Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Jul 15;325(Pt 2):339–349. doi: 10.1042/bj3250339

The cluster of basic amino acids in vitronectin contributes to its binding of plasminogen activator inhibitor-1: evidence from thrombin-, elastase- and plasmin-cleaved vitronectins and anti-peptide antibodies.

Z Gechtman 1, A Belleli 1, S Lechpammer 1, S Shaltiel 1
PMCID: PMC1218566  PMID: 9230112

Abstract

Derivatives of vitronectin obtained by specific cleavage at its cluster of basic amino acids with thrombin, elastase and plasmin are shown to have a decreased ability to bind plasminogen activator inhibitor-1 (PAI-1). The identification and localization of the segment involved in the binding of PAI-1 (Lys348-Arg379) were carried out by purification of these cleaved vitronectins and their subsequent structural characterization (sequence analysis, phosphorylation of Ser378 with cAMP-dependent protein kinase and immunostaining with peptide-specific antibodies), then measurement of the vitronectin-PAI-1 interaction by (a) a two-phase system (ELISA); (b) co-precipitation of the vitronectin-PAI-1 complex out of solution, and (c) analysis of the stereospecific interaction between the active conformation of PAI-1 and a peptide derived from the above-mentioned cluster; this interaction occurs when the peptide is composed of all-l-amino acids but not when it is composed of all-d-amino acids. Our results explain why workers who have used immobilized vitronectin to study this interaction could not have observed the involvement of the cluster of basic amino acids in PAI-1 binding, since the immobilization of vitronectin is shown to render this cluster inaccessible for interaction. We propose that vitronectin binds active PAI-1 by interaction via amino acid residues that originate from distal locations in the N- and C-termini of vitronectin.

Full Text

The Full Text of this article is available as a PDF (517.5 KB).

Selected References

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

  1. Beavo J. A., Bechtel P. J., Krebs E. G. Preparation of homogeneous cyclic AMP-dependent protein kinase(s) and its subunits from rabbit skeletal muscle. Methods Enzymol. 1974;38:299–308. doi: 10.1016/0076-6879(74)38046-9. [DOI] [PubMed] [Google Scholar]
  2. Bieth J. G. L'élastase leucocytaire humaine. Pathol Biol (Paris) 1988 Nov;36(9):1108–1111. [PubMed] [Google Scholar]
  3. Bode W., Meyer E., Jr, Powers J. C. Human leukocyte and porcine pancreatic elastase: X-ray crystal structures, mechanism, substrate specificity, and mechanism-based inhibitors. Biochemistry. 1989 Mar 7;28(5):1951–1963. doi: 10.1021/bi00431a001. [DOI] [PubMed] [Google Scholar]
  4. Chain D., Korc-Grodzicki B., Kreizman T., Shaltiel S. Endogenous cleavage of the Arg-379-Ala-380 bond in vitronectin results in a distinct conformational change which 'buries' Ser-378, its site of phosphorylation by protein kinase A. Biochem J. 1991 Mar 1;274(Pt 2):387–394. doi: 10.1042/bj2740387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chain D., Korc-Grodzicki B., Kreizman T., Shaltiel S. The phosphorylation of the two-chain form of vitronectin by protein kinase A is heparin dependent. FEBS Lett. 1990 Aug 20;269(1):221–225. doi: 10.1016/0014-5793(90)81159-l. [DOI] [PubMed] [Google Scholar]
  6. Chain D., Kreizman T., Shapira H., Shaltiel S. Plasmin cleavage of vitronectin. Identification of the site and consequent attenuation in binding plasminogen activator inhibitor-1. FEBS Lett. 1991 Jul 22;285(2):251–256. doi: 10.1016/0014-5793(91)80810-p. [DOI] [PubMed] [Google Scholar]
  7. Dahlbäck B., Podack E. R. Characterization of human S protein, an inhibitor of the membrane attack complex of complement. Demonstration of a free reactive thiol group. Biochemistry. 1985 Apr 23;24(9):2368–2374. doi: 10.1021/bi00330a036. [DOI] [PubMed] [Google Scholar]
  8. Danø K., Andreasen P. A., Grøndahl-Hansen J., Kristensen P., Nielsen L. S., Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res. 1985;44:139–266. doi: 10.1016/s0065-230x(08)60028-7. [DOI] [PubMed] [Google Scholar]
  9. Declerck P. J., De Mol M., Alessi M. C., Baudner S., Pâques E. P., Preissner K. T., Müller-Berghaus G., Collen D. Purification and characterization of a plasminogen activator inhibitor 1 binding protein from human plasma. Identification as a multimeric form of S protein (vitronectin). J Biol Chem. 1988 Oct 25;263(30):15454–15461. [PubMed] [Google Scholar]
  10. Deng G., Royle G., Seiffert D., Loskutoff D. J. The PAI-1/vitronectin interaction: two cats in a bag? Thromb Haemost. 1995 Jul;74(1):66–70. [PubMed] [Google Scholar]
  11. Deng G., Royle G., Wang S., Crain K., Loskutoff D. J. Structural and functional analysis of the plasminogen activator inhibitor-1 binding motif in the somatomedin B domain of vitronectin. J Biol Chem. 1996 May 31;271(22):12716–12723. doi: 10.1074/jbc.271.22.12716. [DOI] [PubMed] [Google Scholar]
  12. Ehrlich H. J., Gebbink R. K., Keijer J., Linders M., Preissner K. T., Pannekoek H. Alteration of serpin specificity by a protein cofactor. Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties. J Biol Chem. 1990 Aug 5;265(22):13029–13035. [PubMed] [Google Scholar]
  13. Ehrlich H. J., Gebbink R. K., Preissner K. T., Keijer J., Esmon N. L., Mertens K., Pannekoek H. Thrombin neutralizes plasminogen activator inhibitor 1 (PAI-1) that is complexed with vitronectin in the endothelial cell matrix. J Cell Biol. 1991 Dec;115(6):1773–1781. doi: 10.1083/jcb.115.6.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Felding-Habermann B., Cheresh D. A. Vitronectin and its receptors. Curr Opin Cell Biol. 1993 Oct;5(5):864–868. doi: 10.1016/0955-0674(93)90036-p. [DOI] [PubMed] [Google Scholar]
  15. Gechtman Z., Shaltiel S. Phosphorylation of vitronectin on Ser362 by protein kinase C attenuates its cleavage by plasmin. Eur J Biochem. 1997 Jan 15;243(1-2):493–501. doi: 10.1111/j.1432-1033.1997.0493a.x. [DOI] [PubMed] [Google Scholar]
  16. Gechtman Z., Sharma R., Kreizman T., Fridkin M., Shaltiel S. Synthetic peptides derived from the sequence around the plasmin cleavage site in vitronectin. Use in mapping the PAI-1 binding site. FEBS Lett. 1993 Jan 11;315(3):293–297. doi: 10.1016/0014-5793(93)81181-x. [DOI] [PubMed] [Google Scholar]
  17. Hayashi M., Akama T., Kono I., Kashiwagi H. Activation of vitronectin (serum spreading factor) binding of heparin by denaturing agents. J Biochem. 1985 Oct;98(4):1135–1138. doi: 10.1093/oxfordjournals.jbchem.a135363. [DOI] [PubMed] [Google Scholar]
  18. Hayman E. G., Pierschbacher M. D., Ohgren Y., Ruoslahti E. Serum spreading factor (vitronectin) is present at the cell surface and in tissues. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4003–4007. doi: 10.1073/pnas.80.13.4003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hekman C. M., Loskutoff D. J. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem. 1985 Sep 25;260(21):11581–11587. [PubMed] [Google Scholar]
  20. Hekman C. M., Loskutoff D. J. Kinetic analysis of the interactions between plasminogen activator inhibitor 1 and both urokinase and tissue plasminogen activator. Arch Biochem Biophys. 1988 Apr;262(1):199–210. doi: 10.1016/0003-9861(88)90182-8. [DOI] [PubMed] [Google Scholar]
  21. Holmes R. Preparation from human serum of an alpha-one protein which induces the immediate growth of unadapted cells in vitro. J Cell Biol. 1967 Feb;32(2):297–308. doi: 10.1083/jcb.32.2.297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ill C. R., Ruoslahti E. Association of thrombin-antithrombin III complex with vitronectin in serum. J Biol Chem. 1985 Dec 15;260(29):15610–15615. [PubMed] [Google Scholar]
  23. Jang I. K., Lassila R., Fuster V. Atherogenesis and inflammation. Eur Heart J. 1993 Dec;14 (Suppl K):2–6. [PubMed] [Google Scholar]
  24. Jenne D., Stanley K. K. Molecular cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion. EMBO J. 1985 Dec 1;4(12):3153–3157. doi: 10.1002/j.1460-2075.1985.tb04058.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Korc-Grodzicki B., Tauber-Finkelstein M., Chain D., Shaltiel S. Vitronectin is phosphorylated by a cAMP-dependent protein kinase released by activation of human platelets with thrombin. Biochem Biophys Res Commun. 1988 Dec 30;157(3):1131–1138. doi: 10.1016/s0006-291x(88)80991-4. [DOI] [PubMed] [Google Scholar]
  26. Korc-Grodzicki B., Tauber-Finkelstein M., Shaltiel S. Platelet stimulation releases a cAMP-dependent protein kinase that specifically phosphorylates a plasma protein. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7541–7545. doi: 10.1073/pnas.85.20.7541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kost C., Stüber W., Ehrlich H. J., Pannekoek H., Preissner K. T. Mapping of binding sites for heparin, plasminogen activator inhibitor-1, and plasminogen to vitronectin's heparin-binding region reveals a novel vitronectin-dependent feedback mechanism for the control of plasmin formation. J Biol Chem. 1992 Jun 15;267(17):12098–12105. [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. Lindahl T. L., Sigurdardottir O., Wiman B. Stability of plasminogen activator inhibitor 1 (PAI-1). Thromb Haemost. 1989 Sep 29;62(2):748–751. [PubMed] [Google Scholar]
  30. Lindahl T., Wiman B. Purification of high and low molecular weight plasminogen activator inhibitor 1 from fibrosarcoma cell-line HT 1080 conditioned medium. Biochim Biophys Acta. 1989 Feb 23;994(3):253–257. doi: 10.1016/0167-4838(89)90301-4. [DOI] [PubMed] [Google Scholar]
  31. Mehringer J. H., Weigel C. J., Tollefsen D. M. Cyclic AMP-dependent protein kinase phosphorylates serine378 in vitronectin. Biochem Biophys Res Commun. 1991 Aug 30;179(1):655–660. doi: 10.1016/0006-291x(91)91422-9. [DOI] [PubMed] [Google Scholar]
  32. Mimuro J., Loskutoff D. J. Purification of a protein from bovine plasma that binds to type 1 plasminogen activator inhibitor and prevents its interaction with extracellular matrix. Evidence that the protein is vitronectin. J Biol Chem. 1989 Jan 15;264(2):936–939. [PubMed] [Google Scholar]
  33. Mimuro J., Muramatsu S., Kurano Y., Uchida Y., Ikadai H., Watanabe S., Sakata Y. Identification of the plasminogen activator inhibitor-1 binding heptapeptide in vitronectin. Biochemistry. 1993 Mar 9;32(9):2314–2320. doi: 10.1021/bi00060a024. [DOI] [PubMed] [Google Scholar]
  34. Mottonen J., Strand A., Symersky J., Sweet R. M., Danley D. E., Geoghegan K. F., Gerard R. D., Goldsmith E. J. Structural basis of latency in plasminogen activator inhibitor-1. Nature. 1992 Jan 16;355(6357):270–273. doi: 10.1038/355270a0. [DOI] [PubMed] [Google Scholar]
  35. Pipoly D. J., Crouch E. C. Degradation of native type IV procollagen by human neutrophil elastase. Implications for leukocyte-mediated degradation of basement membranes. Biochemistry. 1987 Sep 8;26(18):5748–5754. doi: 10.1021/bi00392a025. [DOI] [PubMed] [Google Scholar]
  36. Podack E. R., Dahlbäck B., Griffin J. H. Interaction of S-protein of complement with thrombin and antithrombin III during coagulation. Protection of thrombin by S-protein from antithrombin III inactivation. J Biol Chem. 1986 Jun 5;261(16):7387–7392. [PubMed] [Google Scholar]
  37. Preissner K. T., Holzhüter S., Justus C., Müller-Berghaus G. Identification of and partial characterization of platelet vitronectin: evidence for complex formation with platelet-derived plasminogen activator inhibitor-1. Blood. 1989 Nov 1;74(6):1989–1996. [PubMed] [Google Scholar]
  38. Preissner K. T., Jenne D. Structure of vitronectin and its biological role in haemostasis. Thromb Haemost. 1991 Jul 12;66(1):123–132. [PubMed] [Google Scholar]
  39. Preissner K. T., Müller-Berghaus G. Neutralization and binding of heparin by S protein/vitronectin in the inhibition of factor Xa by antithrombin III. Involvement of an inducible heparin-binding domain of S protein/vitronectin. J Biol Chem. 1987 Sep 5;262(25):12247–12253. [PubMed] [Google Scholar]
  40. Preissner K. T. Structure and biological role of vitronectin. Annu Rev Cell Biol. 1991;7:275–310. doi: 10.1146/annurev.cb.07.110191.001423. [DOI] [PubMed] [Google Scholar]
  41. Preissner K. T., Zwicker L., Müller-Berghaus G. Formation, characterization and detection of a ternary complex between S protein, thrombin and antithrombin III in serum. Biochem J. 1987 Apr 1;243(1):105–111. doi: 10.1042/bj2430105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Salonen E. M., Vaheri A., Pöllänen J., Stephens R., Andreasen P., Mayer M., Danø K., Gailit J., Ruoslahti E. Interaction of plasminogen activator inhibitor (PAI-1) with vitronectin. J Biol Chem. 1989 Apr 15;264(11):6339–6343. [PubMed] [Google Scholar]
  43. Seiffert D., Ciambrone G., Wagner N. V., Binder B. R., Loskutoff D. J. The somatomedin B domain of vitronectin. Structural requirements for the binding and stabilization of active type 1 plasminogen activator inhibitor. J Biol Chem. 1994 Jan 28;269(4):2659–2666. [PubMed] [Google Scholar]
  44. Seiffert D., Loskutoff D. J. Evidence that type 1 plasminogen activator inhibitor binds to the somatomedin B domain of vitronectin. J Biol Chem. 1991 Feb 15;266(5):2824–2830. [PubMed] [Google Scholar]
  45. Seiffert D., Loskutoff D. J. Kinetic analysis of the interaction between type 1 plasminogen activator inhibitor and vitronectin and evidence that the bovine inhibitor binds to a thrombin-derived amino-terminal fragment of bovine vitronectin. Biochim Biophys Acta. 1991 May 30;1078(1):23–30. doi: 10.1016/0167-4838(91)90087-g. [DOI] [PubMed] [Google Scholar]
  46. Seiffert D., Wagner N. N., Loskutoff D. J. Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor. J Cell Biol. 1990 Sep;111(3):1283–1291. doi: 10.1083/jcb.111.3.1283. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Shaltiel S., Schvartz I., Korc-Grodzicki B., Kreizman T. Evidence for an extra-cellular function for protein kinase A. Mol Cell Biochem. 1993 Nov;127-128:283–291. doi: 10.1007/978-1-4615-2600-1_26. [DOI] [PubMed] [Google Scholar]
  48. Sigurdardottir O., Wiman B. Identification of a PAI-1 binding site in vitronectin. Biochim Biophys Acta. 1994 Sep 21;1208(1):104–110. doi: 10.1016/0167-4838(94)90166-x. [DOI] [PubMed] [Google Scholar]
  49. Su H. R., Boackle R. J. Heparin mediates binding of S-protein/vitronectin to the envelope glycoprotein of the human immunodeficiency virus and CD4. Int Arch Allergy Immunol. 1994 Nov;105(3):238–244. doi: 10.1159/000236763. [DOI] [PubMed] [Google Scholar]
  50. Suzuki S., Oldberg A., Hayman E. G., Pierschbacher M. D., Ruoslahti E. Complete amino acid sequence of human vitronectin deduced from cDNA. Similarity of cell attachment sites in vitronectin and fibronectin. EMBO J. 1985 Oct;4(10):2519–2524. doi: 10.1002/j.1460-2075.1985.tb03965.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Suzuki S., Pierschbacher M. D., Hayman E. G., Nguyen K., Ohgren Y., Ruoslahti E. Domain structure of vitronectin. Alignment of active sites. J Biol Chem. 1984 Dec 25;259(24):15307–15314. [PubMed] [Google Scholar]
  52. Tomasini B. R., Mosher D. F. Vitronectin. Prog Hemost Thromb. 1991;10:269–305. [PubMed] [Google Scholar]
  53. Tucker H. M., Mottonen J., Goldsmith E. J., Gerard R. D. Engineering of plasminogen activator inhibitor-1 to reduce the rate of latency transition. Nat Struct Biol. 1995 Jun;2(6):442–445. doi: 10.1038/nsb0695-442. [DOI] [PubMed] [Google Scholar]
  54. Watanabe H., Hattori S., Katsuda S., Nakanishi I., Nagai Y. Human neutrophil elastase: degradation of basement membrane components and immunolocalization in the tissue. J Biochem. 1990 Nov;108(5):753–759. doi: 10.1093/oxfordjournals.jbchem.a123277. [DOI] [PubMed] [Google Scholar]
  55. Wiman B., Almquist A., Sigurdardottir O., Lindahl T. Plasminogen activator inhibitor 1 (PAI) is bound to vitronectin in plasma. FEBS Lett. 1988 Dec 19;242(1):125–128. doi: 10.1016/0014-5793(88)80999-2. [DOI] [PubMed] [Google Scholar]
  56. 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]
  57. de Boer H. C., Preissner K. T., Bouma B. N., de Groot P. G. Binding of vitronectin-thrombin-antithrombin III complex to human endothelial cells is mediated by the heparin binding site of vitronectin. J Biol Chem. 1992 Feb 5;267(4):2264–2268. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES