A. 1986 Aug;83(16):6002–6006. doi: 10.1073/pnas.83.16.6002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Roman J., LaChance R. M., Broekelmann T. J., Kennedy C. J., Wayner E. A., Carter W. G., McDonald J. A. The fibronectin receptor is organized by extracellular matrix fibronectin: implications for oncogenic transformation and for cell recognition of fibronectin matrices. J Cell Biol. 1989 Jun;108(6):2529–2543. doi: 10.1083/jcb.108.6.2529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Suzuki S., Argraves W. S., Arai H., Languino L. R., Pierschbacher M. D., Ruoslahti E. Amino acid sequence of the vitronectin receptor alpha subunit and comparative expression of adhesion receptor mRNAs. J Biol Chem. 1987 Oct 15;262(29):14080–14085. [PubMed] [Google Scholar]
  35. Uchida N., Smilowitz H., Tanzer M. L. Monovalent ionophores inhibit secretion of procollagen and fibronectin from cultured human fibroblasts. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1868–1872. doi: 10.1073/pnas.76.4.1868. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Weiss H. J., Rogers J. Fibrinogen and platelets in the primary arrest of bleeding. Studies in two patients with congenital afibrinogenemia. N Engl J Med. 1971 Aug 12;285(7):369–374. doi: 10.1056/NEJM197108122850703. [DOI] [PubMed] [Google Scholar]
  37. Yatohgo T., Izumi M., Kashiwagi H., Hayashi M. Novel purification of vitronectin from human plasma by heparin affinity chromatography. Cell Struct Funct. 1988 Aug;13(4):281–292. doi: 10.1247/csf.13.281. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES