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. 1987 Oct 1;105(4):1603–1611. doi: 10.1083/jcb.105.4.1603

Interactions of thrombospondin with endothelial cells: receptor- mediated binding and degradation

PMCID: PMC2114655  PMID: 2444599

Abstract

We studied binding and degradation of labeled platelet thrombospondin (TSP) by normal and variant bovine aorta endothelial (BAE) cells. [125I]-labeled TSP bound to cells at 37 degrees C in a specific, saturable, and time-dependent fashion. Incubation of cell monolayers with fluoresceinated TSP resulted in punctate cellular staining, but no staining of the extracellular matrix. Heparin, fucoidan, chondroitin sulfate, platelet factor 4, beta-thromboglobulin, unlabeled TSP, and serum derived from whole blood all competed for binding of [125I]TSP. [125I]TSP was degraded to TCA-soluble radioactivity, which appeared in the medium after a 60-90-min lag. Degradation was inhibited to the same extent as binding by increasing concentrations of heparin, fucoidan, platelet factor 4, or whole blood serum. Normal BAE cells bound and degraded less [125I]TSP than variant BAE cells. The dissociation constants (Kds) for binding and the constants for degradation (Kms) for degradation by the two cell strains, however, were similar (30-50 nM). The inhibitory effects of heparin and platelet factor 4 were lost when the two inhibitors were present in a 1:1 (wt/wt) ratio. Treatment of suspended cells with trypsin or heparitinase caused less binding of TSP. These results indicate that there is a specific receptor for TSP on endothelial cells which mediates binding and degradation. This receptor may be a heparan sulfate proteoglycan.

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Selected References

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  1. Asch A. S., Barnwell J., Silverstein R. L., Nachman R. L. Isolation of the thrombospondin membrane receptor. J Clin Invest. 1987 Apr;79(4):1054–1061. doi: 10.1172/JCI112918. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Asch A. S., Leung L. L., Shapiro J., Nachman R. L. Human brain glial cells synthesize thrombospondin. Proc Natl Acad Sci U S A. 1986 May;83(9):2904–2908. doi: 10.1073/pnas.83.9.2904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Baenziger N. L., Brodie G. N., Majerus P. W. Isolation and properties of a thrombin-sensitive protein of human platelets. J Biol Chem. 1972 May 10;247(9):2723–2731. [PubMed] [Google Scholar]
  5. Bale M. D., Mosher D. F. Effects of thrombospondin on fibrin polymerization and structure. J Biol Chem. 1986 Jan 15;261(2):862–868. [PubMed] [Google Scholar]
  6. Bale M. D., Westrick L. G., Mosher D. F. Incorporation of thrombospondin into fibrin clots. J Biol Chem. 1985 Jun 25;260(12):7502–7508. [PubMed] [Google Scholar]
  7. Bar R. S., Hoak J. C., Peacock M. L. Insulin receptors in human endothelial cells: identification and characterization. J Clin Endocrinol Metab. 1978 Sep;47(3):699–702. doi: 10.1210/jcem-47-3-699. [DOI] [PubMed] [Google Scholar]
  8. Bar R. S., Peacock M. L., Rechler M. M., Nissley S. P. Receptors for multiplication-stimulating activity on human arterial and venous endothelial cells. J Clin Endocrinol Metab. 1981 Apr;52(4):814–816. doi: 10.1210/jcem-52-4-814. [DOI] [PubMed] [Google Scholar]
  9. Booyse F. M., Sedlak B. J., Rafelson M. E., Jr Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. Thromb Diath Haemorrh. 1975 Dec 15;34(3):825–839. [PubMed] [Google Scholar]
  10. Busch C., Dawes J., Pepper D. S., Wasteson A. Binding of platelet factor 4 to cultured human umbilical vein endothelial cells. Thromb Res. 1980 Jul 1;19(1-2):129–137. doi: 10.1016/0049-3848(80)90412-0. [DOI] [PubMed] [Google Scholar]
  11. Castor C. W., Miller J. W., Walz D. A. Structural and biological characteristics of connective tissue activating peptide (CTAP-III), a major human platelet-derived growth factor. Proc Natl Acad Sci U S A. 1983 Feb;80(3):765–769. doi: 10.1073/pnas.80.3.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Coligan J. E., Slayter H. S. Structure of thrombospondin. J Biol Chem. 1984 Mar 25;259(6):3944–3948. [PubMed] [Google Scholar]
  13. Danishefsky K. J., Alexander R. J., Detwiler T. C. Formation of a stable complex of thrombin and the secreted platelet protein glycoprotein G (thrombin-sensitive protein, thrombospondin) by thiol-disulfide exchange. Biochemistry. 1984 Oct 9;23(21):4984–4990. doi: 10.1021/bi00316a024. [DOI] [PubMed] [Google Scholar]
  14. Dejana E., Languino L. R., Polentarutti N., Balconi G., Ryckewaert J. J., Larrieu M. J., Donati M. B., Mantovani A., Marguerie G. Interaction between fibrinogen and cultured endothelial cells. Induction of migration and specific binding. J Clin Invest. 1985 Jan;75(1):11–18. doi: 10.1172/JCI111661. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dixit V. M., Galvin N. J., O'Rourke K. M., Frazier W. A. Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes. J Biol Chem. 1986 Feb 5;261(4):1962–1968. [PubMed] [Google Scholar]
  16. Dixit V. M., Grant G. A., Frazier W. A., Santoro S. A. Isolation of the fibrinogen-binding region of platelet thrombospondin. Biochem Biophys Res Commun. 1984 Mar 30;119(3):1075–1081. doi: 10.1016/0006-291x(84)90884-2. [DOI] [PubMed] [Google Scholar]
  17. Dixit V. M., Grant G. A., Santoro S. A., Frazier W. A. Isolation and characterization of a heparin-binding domain from the amino terminus of platelet thrombospondin. J Biol Chem. 1984 Aug 25;259(16):10100–10105. [PubMed] [Google Scholar]
  18. Dixit V. M., Haverstick D. M., O'Rourke K. M., Hennessy S. W., Grant G. A., Santoro S. A., Frazier W. A. Effects of anti-thrombospondin monoclonal antibodies on the agglutination of erythrocytes and fixed, activated platelets by purified thrombospondin. Biochemistry. 1985 Jul 30;24(16):4270–4275. doi: 10.1021/bi00337a003. [DOI] [PubMed] [Google Scholar]
  19. Galvin N. J., Dixit V. M., O'Rourke K. M., Santoro S. A., Grant G. A., Frazier W. A. Mapping of epitopes for monoclonal antibodies against human platelet thrombospondin with electron microscopy and high sensitivity amino acid sequencing. J Cell Biol. 1985 Oct;101(4):1434–1441. doi: 10.1083/jcb.101.4.1434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gartner T. K., Dockter M. E. Secreted platelet thrombospondin binds monovalently to platelets and erythrocytes in the absence of free Ca2+. Thromb Res. 1984 Jan 1;33(1):19–30. doi: 10.1016/0049-3848(84)90151-8. [DOI] [PubMed] [Google Scholar]
  21. Gartner T. K., Doyle M. J., Mosher D. F. Effect of anti-thrombospondin antibodies on the hemagglutination activities of the endogenous platelet lectin and thrombospondin. Thromb Haemost. 1984 Dec 29;52(3):354–357. [PubMed] [Google Scholar]
  22. Glimelius B., Busch C., Hök M. Binding of heparin on the surface of cultured human endothelial cells. Thromb Res. 1978 May;12(5):773–782. doi: 10.1016/0049-3848(78)90271-2. [DOI] [PubMed] [Google Scholar]
  23. Gown A. M., Vogel A. M., Gordon D., Lu P. L. A smooth muscle-specific monoclonal antibody recognizes smooth muscle actin isozymes. J Cell Biol. 1985 Mar;100(3):807–813. doi: 10.1083/jcb.100.3.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hope W., Martin T. J., Chesterman C. N., Morgan F. J. Human beta-thromboglobulin inhibits PGI2 production and binds to a specific site in bovine aortic endothelial cells. Nature. 1979 Nov 8;282(5735):210–212. doi: 10.1038/282210a0. [DOI] [PubMed] [Google Scholar]
  25. Hunter N. R., Dawes J., MacGregor I. R., Pepper D. S. Quantitation by radioimmunoassay of thrombospondin synthesised and secreted by human endothelial cells. Thromb Haemost. 1984 Dec 29;52(3):288–291. [PubMed] [Google Scholar]
  26. Jaffe E. A., Ruggiero J. T., Leung L. K., Doyle M. J., McKeown-Longo P. J., Mosher D. F. Cultured human fibroblasts synthesize and secrete thrombospondin and incorporate it into extracellular matrix. Proc Natl Acad Sci U S A. 1983 Feb;80(4):998–1002. doi: 10.1073/pnas.80.4.998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kramer R. H., Fuh G. M., Bensch K. G., Karasek M. A. Synthesis of extracellular matrix glycoproteins by cultured microvascular endothelial cells isolated from the dermis of neonatal and adult skin. J Cell Physiol. 1985 Apr;123(1):1–9. doi: 10.1002/jcp.1041230102. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Lahav J., Lawler J., Gimbrone M. A. Thrombospondin interactions with fibronectin and fibrinogen. Mutual inhibition in binding. Eur J Biochem. 1984 Nov 15;145(1):151–156. doi: 10.1111/j.1432-1033.1984.tb08534.x. [DOI] [PubMed] [Google Scholar]
  30. Lahav J., Schwartz M. A., Hynes R. O. Analysis of platelet adhesion with a radioactive chemical crosslinking reagent: interaction of thrombospondin with fibronectin and collagen. Cell. 1982 Nov;31(1):253–262. doi: 10.1016/0092-8674(82)90425-1. [DOI] [PubMed] [Google Scholar]
  31. Lawler J. W., Slayter H. S., Coligan J. E. Isolation and characterization of a high molecular weight glycoprotein from human blood platelets. J Biol Chem. 1978 Dec 10;253(23):8609–8616. [PubMed] [Google Scholar]
  32. Lawler J., Derick L. H., Connolly J. E., Chen J. H., Chao F. C. The structure of human platelet thrombospondin. J Biol Chem. 1985 Mar 25;260(6):3762–3772. [PubMed] [Google Scholar]
  33. Lawler J., Hynes R. O. The structure of human thrombospondin, an adhesive glycoprotein with multiple calcium-binding sites and homologies with several different proteins. J Cell Biol. 1986 Nov;103(5):1635–1648. doi: 10.1083/jcb.103.5.1635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Leung L. L., Nachman R. L. Complex formation of platelet thrombospondin with fibrinogen. J Clin Invest. 1982 Sep;70(3):542–549. doi: 10.1172/JCI110646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Leung L. L., Nachman R. L., Harpel P. C. Complex formation of platelet thrombospondin with histidine-rich glycoprotein. J Clin Invest. 1984 Jan;73(1):5–12. doi: 10.1172/JCI111206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Majack R. A., Cook S. C., Bornstein P. Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9050–9054. doi: 10.1073/pnas.83.23.9050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Majack R. A., Cook S. C., Bornstein P. Platelet-derived growth factor and heparin-like glycosaminoglycans regulate thrombospondin synthesis and deposition in the matrix by smooth muscle cells. J Cell Biol. 1985 Sep;101(3):1059–1070. doi: 10.1083/jcb.101.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Marcum J. A., Atha D. H., Fritze L. M., Nawroth P., Stern D., Rosenberg R. D. Cloned bovine aortic endothelial cells synthesize anticoagulantly active heparan sulfate proteoglycan. J Biol Chem. 1986 Jun 5;261(16):7507–7517. [PubMed] [Google Scholar]
  39. Margossian S. S., Lawler J. W., Slayter H. S. Physical characterization of platelet thrombospondin. J Biol Chem. 1981 Jul 25;256(14):7495–7500. [PubMed] [Google Scholar]
  40. McKeown-Longo P. J., Hanning R., Mosher D. F. Binding and degradation of platelet thrombospondin by cultured fibroblasts. J Cell Biol. 1984 Jan;98(1):22–28. doi: 10.1083/jcb.98.1.22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. McLaren K. M. Immunohistochemical localisation of thrombospondin in human megakaryocytes and platelets. J Clin Pathol. 1983 Feb;36(2):197–199. doi: 10.1136/jcp.36.2.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. 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]
  43. Mosher D. F. Cross-linking of cold-insoluble globulin by fibrin-stabilizing factor. J Biol Chem. 1975 Aug 25;250(16):6614–6621. [PubMed] [Google Scholar]
  44. Mosher D. F., Doyle M. J., Jaffe E. A. Synthesis and secretion of thrombospondin by cultured human endothelial cells. J Cell Biol. 1982 May;93(2):343–348. doi: 10.1083/jcb.93.2.343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Mosher D. F., Johnson R. B. In vitro formation of disulfide-bonded fibronectin multimers. J Biol Chem. 1983 May 25;258(10):6595–6601. [PubMed] [Google Scholar]
  46. Mumby S. M., Abbott-Brown D., Raugi G. J., Bornstein P. Regulation of thrombospondin secretion by cells in culture. J Cell Physiol. 1984 Sep;120(3):280–288. doi: 10.1002/jcp.1041200304. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Murphy-Ullrich J. E., Mosher D. F. Localization of thrombospondin in clots formed in situ. Blood. 1985 Nov;66(5):1098–1104. [PubMed] [Google Scholar]
  49. Niewiarowski S., Walz D. A., James P., Rucinski B., Kueppers F. Identification and separation of secreted platelet proteins by isoelectric focusing. Evidence that low-affinity platelet factor 4 is converted to beta-thromboglobulin by limited proteolysis. Blood. 1980 Mar;55(3):453–456. [PubMed] [Google Scholar]
  50. Oldberg A., Heldin C. H., Wasteson A., Busch C., Hök M. Characterization of a platelet endoglycosidase degrading heparin-like polysaccharides. Biochemistry. 1980 Dec 9;19(25):5755–5762. doi: 10.1021/bi00566a014. [DOI] [PubMed] [Google Scholar]
  51. Phillips D. R., Jennings L. K., Prasanna H. R. Ca2+-mediated association of glycoprotein G (thrombinsensitive protein, thrombospondin) with human platelets. J Biol Chem. 1980 Dec 25;255(24):11629–11632. [PubMed] [Google Scholar]
  52. Rapraeger A., Bernfield M. Cell surface proteoglycan of mammary epithelial cells. Protease releases a heparan sulfate-rich ectodomain from a putative membrane-anchored domain. J Biol Chem. 1985 Apr 10;260(7):4103–4109. [PubMed] [Google Scholar]
  53. Raugi G. J., Mumby S. M., Abbott-Brown D., Bornstein P. Thrombospondin: synthesis and secretion by cells in culture. J Cell Biol. 1982 Oct;95(1):351–354. doi: 10.1083/jcb.95.1.351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Roberts D. D., Haverstick D. M., Dixit V. M., Frazier W. A., Santoro S. A., Ginsburg V. The platelet glycoprotein thrombospondin binds specifically to sulfated glycolipids. J Biol Chem. 1985 Aug 5;260(16):9405–9411. [PubMed] [Google Scholar]
  55. Roberts D. D., Rao C. N., Liotta L. A., Gralnick H. R., Ginsburg V. Comparison of the specificities of laminin, thrombospondin, and von Willebrand factor for binding to sulfated glycolipids. J Biol Chem. 1986 May 25;261(15):6872–6877. [PubMed] [Google Scholar]
  56. Schwartz S. M. Selection and characterization of bovine aortic endothelial cells. In Vitro. 1978 Dec;14(12):966–980. doi: 10.1007/BF02616210. [DOI] [PubMed] [Google Scholar]
  57. Silverstein R. L., Leung L. L., Harpel P. C., Nachman R. L. Complex formation of platelet thrombospondin with plasminogen. Modulation of activation by tissue activator. J Clin Invest. 1984 Nov;74(5):1625–1633. doi: 10.1172/JCI111578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Switalska H. I., Niewiarowski S., Tuszynski G. P., Rucinski B., Schmaier A. H., Morinelli T. A., Cierniewski C. S. Radioimmunoassay of human platelet thrombospondin: different patterns of thrombospondin and beta-thromboglobulin antigen secretion and clearance from the circulation. J Lab Clin Med. 1985 Dec;106(6):690–700. [PubMed] [Google Scholar]
  59. Voyta J. C., Via D. P., Butterfield C. E., Zetter B. R. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol. 1984 Dec;99(6):2034–2040. doi: 10.1083/jcb.99.6.2034. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wencel-Drake J. D., Plow E. F., Zimmerman T. S., Painter R. G., Ginsberg M. H. Immunofluorescent localization of adhesive glycoproteins in resting and thrombin-stimulated platelets. Am J Pathol. 1984 May;115(2):156–164. [PMC free article] [PubMed] [Google Scholar]
  61. Wight T. N., Raugi G. J., Mumby S. M., Bornstein P. Light microscopic immunolocation of thrombospondin in human tissues. J Histochem Cytochem. 1985 Apr;33(4):295–302. doi: 10.1177/33.4.3884704. [DOI] [PubMed] [Google Scholar]
  62. Wolff R., Plow E. F., Ginsberg M. H. Interaction of thrombospondin with resting and stimulated human platelets. J Biol Chem. 1986 May 25;261(15):6840–6846. [PubMed] [Google Scholar]

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