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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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A novel variant of t-PA resistant to plasminogen activator inhibitor-1; expression in CHO cells based on In Silico experiments

  • Davami, Fatemeh (Biotechnology Research center, Pasteur Institute of Iran Tehran) ;
  • Sardari, Soroush (Biotechnology Research center, Pasteur Institute of Iran Tehran) ;
  • Majidzadeh-A, Keivan (Biotechnology Research center, Pasteur Institute of Iran Tehran) ;
  • Hemayatkar, Mahdi (Biotechnology Research center, Pasteur Institute of Iran Tehran) ;
  • Barkhordari, Farzaneh (Biotechnology Research center, Pasteur Institute of Iran Tehran) ;
  • Enayati, Somayeh (Biotechnology Research center, Pasteur Institute of Iran Tehran) ;
  • Adeli, Ahmad (Biotechnology Research center, Pasteur Institute of Iran Tehran) ;
  • Mahboudi, Fereidoun (Biotechnology Research center, Pasteur Institute of Iran Tehran)
  • Received : 2010.10.21
  • Accepted : 2010.11.20
  • Published : 2011.01.31
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Abstract

Resistance to PAI-1 is a factor which confers clinical benefits in thrombolytic therapy. The only US FDA approved PAI-1 resistant drug is Tenecteplase$^{(R)}$. Deletion variants of t-PA have the advantage of fewer disulfide bonds in addition to higher plasma half lives. A new variant was developed by deletion of the first three domains in t-PA in addition to substitution of KHRR 128-131 amino acids with AAAA in truncated t-PA. The specific activity of this new variant, $570\;IU/{\mu}g$, was found to be similar to those found in full length t-PA (Alteplase$^{(R)}$), $580\;IU/{\mu}g$. A 65% and 85% residual activity after inhibition by rPAI-1 was observed for full length and truncated-mutant form, respectively. This new variant as the first PAI-1 resistant truncated t-PA may offer more advantages in clinical conditions in which high PAI-1 levels makes the thrombolytic system prone to re-occlusion.

Keywords

References

  1. American Heart Association. (2008) Heart Disease andStroke Statistics, http://www.americanheart.org/downloadable/heart/1200082005246HS_Stats%202008.final.pdf.
  2. Ranby, M., Bergsdorf, N., Pohl, G. and Wallen, P. (1982)Isolation of two variants of native one-chain tissue plasminogenactivator. FEBS Lett. 146, 289-292. https://doi.org/10.1016/0014-5793(82)80936-8
  3. Benchenane, K., Lopez-Atalaya, J. P., Fernandez-Monreal,M., Touzani, O. and Vivien, D. (2004) Equivocal roles oftissue-type plasminogen activator in stroke-induced injury.Trends Neurosci. 27, 155-160. https://doi.org/10.1016/j.tins.2003.12.011
  4. Travis, J. and Salvesen, G. S. (1983) Human plasma proteinaseinhibitors. Annu. Rev. Biochem. 52, 655-709. https://doi.org/10.1146/annurev.bi.52.070183.003255
  5. Carrell, R. and Boswell, D. R. (1986) Serpins: the superfamilyof plasma serine proteinase inhibitors; in ProteinaseInhibitors, Barrelland, A. and Salvesen, G. (eds.). pp. 403-420, Elsevier, North Holland, Amsterdam, Netherland.
  6. Erickson, L. A., Ginsberg, M. H. and Loskutoff, D. J.(1984) Detection and partial characterization of an inhibitorof plasminogen activator in human platelets. J.Clin. Invest. 74, 1465-1472. https://doi.org/10.1172/JCI111559
  7. Hekman, C. M. and Loskutoff, D. J. (1988) Kinetic analysisof the interactions between plasminogen activator inhibitor1 and both urokinase and tissue plasminogenactivator. Arch. Biochem. Biophys. 262, 199-210. https://doi.org/10.1016/0003-9861(88)90182-8
  8. Higgins, D. L. and Vehar, G. A. (1987) Interaction of onechain and two-chain tissue plasminogen activator with intact and plasmin-degraded fibrin. Biochemistry 26, 7786-7791. https://doi.org/10.1021/bi00398a038
  9. Wagner, O. F., de Vries, C., Hohmann, C., Veerman, H.and Pannekoek, H. (1989) Interaction between plasminogenactivator inhibitor type 1 (PAI-1) bound to fibrin andeither tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA). Binding oft-PA/PAI-1 complexes to fibrin mediated by both the fingerand the kringle-2 domain of t-PA. J. Clin. Invest. 84,647-655. https://doi.org/10.1172/JCI114211
  10. Shohet, R. V., Spitzer, S., Madison, E. L., Bassel-Duby, R.,Gething, M. J. and Sambrook, J. F. (1994) Inhibitor-resistanttissue-type plasminogen activator: an improvedthrombolytic agent in vitro. Thromb. Haemost. 71, 124-128.
  11. Paoni, N. F., Keyt, B. A., Refino, C. J., Chow, A. M.,Nguyen, H. V., Berleau, L. T., Badillo, J., Pena, L. C.,Brady, K. and Wurm, F. M. (1993) A slow clearing, fibrin-specific, PAI-1 resistant variant of t-PA (T103N ,KHRR296-299 AAAA). Thromb. Haemost. 70, 307-312.
  12. Li, X. K., Lijnen, H. R., Nelles, L., Van, H. B., Stassen, J.M. and Collen, D. (1992) Biochemical and biologic propertiesof rt-PA del (K296-G302), a recombinant human tissue-type plasminogen activator deletion mutant resistantto plasminogen activator inhibitor-1. Blood 79, 417-429.
  13. Nordt, T. K. and Bode, C. (2003) Thrombolysis: newerthrombolytic agents and their role in clinical medicine.Heart 89, 1358-1362. https://doi.org/10.1136/heart.89.11.1358
  14. Baruah, D. B., Dash, R. N., Chaudhari, M. R. and Kadam,S. S. (2006) Plasminogen activators: a comparison. Vascul.Pharmacol. 44, 1-9. https://doi.org/10.1016/j.vph.2005.09.003
  15. Weaver, W. D. (1996) The role of thrombolytic drugs inthe management of myocardial infarction. Comparativeclinical trials. Eur. Heart J. 17(Suppl F), 9-15. https://doi.org/10.1093/eurheartj/17.suppl_F.9
  16. Davami, F., Sardari, S., Majidzadeh, A., Hemayatkar, M.,Barkhrdari, F., Omidi, M., Azami, M., Adeli, A., Davoudi,N. and Mahboudi, F. (2010) Expression of a novel chimerictruncated t-PA in CHO cells based on in silicoexperiments. J. Biomed. Biotechnol. 2010, 108-159.
  17. Burck, P. J., Berg, D. H., Warrick, M. W., Berg, D. T.,Walls, J. D., Jaskunas, S. R., Crisel, R. M., Weigel, B.,Vlahos, C. J. and McClure, D. B. (1990) Characterizationof a modified human tissue plasminogen activator comprisinga kringle-2 and a protease domain. J. Biol. Chem.265, 5170-5177.
  18. Martin, U., Dorge, L. and Fischer, S. Comparison of desulfatohirudin(REVASC) and heparin as adjuncts to thrombolytictherapy with reteplase in a canine model of coronarythrombosis. Br. J. Pharmacol. 118, 271-276.
  19. http://www.gene.com/gene/products/information/pdf/activase-prescribing.pdf. 2010.
  20. Levine, G. N., Ali, M. N. and Schafer, A. I. (2001) Antithrombotictherapy in patients with acute coronary syndromes.Arch. Intern. Med. 161, 937-948. https://doi.org/10.1001/archinte.161.7.937
  21. Emeis, J. J., van den Hoogen, C. M. and Jense, D. (1985)Hepatic clearance of tissue-type plasminogen activator inrats. Thromb. Haemost. 54, 661-664.
  22. Ouriel, K., Katzen, B., Mewissen, M., Flick, P., Clair, D.G., Benenati, J., McNamara, T. O. and Gibbens, D. (2000)Reteplase in the treatment of peripheral arterial and venousocclusions: a pilot study. J. Vasc. Interv. Radiol. 11,849-854. https://doi.org/10.1016/S1051-0443(07)61799-0
  23. Madison, E. L., Goldsmith, E. J., Gerard, R. D., Gething,M. J., Sambrook, J. F. and Bassel-Duby, R. S. (1990)Amino acid residues that affect interaction of tissue-typeplasminogen activator with plasminogen activator inhibitor1. Proc. Natl. Acad. Sci. U.S.A. 87, 3530-3533. https://doi.org/10.1073/pnas.87.9.3530
  24. Bagchi, P., Mahesh, M. and Somashekhar, R. (2009) Pharmaco-Informatics: Homology Modelling of the TargetProtein (GP1, 2) for Ebola Hemorrhagic Fever and Predictingan Ayurvedic Remediation of the Disease. J. Proteomics.Bioinform. 2, 287-294. https://doi.org/10.4172/jpb.1000088
  25. Pennica, D., Holmes, W. E., Kohr, W. J., Harkins, R. N.,Vehar, G. A., Ward, C. A., Bennett, W. F., Yelverton, E.,Seeburg, P. H., Heyneker, H. L., Goeddel, D. V. andCollen, D. (1983) Cloning and expression of human tissue-type plasminogen activator cDNA in E. coli. Nature301, 214-221. https://doi.org/10.1038/301214a0
  26. Rouf, S. A., Moo-Young, M. and Chisti, Y. (1996) Tissuetypeplasminogen activator: characteristics, applicationsand production technology. Biotechnol. Adv. 14, 239-266. https://doi.org/10.1016/0734-9750(96)00019-5
  27. Upshall, A., Kumar, A. A., Bailey, M. C., Parker, M. D.,Favreau, M. A., Lewison, K. P., Joseph, M. L., Maraganore,J. M. and McKnight, G. L. (1987) Secretion of active humantissue plasminogen activator from the filamentousfungus Aspergillus nidulans. Nature Biotechnology 5, 1301-1304. https://doi.org/10.1038/nbt1287-1301
  28. Soleimani, M., Mahboudi, F., Davoudi, N., Amanzadeh,A., Azizi, M., Adeli, A., Rastegar, H., Barkhordari, F. andMohajer-Maghari, B. (2007) Expression of human tissueplasminogen activator in the trypanosomatid protozoanLeishmania tarentolae. Biotechnol. Appl. Biochem. 48,55-61. https://doi.org/10.1042/BA20060217
  29. Opdenakker, G. M. M. and It, P. (1996) Protease inhibitors,a DNA construct for the expression of a proteaseand a process for measuring proteases and/or protease inhibitors.(EP0736302).
  30. Hamsten, A., de, F. U., Walldius, G., Dahlen, G., Szamosi,A., Landou, C., Blomback, M. and Wiman, B. (1987) Plasminogenactivator inhibitor in plasma: risk factor for recurrentmyocardial infarction. Lancet 2, 3-9.
  31. Madison, E. L., Goldsmith, E. J., Gerard, R. D., Gething,M. J. and Sambrook, J. F. (1989) Serpin-resistant mutantsof human tissue-type plasminogen activator. Nature 339,721-724. https://doi.org/10.1038/339721a0
  32. Deleage, G. and Roux, B. (1987) An algorithm for proteinsecondary structure prediction based on class prediction.Protein Eng. 1, 289-294. https://doi.org/10.1093/protein/1.4.289
  33. Heussen, C. and Dowdle, E. B. (1980) Electrophoreticanalysis of plasminogen activators in polyacrylamide gelscontaining sodium dodecyl sulfate and copolymerizedsubstrates. Anal. Biochem. 102, 196-202. https://doi.org/10.1016/0003-2697(80)90338-3
  34. Pandya, B. V., Gabriel, J. L., O'Brien, J. and Budzynski, A.Z. (1991) Polymerization site in the beta chain of fibrin:mapping of the B beta 1-55 sequence. Biochemistry 30,162-168. https://doi.org/10.1021/bi00215a024
  35. Chmielewska, J., Ranby, M. and Wiman, B. (1983) Evidencefor a rapid inhibitor to tissue plasminogen activator inplasma. Thromb. Res. 31, 427-436. https://doi.org/10.1016/0049-3848(83)90407-3
  36. Krishnamurti, C., Keyt, B., Maglasang, P. and Alving, B.M. (1996) PAI-1-resistant t-PA: low doses prevent fibrindeposition in rabbits with increased PAI-1 activity. Blood87, 14-19.
  37. Green George, D. J. (1989) Purified type I and type II t-PA.(WO 89/09820).
  38. Kohnert, U., Rudolph, R., Verheijen, J. H., Weening-Verhoeff, E. J., Stern, A., Opitz, U., Martin, U., Lill, H., Prinz,H., Lechner, M. and Lechner, M. (1992) Biochemicalproperties of the kringle 2 and protease domains are maintainedin the refolded t-PA deletion variant BM 06.022.Protein Eng. 5, 93-100. https://doi.org/10.1093/protein/5.1.93

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