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. 1995 Oct 15;311(Pt 2):461–469. doi: 10.1042/bj3110461

Identification of a heparin-binding protein using monoclonal antibodies that block heparin binding to porcine aortic endothelial cells.

W A Patton 2nd 1, C A Granzow 1, L A Getts 1, S C Thomas 1, L M Zotter 1, K A Gunzel 1, L J Lowe-Krentz 1
PMCID: PMC1136022  PMID: 7487882

Abstract

The binding of heparin or heparan sulphate to a variety of cell types results in specific changes in cell function. Endothelial cells treated with heparin alter their synthesis of heparan sulphate proteoglycans and extracellular matrix proteins. In order to identify a putative endothelial cell heparin receptor that could be involved in heparin signalling, anti-(endothelial cell) monoclonal antibodies that significantly inhibit heparin binding to endothelial cells were prepared. Four of these antibodies were employed in affinity-chromatographic isolation of a heparin-binding protein from detergent-solubilized endothelial cells. The heparin-binding protein isolated from porcine aortic endothelial cells using four different monoclonal antibodies has an M(r) of 45,000 assessed by SDS/PAGE. The 45,000-M(r) heparin-binding polypeptide is isolated as a multimer. The antibody-isolated protein binds to heparin-affinity columns as does the pure 45,000-M(r) polypeptide, consistent with its identification as a putative endothelial heparin receptor.

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Selected References

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  1. Au Y. P., Kenagy R. D., Clowes A. W. Heparin selectively inhibits the transcription of tissue-type plasminogen activator in primate arterial smooth muscle cells during mitogenesis. J Biol Chem. 1992 Feb 15;267(5):3438–3444. [PubMed] [Google Scholar]
  2. Benitz W. E., Kelley R. T., Anderson C. M., Lorant D. E., Bernfield M. Endothelial heparan sulfate proteoglycan. I. Inhibitory effects on smooth muscle cell proliferation. Am J Respir Cell Mol Biol. 1990 Jan;2(1):13–24. doi: 10.1165/ajrcmb/2.1.13. [DOI] [PubMed] [Google Scholar]
  3. Berenson G. S., Radhakrishnamurthy B., Srinivasan S. R., Vijayagopal P., Dalferes E. R., Jr Arterial wall injury and proteoglycan changes in atherosclerosis. Arch Pathol Lab Med. 1988 Oct;112(10):1002–1010. [PubMed] [Google Scholar]
  4. Busch S. J., Martin G. A., Barnhart R. L., Mano M., Cardin A. D., Jackson R. L. Trans-repressor activity of nuclear glycosaminoglycans on Fos and Jun/AP-1 oncoprotein-mediated transcription. J Cell Biol. 1992 Jan;116(1):31–42. doi: 10.1083/jcb.116.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bârzu T., Molho P., Tobelem G., Petitou M., Caen J. Binding and endocytosis of heparin by human endothelial cells in culture. Biochim Biophys Acta. 1985 May 30;845(2):196–203. doi: 10.1016/0167-4889(85)90177-6. [DOI] [PubMed] [Google Scholar]
  6. Bârzu T., Van Rijn J. L., Petitou M., Molho P., Tobelem G., Caen J. P. Endothelial binding sites for heparin. Specificity and role in heparin neutralization. Biochem J. 1986 Sep 15;238(3):847–854. doi: 10.1042/bj2380847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Castellot J. J., Jr, Addonizio M. L., Rosenberg R., Karnovsky M. J. Cultured endothelial cells produce a heparinlike inhibitor of smooth muscle cell growth. J Cell Biol. 1981 Aug;90(2):372–379. doi: 10.1083/jcb.90.2.372. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Castellot J. J., Jr, Wong K., Herman B., Hoover R. L., Albertini D. F., Wright T. C., Caleb B. L., Karnovsky M. J. Binding and internalization of heparin by vascular smooth muscle cells. J Cell Physiol. 1985 Jul;124(1):13–20. doi: 10.1002/jcp.1041240104. [DOI] [PubMed] [Google Scholar]
  9. Cheng C. F., Oosta G. M., Bensadoun A., Rosenberg R. D. Binding of lipoprotein lipase to endothelial cells in culture. J Biol Chem. 1981 Dec 25;256(24):12893–12898. [PubMed] [Google Scholar]
  10. Cohen M. P., Surma M. L. Effect of diabetes on in vivo metabolism of [35S]-labeled glomerular basement membrane. Diabetes. 1984 Jan;33(1):8–12. doi: 10.2337/diab.33.1.8. [DOI] [PubMed] [Google Scholar]
  11. Colburn P., Buonassisi V. Anti-clotting activity of endothelial cell cultures and heparan sulfate proteoglycans. Biochem Biophys Res Commun. 1982 Jan 15;104(1):220–227. doi: 10.1016/0006-291x(82)91962-3. [DOI] [PubMed] [Google Scholar]
  12. Damon D. H., Lobb R. R., D'Amore P. A., Wagner J. A. Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life. J Cell Physiol. 1989 Feb;138(2):221–226. doi: 10.1002/jcp.1041380202. [DOI] [PubMed] [Google Scholar]
  13. DeLisser H. M., Yan H. C., Newman P. J., Muller W. A., Buck C. A., Albelda S. M. Platelet/endothelial cell adhesion molecule-1 (CD31)-mediated cellular aggregation involves cell surface glycosaminoglycans. J Biol Chem. 1993 Jul 25;268(21):16037–16046. [PubMed] [Google Scholar]
  14. Fedarko N. S., Conrad H. E. A unique heparan sulfate in the nuclei of hepatocytes: structural changes with the growth state of the cells. J Cell Biol. 1986 Feb;102(2):587–599. doi: 10.1083/jcb.102.2.587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Gordon P. B., Choi H. U., Conn G., Ahmed A., Ehrmann B., Rosenberg L., Hatcher V. B. Extracellular matrix heparan sulfate proteoglycans modulate the mitogenic capacity of acidic fibroblast growth factor. J Cell Physiol. 1989 Sep;140(3):584–592. doi: 10.1002/jcp.1041400325. [DOI] [PubMed] [Google Scholar]
  17. Imamura T., Mitsui Y. Heparan sulfate and heparin as a potentiator or a suppressor of growth of normal and transformed vascular endothelial cells. Exp Cell Res. 1987 Sep;172(1):92–100. doi: 10.1016/0014-4827(87)90096-6. [DOI] [PubMed] [Google Scholar]
  18. Ishihara M., Fedarko N. S., Conrad H. E. Transport of heparan sulfate into the nuclei of hepatocytes. J Biol Chem. 1986 Oct 15;261(29):13575–13580. [PubMed] [Google Scholar]
  19. Jackson R. L., Busch S. J., Cardin A. D. Glycosaminoglycans: molecular properties, protein interactions, and role in physiological processes. Physiol Rev. 1991 Apr;71(2):481–539. doi: 10.1152/physrev.1991.71.2.481. [DOI] [PubMed] [Google Scholar]
  20. Kanwar Y. S., Rosenzweig L. J., Linker A., Jakubowski M. L. Decreased de novo synthesis of glomerular proteoglycans in diabetes: biochemical and autoradiographic evidence. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2272–2275. doi: 10.1073/pnas.80.8.2272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kiefer M. C., Stephans J. C., Crawford K., Okino K., Barr P. J. Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor. Proc Natl Acad Sci U S A. 1990 Sep;87(18):6985–6989. doi: 10.1073/pnas.87.18.6985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kinsella M. G., Wight T. N. Modulation of sulfated proteoglycan synthesis by bovine aortic endothelial cells during migration. J Cell Biol. 1986 Mar;102(3):679–687. doi: 10.1083/jcb.102.3.679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kjellén L., Bielefeld D., Hook M. Reduced sulfation of liver heparan sulfate in experimentally diabetic rats. Diabetes. 1983 Apr;32(4):337–342. doi: 10.2337/diab.32.4.337. [DOI] [PubMed] [Google Scholar]
  24. Kjellén L., Lindahl U. Proteoglycans: structures and interactions. Annu Rev Biochem. 1991;60:443–475. doi: 10.1146/annurev.bi.60.070191.002303. [DOI] [PubMed] [Google Scholar]
  25. Klagsbrun M. The affinity of fibroblast growth factors (FGFs) for heparin; FGF-heparan sulfate interactions in cells and extracellular matrix. Curr Opin Cell Biol. 1990 Oct;2(5):857–863. doi: 10.1016/0955-0674(90)90084-r. [DOI] [PubMed] [Google Scholar]
  26. Kojima T., Shworak N. W., Rosenberg R. D. Molecular cloning and expression of two distinct cDNA-encoding heparan sulfate proteoglycan core proteins from a rat endothelial cell line. J Biol Chem. 1992 Mar 5;267(7):4870–4877. [PubMed] [Google Scholar]
  27. 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]
  28. Lisanti M. P., Le Bivic A., Sargiacomo M., Rodriguez-Boulan E. Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery. J Cell Biol. 1989 Nov;109(5):2117–2127. doi: 10.1083/jcb.109.5.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lowe-Krentz L. J., Joyce J. G. Venous and aortic porcine endothelial cells cultured under standardized conditions synthesize heparan sulfate chains which differ in charge. Anal Biochem. 1991 Mar 2;193(2):155–163. doi: 10.1016/0003-2697(91)90001-a. [DOI] [PubMed] [Google Scholar]
  30. Lowe-Krentz L. J., Thompson K., Patton W. A., 2nd Heparin releasable and nonreleasable forms of heparan sulfate proteoglycan are found on the surfaces of cultured porcine aortic endothelial cells. Mol Cell Biochem. 1992 Jan 15;109(1):51–60. doi: 10.1007/BF00230873. [DOI] [PubMed] [Google Scholar]
  31. Lyons-Giordano B., Brinker J. M., Kefalides N. A. The effect of heparin on fibronectin and thrombospondin synthesis and mRNA levels in cultured human endothelial cells. Exp Cell Res. 1990 Jan;186(1):39–46. doi: 10.1016/0014-4827(90)90207-q. [DOI] [PubMed] [Google Scholar]
  32. López-Casillas F., Cheifetz S., Doody J., Andres J. L., Lane W. S., Massagué J. Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-beta receptor system. Cell. 1991 Nov 15;67(4):785–795. doi: 10.1016/0092-8674(91)90073-8. [DOI] [PubMed] [Google Scholar]
  33. Majack R. A., Bornstein P. Heparin regulates the collagen phenotype of vascular smooth muscle cells: induced synthesis of an Mr 60,000 collagen. J Cell Biol. 1985 Feb;100(2):613–619. doi: 10.1083/jcb.100.2.613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Marcum J. A., Rosenberg R. D. Heparinlike molecules with anticoagulant activity are synthesized by cultured endothelial cells. Biochem Biophys Res Commun. 1985 Jan 16;126(1):365–372. doi: 10.1016/0006-291x(85)90615-1. [DOI] [PubMed] [Google Scholar]
  36. Mertens G., Cassiman J. J., Van den Berghe H., Vermylen J., David G. Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties. J Biol Chem. 1992 Oct 5;267(28):20435–20443. [PubMed] [Google Scholar]
  37. Morrison P., Lowe-Krentz L. J. Heparin induces changes in the synthesis of porcine aortic endothelial cell heparan sulfate proteoglycans. Exp Cell Res. 1989 Oct;184(2):304–315. doi: 10.1016/0014-4827(89)90330-3. [DOI] [PubMed] [Google Scholar]
  38. Nader H. B., Buonassisi V., Colburn P., Dietrich C. P. Heparin stimulates the synthesis and modifies the sulfation pattern of heparan sulfate proteoglycan from endothelial cells. J Cell Physiol. 1989 Aug;140(2):305–310. doi: 10.1002/jcp.1041400216. [DOI] [PubMed] [Google Scholar]
  39. Nader H. B., Toma L., Pinhal M. A., Buonassisi V., Colburn P., Dietrich C. P. Effect of heparin and dextran sulfate on the synthesis and structure of heparan sulfate from cultured endothelial cells. Semin Thromb Hemost. 1991;17 (Suppl 1):47–56. [PubMed] [Google Scholar]
  40. Parthasarathy N., Spiro R. G. Effect of diabetes on the glycosaminoglycan component of the human glomerular basement membrane. Diabetes. 1982 Aug;31(8 Pt 1):738–741. doi: 10.2337/diab.31.8.738. [DOI] [PubMed] [Google Scholar]
  41. Pirrotta V., Bickle T. A. General purification schemes for restriction endonucleases. Methods Enzymol. 1980;65(1):89–95. doi: 10.1016/s0076-6879(80)65013-7. [DOI] [PubMed] [Google Scholar]
  42. Pukac L. A., Castellot J. J., Jr, Wright T. C., Jr, Caleb B. L., Karnovsky M. J. Heparin inhibits c-fos and c-myc mRNA expression in vascular smooth muscle cells. Cell Regul. 1990 Apr;1(5):435–443. doi: 10.1091/mbc.1.5.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pukac L. A., Ottlinger M. E., Karnovsky M. J. Heparin suppresses specific second messenger pathways for protooncogene expression in rat vascular smooth muscle cells. J Biol Chem. 1992 Feb 25;267(6):3707–3711. [PubMed] [Google Scholar]
  44. Raboudi N., Julian J., Rohde L. H., Carson D. D. Identification of cell-surface heparin/heparan sulfate-binding proteins of a human uterine epithelial cell line (RL95). J Biol Chem. 1992 Jun 15;267(17):11930–11939. [PubMed] [Google Scholar]
  45. Rapraeger A. C., Krufka A., Olwin B. B. Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science. 1991 Jun 21;252(5013):1705–1708. doi: 10.1126/science.1646484. [DOI] [PubMed] [Google Scholar]
  46. Robinson J., Gospodarowicz D. Effect of p-nitrophenyl-beta-D-xyloside on proteoglycan synthesis and extracellular matrix formation by bovine corneal endothelial cell cultures. J Biol Chem. 1984 Mar 25;259(6):3818–3824. [PubMed] [Google Scholar]
  47. Robinson J., Gospodarowicz D. Glycosaminoglycans synthesized by cultured bovine corneal endothelial cells. J Cell Physiol. 1983 Dec;117(3):368–376. doi: 10.1002/jcp.1041170312. [DOI] [PubMed] [Google Scholar]
  48. Rohrbach D. H., Hassell J. R., Kleinman H. K., Martin G. R. Alterations in the basement membrane (heparan sulfate) proteoglycan in diabetic mice. Diabetes. 1982 Feb;31(2):185–188. doi: 10.2337/diab.31.2.185. [DOI] [PubMed] [Google Scholar]
  49. Sakaguchi K., Yanagishita M., Takeuchi Y., Aurbach G. D. Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line. J Biol Chem. 1991 Apr 15;266(11):7270–7278. [PubMed] [Google Scholar]
  50. Saxena U., Klein M. G., Goldberg I. J. Identification and characterization of the endothelial cell surface lipoprotein lipase receptor. J Biol Chem. 1991 Sep 15;266(26):17516–17521. [PubMed] [Google Scholar]
  51. Saxena U., Klein M. G., Goldberg I. J. Transport of lipoprotein lipase across endothelial cells. Proc Natl Acad Sci U S A. 1991 Mar 15;88(6):2254–2258. doi: 10.1073/pnas.88.6.2254. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Shimada K., Gill P. J., Silbert J. E., Douglas W. H., Fanburg B. L. Involvement of cell surface heparin sulfate in the binding of lipoprotein lipase to cultured bovine endothelial cells. J Clin Invest. 1981 Oct;68(4):995–1002. doi: 10.1172/JCI110354. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Stamatoglou S. C., Keller J. M. Correlation between cell substrate attachment in vitro and cell surface heparan sulfate affinity for fibronectin and collagen. J Cell Biol. 1983 Jun;96(6):1820–1823. doi: 10.1083/jcb.96.6.1820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stevens R. L., Colombo M., Gonzales J. J., Hollander W., Schmid K. The glycosaminoglycans of the human artery and their changes in atherosclerosis. J Clin Invest. 1976 Aug;58(2):470–481. doi: 10.1172/JCI108491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Vannucchi S., Pasquali F., Chiarugi V., Ruggiero M. Internalization and metabolism of endogenous heparin by cultured endothelial cells. Biochem Biophys Res Commun. 1986 Oct 15;140(1):294–301. doi: 10.1016/0006-291x(86)91089-2. [DOI] [PubMed] [Google Scholar]
  56. Vlodavsky I., Bar-Shavit R., Ishai-Michaeli R., Bashkin P., Fuks Z. Extracellular sequestration and release of fibroblast growth factor: a regulatory mechanism? Trends Biochem Sci. 1991 Jul;16(7):268–271. doi: 10.1016/0968-0004(91)90102-2. [DOI] [PubMed] [Google Scholar]
  57. Völker W., Schmidt A., Oortmann W., Broszey T., Faber V., Buddecke E. Mapping of proteoglycans in atherosclerotic lesions. Eur Heart J. 1990 Aug;11 (Suppl E):29–40. doi: 10.1093/eurheartj/11.suppl_e.29. [DOI] [PubMed] [Google Scholar]
  58. Wang X. F., Lin H. Y., Ng-Eaton E., Downward J., Lodish H. F., Weinberg R. A. Expression cloning and characterization of the TGF-beta type III receptor. Cell. 1991 Nov 15;67(4):797–805. doi: 10.1016/0092-8674(91)90074-9. [DOI] [PubMed] [Google Scholar]
  59. Wight T. N., Curwen K. D., Litrenta M. M., Alonso D. R., Minick C. R. Effect of endothelium on glycosaminoglycan accumulation in injured rabbit aorta. Am J Pathol. 1983 Nov;113(2):156–164. [PMC free article] [PubMed] [Google Scholar]
  60. Wight T. N. Proteoglycans in pathological conditions: atherosclerosis. Fed Proc. 1985 Feb;44(2):381–385. [PubMed] [Google Scholar]
  61. Williams M. P., Streeter H. B., Wusteman F. S., Cryer A. Heparan sulphate and the binding of lipoprotein lipase to porcine thoracic aorta endothelium. Biochim Biophys Acta. 1983 Mar 15;756(1):83–91. doi: 10.1016/0304-4165(83)90027-2. [DOI] [PubMed] [Google Scholar]
  62. Yayon A., Klagsbrun M., Esko J. D., Leder P., Ornitz D. M. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell. 1991 Feb 22;64(4):841–848. doi: 10.1016/0092-8674(91)90512-w. [DOI] [PubMed] [Google Scholar]
  63. de Agostini A. I., Watkins S. C., Slayter H. S., Youssoufian H., Rosenberg R. D. Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta. J Cell Biol. 1990 Sep;111(3):1293–1304. doi: 10.1083/jcb.111.3.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]

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