Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2003 Aug 1;373(Pt 3):723–732. doi: 10.1042/BJ20021880

Biochemical mechanism of action of a diketopiperazine inactivator of plasminogen activator inhibitor-1.

Anja P Einholm 1, Katrine E Pedersen 1, Troels Wind 1, Paulina Kulig 1, Michael T Overgaard 1, Jan K Jensen 1, Julie S Bødker 1, Anni Christensen 1, Peter Charlton 1, Peter A Andreasen 1
PMCID: PMC1223537  PMID: 12723974

Abstract

XR5118 [(3 Z,6 Z )-6-benzylidine-3-(5-(2-dimethylaminoethyl-thio-))-2-(thienyl)methylene-2,5-dipiperazinedione hydrochloride] can inactivate the anti-proteolytic activity of the serpin plasminogen activator inhibitor-1 (PAI-1), a potential therapeutic target in cancer and cardiovascular diseases. Serpins inhibit their target proteases by the P(1) residue of their reactive centre loop (RCL) forming an ester bond with the active-site serine residue of the protease, followed by insertion of the RCL into the serpin's large central beta-sheet A. In the present study, we show that the RCL of XR5118-inactivated PAI-1 is inert to reaction with its target proteases and has a decreased susceptibility to non-target proteases, in spite of a generally increased proteolytic susceptibility of specific peptide bonds elsewhere in PAI-1. The properties of XR5118-inactivated PAI-1 were different from those of the so-called latent form of PAI-1. Alanine substitution of several individual residues decreased the susceptibility of PAI-1 to XR5118. The localization of these residues in the three-dimensional structure of PAI-1 suggested that the XR5118-induced inactivating conformational change requires mobility of alpha-helix F, situated above beta-sheet A, and is in agreement with the hypothesis that XR5118 binds laterally to beta-sheet A. These results improve our understanding of the unique conformational flexibility of serpins and the biochemical basis for using PAI-1 as a therapeutic target.

Full Text

The Full Text of this article is available as a PDF (310.3 KB).

Selected References

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

  1. Andreasen P. A., Egelund R., Jensen S., Rodenburg K. W. Solvent effects on activity and conformation of plasminogen activator inhibitor-1. Thromb Haemost. 1999 Mar;81(3):407–414. [PubMed] [Google Scholar]
  2. Andreasen P. A., Egelund R., Petersen H. H. The plasminogen activation system in tumor growth, invasion, and metastasis. Cell Mol Life Sci. 2000 Jan 20;57(1):25–40. doi: 10.1007/s000180050497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Andreasen P. A., Kjøller L., Christensen L., Duffy M. J. The urokinase-type plasminogen activator system in cancer metastasis: a review. Int J Cancer. 1997 Jul 3;72(1):1–22. doi: 10.1002/(sici)1097-0215(19970703)72:1<1::aid-ijc1>3.0.co;2-z. [DOI] [PubMed] [Google Scholar]
  4. Andreasen P. A., Riccio A., Welinder K. G., Douglas R., Sartorio R., Nielsen L. S., Oppenheimer C., Blasi F., Danø K. Plasminogen activator inhibitor type-1: reactive center and amino-terminal heterogeneity determined by protein and cDNA sequencing. FEBS Lett. 1986 Dec 15;209(2):213–218. doi: 10.1016/0014-5793(86)81113-9. [DOI] [PubMed] [Google Scholar]
  5. Bajou K., Masson V., Gerard R. D., Schmitt P. M., Albert V., Praus M., Lund L. R., Frandsen T. L., Brunner N., Dano K. The plasminogen activator inhibitor PAI-1 controls in vivo tumor vascularization by interaction with proteases, not vitronectin. Implications for antiangiogenic strategies. J Cell Biol. 2001 Feb 19;152(4):777–784. doi: 10.1083/jcb.152.4.777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bajou K., Noël A., Gerard R. D., Masson V., Brunner N., Holst-Hansen C., Skobe M., Fusenig N. E., Carmeliet P., Collen D. Absence of host plasminogen activator inhibitor 1 prevents cancer invasion and vascularization. Nat Med. 1998 Aug;4(8):923–928. doi: 10.1038/nm0898-923. [DOI] [PubMed] [Google Scholar]
  7. Berkenpas M. B., Lawrence D. A., Ginsburg D. Molecular evolution of plasminogen activator inhibitor-1 functional stability. EMBO J. 1995 Jul 3;14(13):2969–2977. doi: 10.1002/j.1460-2075.1995.tb07299.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Björquist P., Ehnebom J., Inghardt T., Hansson L., Lindberg M., Linschoten M., Strömqvist M., Deinum J. Identification of the binding site for a low-molecular-weight inhibitor of plasminogen activator inhibitor type 1 by site-directed mutagenesis. Biochemistry. 1998 Feb 3;37(5):1227–1234. doi: 10.1021/bi971554q. [DOI] [PubMed] [Google Scholar]
  9. Booth N. A. Fibrinolysis and thrombosis. Baillieres Best Pract Res Clin Haematol. 1999 Sep;12(3):423–433. doi: 10.1053/beha.1999.0034. [DOI] [PubMed] [Google Scholar]
  10. Cabrita Lisa D., Whisstock James C., Bottomley Stephen P. Probing the role of the F-helix in serpin stability through a single tryptophan substitution. Biochemistry. 2002 Apr 9;41(14):4575–4581. doi: 10.1021/bi0158932. [DOI] [PubMed] [Google Scholar]
  11. Charlton P. A., Faint R. W., Bent F., Bryans J., Chicarelli-Robinson I., Mackie I., Machin S., Bevan P. Evaluation of a low molecular weight modulator of human plasminogen activator inhibitor-1 activity. Thromb Haemost. 1996 May;75(5):808–815. [PubMed] [Google Scholar]
  12. Christensen J. H., Hansen P. K., Lillelund O., Thøgersen H. C. Sequence-specific binding of the N-terminal three-finger fragment of Xenopus transcription factor IIIA to the internal control region of a 5S RNA gene. FEBS Lett. 1991 Apr 9;281(1-2):181–184. doi: 10.1016/0014-5793(91)80388-j. [DOI] [PubMed] [Google Scholar]
  13. Devy Laetitia, Blacher Silvia, Grignet-Debrus Christine, Bajou Khalid, Masson Veronique, Gerard Robert D., Gils Ann, Carmeliet Geert, Carmeliet Peter, Declerck Paul J. The pro- or antiangiogenic effect of plasminogen activator inhibitor 1 is dose dependent. FASEB J. 2002 Feb;16(2):147–154. doi: 10.1096/fj.01-0552com. [DOI] [PubMed] [Google Scholar]
  14. Egelund R., Einholm A. P., Pedersen K. E., Nielsen R. W., Christensen A., Deinum J., Andreasen P. A. A regulatory hydrophobic area in the flexible joint region of plasminogen activator inhibitor-1, defined with fluorescent activity-neutralizing ligands. Ligand-induced serpin polymerization. J Biol Chem. 2001 Jan 25;276(16):13077–13086. doi: 10.1074/jbc.M009024200. [DOI] [PubMed] [Google Scholar]
  15. Egelund R., Petersen T. E., Andreasen P. A. A serpin-induced extensive proteolytic susceptibility of urokinase-type plasminogen activator implicates distortion of the proteinase substrate-binding pocket and oxyanion hole in the serpin inhibitory mechanism. Eur J Biochem. 2001 Feb;268(3):673–685. doi: 10.1046/j.1432-1327.2001.01921.x. [DOI] [PubMed] [Google Scholar]
  16. Egelund R., Rodenburg K. W., Andreasen P. A., Rasmussen M. S., Guldberg R. E., Petersen T. E. An ester bond linking a fragment of a serine proteinase to its serpin inhibitor. Biochemistry. 1998 May 5;37(18):6375–6379. doi: 10.1021/bi973043+. [DOI] [PubMed] [Google Scholar]
  17. Egelund R., Schousboe S. L., Sottrup-Jensen L., Rodenburg K. W., Andreasen P. A. Type-1 plasminogen-activator inhibitor -- conformational differences between latent, active, reactive-centre-cleaved and plasminogen-activator-complexed forms, as probed by proteolytic susceptibility. Eur J Biochem. 1997 Sep 15;248(3):775–785. doi: 10.1111/j.1432-1033.1997.t01-1-00775.x. [DOI] [PubMed] [Google Scholar]
  18. Eren Mesut, Painter Corrie A., Atkinson James B., Declerck Paul J., Vaughan Douglas E. Age-dependent spontaneous coronary arterial thrombosis in transgenic mice that express a stable form of human plasminogen activator inhibitor-1. Circulation. 2002 Jul 23;106(4):491–496. doi: 10.1161/01.cir.0000023186.60090.fb. [DOI] [PubMed] [Google Scholar]
  19. Erickson L. A., Fici G. J., Lund J. E., Boyle T. P., Polites H. G., Marotti K. R. Development of venous occlusions in mice transgenic for the plasminogen activator inhibitor-1 gene. Nature. 1990 Jul 5;346(6279):74–76. doi: 10.1038/346074a0. [DOI] [PubMed] [Google Scholar]
  20. Fa M., Bergström F., Hägglöf P., Wilczynska M., Johansson L. B., Ny T. The structure of a serpin-protease complex revealed by intramolecular distance measurements using donor-donor energy migration and mapping of interaction sites. Structure. 2000 Apr 15;8(4):397–405. doi: 10.1016/s0969-2126(00)00121-0. [DOI] [PubMed] [Google Scholar]
  21. Friederich P. W., Levi M., Biemond B. J., Charlton P., Templeton D., van Zonneveld A. J., Bevan P., Pannekoek H., ten Cate J. W. Novel low-molecular-weight inhibitor of PAI-1 (XR5118) promotes endogenous fibrinolysis and reduces postthrombolysis thrombus growth in rabbits. Circulation. 1997 Aug 5;96(3):916–921. [PubMed] [Google Scholar]
  22. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  23. Gils A., Declerck P. J. Modulation of plasminogen activator inhibitor 1 by Triton X-100--identification of two consecutive conformational transitions. Thromb Haemost. 1998 Aug;80(2):286–291. [PubMed] [Google Scholar]
  24. Gils Ann, Stassen Jean-Marie, Nar Herbert, Kley Joerg T., Wienen Wolfgang, Ries Uwe J., Declerck Paul J. Characterization and comparative evaluation of a novel PAI-1 inhibitor. Thromb Haemost. 2002 Jul;88(1):137–143. [PubMed] [Google Scholar]
  25. Hansen M., Busse M. N., Andreasen P. A. Importance of the amino-acid composition of the shutter region of plasminogen activator inhibitor-1 for its transitions to latent and substrate forms. Eur J Biochem. 2001 Dec;268(23):6274–6283. doi: 10.1046/j.0014-2956.2001.02582.x. [DOI] [PubMed] [Google Scholar]
  26. Huber K., Christ G., Wojta J., Gulba D. Plasminogen activator inhibitor type-1 in cardiovascular disease. Status report 2001. Thromb Res. 2001 Sep 30;103 (Suppl 1):S7–19. doi: 10.1016/s0049-3848(01)00293-6. [DOI] [PubMed] [Google Scholar]
  27. Huntington J. A., Read R. J., Carrell R. W. Structure of a serpin-protease complex shows inhibition by deformation. Nature. 2000 Oct 19;407(6806):923–926. doi: 10.1038/35038119. [DOI] [PubMed] [Google Scholar]
  28. Irving J. A., Pike R. N., Lesk A. M., Whisstock J. C. Phylogeny of the serpin superfamily: implications of patterns of amino acid conservation for structure and function. Genome Res. 2000 Dec;10(12):1845–1864. doi: 10.1101/gr.gr-1478r. [DOI] [PubMed] [Google Scholar]
  29. Jensen Jan K., Wind Troels, Andreasen Peter A. The vitronectin binding area of plasminogen activator inhibitor-1, mapped by mutagenesis and protection against an inactivating organochemical ligand. FEBS Lett. 2002 Jun 19;521(1-3):91–94. doi: 10.1016/s0014-5793(02)02830-2. [DOI] [PubMed] [Google Scholar]
  30. Jensen Signe, Kirkegaard Tove, Pedersen Katrine E., Busse Marta, Preissner Klaus T., Rodenburg Kees W., Andreasen Peter A. The role of beta-strand 5A of plasminogen activator inhibitor-1 in regulation of its latency transition and inhibitory activity by vitronectin. Biochim Biophys Acta. 2002 Jun 3;1597(2):301–310. doi: 10.1016/s0167-4838(02)00312-6. [DOI] [PubMed] [Google Scholar]
  31. Kjøller L., Martensen P. M., Sottrup-Jensen L., Justesen J., Rodenburg K. W., Andreasen P. A. Conformational changes of the reactive-centre loop and beta-strand 5A accompany temperature-dependent inhibitor-substrate transition of plasminogen-activator inhibitor 1. Eur J Biochem. 1996 Oct 1;241(1):38–46. doi: 10.1111/j.1432-1033.1996.0038t.x. [DOI] [PubMed] [Google Scholar]
  32. Lambert V., Munaut C., Noël A., Frankenne F., Bajou K., Gerard R., Carmeliet P., Defresne M. P., Foidart J. M., Rakic J. M. Influence of plasminogen activator inhibitor type 1 on choroidal neovascularization. FASEB J. 2001 Apr;15(6):1021–1027. doi: 10.1096/fj.00-0393com. [DOI] [PubMed] [Google Scholar]
  33. Lawrence D. A., Ginsburg D., Day D. E., Berkenpas M. B., Verhamme I. M., Kvassman J. O., Shore J. D. Serpin-protease complexes are trapped as stable acyl-enzyme intermediates. J Biol Chem. 1995 Oct 27;270(43):25309–25312. doi: 10.1074/jbc.270.43.25309. [DOI] [PubMed] [Google Scholar]
  34. McMahon G. A., Petitclerc E., Stefansson S., Smith E., Wong M. K., Westrick R. J., Ginsburg D., Brooks P. C., Lawrence D. A. Plasminogen activator inhibitor-1 regulates tumor growth and angiogenesis. J Biol Chem. 2001 Jul 5;276(36):33964–33968. doi: 10.1074/jbc.M105980200. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Nar H., Bauer M., Stassen J. M., Lang D., Gils A., Declerck P. J. Plasminogen activator inhibitor 1. Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation. J Mol Biol. 2000 Mar 31;297(3):683–695. doi: 10.1006/jmbi.2000.3604. [DOI] [PubMed] [Google Scholar]
  37. Neve J., Leone P. A., Carroll A. R., Moni R. W., Paczkowski N. J., Pierens G., Björquist P., Deinum J., Ehnebom J., Inghardt T. Sideroxylonal C, a new inhibitor of human plasminogen activator inhibitor type-1, from the flowers of Eucalyptus albens. J Nat Prod. 1999 Feb;62(2):324–326. doi: 10.1021/np980286+. [DOI] [PubMed] [Google Scholar]
  39. Schousboe S. L., Egelund R., Kirkegaard T., Preissner K. T., Rodenburg K. W., Andreasen P. A. Vitronectin and substitution of a beta-strand 5A lysine residue potentiate activity-neutralization of PA inhibitor-1 by monoclonal antibodies against alpha-helix F. Thromb Haemost. 2000 May;83(5):742–751. [PubMed] [Google Scholar]
  40. Schägger H., von Jagow G. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem. 1991 Dec;199(2):223–231. doi: 10.1016/0003-2697(91)90094-a. [DOI] [PubMed] [Google Scholar]
  41. Sharp A. M., Stein P. E., Pannu N. S., Carrell R. W., Berkenpas M. B., Ginsburg D., Lawrence D. A., Read R. J. The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion. Structure. 1999 Feb 15;7(2):111–118. doi: 10.1016/S0969-2126(99)80018-5. [DOI] [PubMed] [Google Scholar]
  42. Shore J. D., Day D. E., Francis-Chmura A. M., Verhamme I., Kvassman J., Lawrence D. A., Ginsburg D. A fluorescent probe study of plasminogen activator inhibitor-1. Evidence for reactive center loop insertion and its role in the inhibitory mechanism. J Biol Chem. 1995 Mar 10;270(10):5395–5398. doi: 10.1074/jbc.270.10.5395. [DOI] [PubMed] [Google Scholar]
  43. Stratikos E., Gettins P. G. Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 A and full insertion of the reactive center loop into beta-sheet A. Proc Natl Acad Sci U S A. 1999 Apr 27;96(9):4808–4813. doi: 10.1073/pnas.96.9.4808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wilczynska M., Fa M., Ohlsson P. I., Ny T. The inhibition mechanism of serpins. Evidence that the mobile reactive center loop is cleaved in the native protease-inhibitor complex. J Biol Chem. 1995 Dec 15;270(50):29652–29655. doi: 10.1074/jbc.270.50.29652. [DOI] [PubMed] [Google Scholar]
  45. Wind T., Jensen M. A., Andreasen P. A. Epitope mapping for four monoclonal antibodies against human plasminogen activator inhibitor type-1: implications for antibody-mediated PAI-1-neutralization and vitronectin-binding. Eur J Biochem. 2001 Feb;268(4):1095–1106. doi: 10.1046/j.1432-1327.2001.2680041095.x. [DOI] [PubMed] [Google Scholar]
  46. Wind Troels, Hansen Martin, Jensen Jan K., Andreasen Peter A. The molecular basis for anti-proteolytic and non-proteolytic functions of plasminogen activator inhibitor type-1: roles of the reactive centre loop, the shutter region, the flexible joint region and the small serpin fragment. Biol Chem. 2002 Jan;383(1):21–36. doi: 10.1515/BC.2002.003. [DOI] [PubMed] [Google Scholar]
  47. Wind Troels, Jensen Jan K., Dupont Daniel M., Kulig Paulina, Andreasen Peter A. Mutational analysis of plasminogen activator inhibitor-1. Eur J Biochem. 2003 Apr;270(8):1680–1688. doi: 10.1046/j.1432-1033.2003.03524.x. [DOI] [PubMed] [Google Scholar]
  48. Ye S., Cech A. L., Belmares R., Bergstrom R. C., Tong Y., Corey D. R., Kanost M. R., Goldsmith E. J. The structure of a Michaelis serpin-protease complex. Nat Struct Biol. 2001 Nov;8(11):979–983. doi: 10.1038/nsb1101-979. [DOI] [PubMed] [Google Scholar]
  49. Zhou A., Faint R., Charlton P., Dafforn T. R., Carrell R. W., Lomas D. A. Polymerization of plasminogen activator inhibitor-1. J Biol Chem. 2000 Dec 1;276(12):9115–9122. doi: 10.1074/jbc.M010631200. [DOI] [PubMed] [Google Scholar]

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

RESOURCES