Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Paper
  • Published:

Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins

Gene Therapy volume 6, pages 385–392 (1999)Cite this article

Abstract

We constructed a hirudin cDNA cassette, HV-1.1, that encodes mature hirudin variant-1 fused to the signal peptide of human tissue-type plasminogen activator (t-PA). The cassette was subcloned into retroviral vectors and used to transduce human vascular endothelial cells in vitro. Hirudin antigen and activity were measured by ELISA and thrombin inhibition assays, respectively. Transduced cells secreted up to 35 ± 2 ng/106 cells/24 h of biologically active hirudin; expression was stable for at least 7 weeks. Recombinant hirudin, expressed from the HV-1.1 cassette, had a specific activity of 7.1 ± 0.2 antithrombin units per microgram (ATU/μg), compared with specific activities of approximately 12 ATU/μg for both native leech hirudin and recombinant hirudin produced in yeast. Protein sequencing and mass spectroscopic analysis revealed the presence of an extra N-terminal serine residue, indicating aberrant cleavage of the t-PA signal peptide and likely accounting for the diminished activity. We therefore constructed a second cDNA cassette, HV-1.2, in which hirudin secretion was directed by the signal peptide of human growth hormone. Hirudin expressed from the HV-1.2 cassette had a specific activity of 13.5 ± 0.2 ATU/μg. Protein sequencing and mass spectroscopic analysis demonstrated proper cleavage of the growth hormone signal peptide. Thus, we achieved high level retrovirus-mediated secretion of biologically active hirudin from endothelial cells in vitro. Use of these vectors may permit sustained local antagonism of thrombin activity in vivo.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Mann KG . Prothrombin and thrombin. In: Colman RW, Hirsh J, Marder VJ, Salzman EW (eds) . Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 3rd edn JB Lippincott: Philadelphia 1994 184–199

    Google Scholar 

  2. Sugama Y et al. Thrombin-induced expression of endothelial P-selectin and intercellular adhesion molecule-1: a mechanism for stabilizing neutrophil adhesion J Cell Biol 1992 119: 935–944

    Article  CAS  PubMed  Google Scholar 

  3. Harker LA, Hanson SR, Runge MS . Thrombin hypothesis of thrombus generation and vascular lesion formation Am J Cardiol 1995 75: 12B–17B

    Article  CAS  PubMed  Google Scholar 

  4. McNamara CA et al. Thrombin stimulates proliferation of cultured rat aortic smooth muscle cells by a proteolytically activated receptor J Clin Invest 1993 91: 94–98

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Okazaki H, Majesky MW, Harker LA, Schwartz SM . Regulation of platelet-derived growth factor ligand and receptor gene expression by α-thrombin in vascular smooth muscle cells Circ Res 1992 71: 1285–1293

    Article  CAS  PubMed  Google Scholar 

  6. Markwardt F . Hirudin as an inhibitor of thrombin Meth Enzymol 1970 19: 924–932

    Article  Google Scholar 

  7. Talbot MD et al. The effects of recombinant desulphatohirudin on arterial thrombosis in rats Haemostasis 1991 21 (Suppl. 1): 73–79

    Google Scholar 

  8. Just M, Tripier D, Seiffge D . Antithrombotic effects of recombinant hirudin in different animal models Haemostasis 1991 21 (Suppl. 1): 80–87

    Google Scholar 

  9. Brill-Edwards P et al. Prevention of thrombus growth by antithrombin III-dependent and two direct thrombin inhibitors in rabbits: implications for antithrombotic therapy Thromb Haemost 1992 68: 424–427

    Article  CAS  PubMed  Google Scholar 

  10. Sarembock IJ et al. Effectiveness of recombinant desulphatohirudin in reducing restenosis after balloon angioplasty of atherosclerotic femoral arteries in rabbits Circulation 1991 84: 232–243

    Article  CAS  PubMed  Google Scholar 

  11. Topol EJ et al. Recombinant hirudin for unstable angina pectoris. A multicenter, randomized angiographic trial Circulation 1994 89: 1557–1566

    Article  CAS  PubMed  Google Scholar 

  12. Serruys PW et al. A comparison of hirudin with heparin in the prevention of restenosis after coronary angioplasty New Engl J Med 1995 333: 757–763

    Article  CAS  PubMed  Google Scholar 

  13. van den Bos AA et al. Safety and efficacy of recombinant hirudin (CGP 39 393) versus heparin in patients with stable angina undergoing coronary angioplasty Circulation 1993 88: 2058–2066

    Article  CAS  PubMed  Google Scholar 

  14. The Global Use of Strategies to Open Occluded Coronary Arteries (GUSTO) IIa Investigators . Randomized trial of intravenous heparin versus recombinant hirudin for acute coronary syndromes Circulation 1994 90: 1631–1637

    Article  Google Scholar 

  15. Rade JJ, Schulick AH, Virmani R, Dichek DA . Local adenoviral-mediated expression of recombinant hirudin reduces neointimal formation after arterial injury Nature Med 1996 2: 293–298

    Article  CAS  PubMed  Google Scholar 

  16. Newman KD et al. Adenovirus-mediated gene transfer into normal rabbit arteries results in prolonged vascular cell activation, inflammation, and neointimal hyperplasia J Clin Invest 1995 96: 2955–2965

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Schulick AH et al. Established immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. Potential for immunosuppression and vector engineering to overcome barriers of immunity J Clin Invest 1997 99: 209–219

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Yang Y et al. Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy Proc Natl Acad Sci USA 1994 91: 4407–4411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Flugelman MY et al. Low level in vivo gene transfer into the arterial wall through a perforated balloon catheter Circulation 1992 85: 1110–1117

    Article  CAS  PubMed  Google Scholar 

  20. Nabel EG . Gene therapy for cardiovascular disease Circulation 1995 91: 541–548

    Article  CAS  PubMed  Google Scholar 

  21. Zhu NL et al. Downregulation of cyclin G1 expression by retrovirus-mediated antisense gene transfer inhibits vascular smooth muscle cell proliferation and neointima formation Circulation 1997 96: 628–635

    Article  CAS  PubMed  Google Scholar 

  22. Wilson JM et al. Implantation of vascular grafts lined with genetically modified endothelial cells Science 1989 244: 1344–1346

    Article  CAS  PubMed  Google Scholar 

  23. Dichek DA et al. Seeding of intravascular stents with genetically engineered endothelial cells Circulation 1989 80: 1347–1353

    Article  CAS  PubMed  Google Scholar 

  24. Nabel EG et al. Recombinant gene expression in vivo within endothelial cells of the arterial wall Science 1989 244: 1342–1344

    Article  CAS  PubMed  Google Scholar 

  25. Konno S, Fenton JW II, Villanueva GB . Analysis of the secondary structure of hirudin and the mechanism of its interaction with thrombin Arch Biochem Biophys 1988 267: 158–166

    Article  CAS  PubMed  Google Scholar 

  26. Walsmann P . Isolation and characterization of hirudin from Hirudo medicinalis Semin Thromb Hemost 1991 17: 83–87

    Article  CAS  PubMed  Google Scholar 

  27. Braun PJ . Binding properties of hirudin determined by gel filtration and gel electrophoresis Thromb Res 1990 59: 657–662

    Article  CAS  PubMed  Google Scholar 

  28. Meyer BJ et al. Dissolution of mural thrombus by specific thrombin inhibition with r-hirudin Circulation 1998 97: 681–685

    Article  CAS  PubMed  Google Scholar 

  29. White HD et al. Randomized, double-blind comparison of hirulog versus heparin in patients receiving streptokinase and aspirin for acute myocardial infarction (HERO) Circulation 1997 96: 2155–2161

    Article  CAS  PubMed  Google Scholar 

  30. Chesebro JH . Direct thrombin inhibition superior to heparin during and after thrombolysis Circulation 1997 96: 2118–2120

    Article  CAS  PubMed  Google Scholar 

  31. Bergmann C et al. Chemical synthesis and expression of a gene coding for hirudin, the thrombin-specific inhibitor from the leech Hirudo medicinalis Biol Chem Hoppe Seyler 1986 367: 731–740

    Article  CAS  PubMed  Google Scholar 

  32. Meyhack B et al. Desulfatohirudin, a specific thrombin inhibitor: expression and secretion in yeast Thromb Res 1987 48 (Suppl. VII): 33

    Google Scholar 

  33. Benatti L, Scacheri E, Bishop DHL, Sarmientos P . Secretion of biologically active leech hirudin from baculovirus-infected insect eggs Gene 1991 101: 225–260

    Article  Google Scholar 

  34. Bird P, Gething M-J, Sambrook J . Translocation in yeast and mammalian cells: not all signal sequences are functionally equivalent J Cell Biol 1987 105: 2905–2914

    Article  CAS  PubMed  Google Scholar 

  35. Tessier DC et al. Enhanced secretion from insect cells of a foreign protein fused to the honeybee melittin signal peptide Gene 1991 98: 177–183

    Article  CAS  PubMed  Google Scholar 

  36. Levin EG . Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzyme–inhibitor complex Proc Natl Acad Sci USA 1983 80: 6804–6808

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. van Zonneveld A-J, Veerman H, Pannekoek H . Autonomous functions of structural domains on human tissue-type plasminogen activator Proc Natl Acad Sci USA 1986 83: 4670–4674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Devlin JJ et al. Novel expression of chimeric plasminogen activators in insect cells Biotechnology 1989 7: 286–292

    CAS  Google Scholar 

  39. Kopfler WP et al. Adenovirus-mediated transfer of a gene encoding human apolipoprotein A-I into normal mice increases circulating high-density lipoprotein cholesterol Circulation 1994 90: 1319–1327

    Article  CAS  PubMed  Google Scholar 

  40. von Heijne G . Patterns of amino acids near signal-sequence cleavage sites Eur J Biochem 1983 133: 17–21

    Article  CAS  PubMed  Google Scholar 

  41. Wiren KW, Potts JT Jr, Kronenberg HM . Importance of the propeptide sequence of human preproparathyroid hormone for signal sequence function J Biol Chem 1988 263: 19771–19777

    CAS  PubMed  Google Scholar 

  42. Matsuoka K, Nakamura K . Propeptide of a precursor to a plant vacuolar protein required for vacuolar targeting Proc Natl Acad Sci USA 1991 88: 834–838

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Goeddel DV et al. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone Nature 1979 281: 544–548

    Article  CAS  PubMed  Google Scholar 

  44. Wallace A, Dennis S, Hofsteenge J, Stone SR . Contribution of the N-terminal region of hirudin to its interaction with thrombin Biochemistry 1989 28: 10079–10084

    Article  CAS  PubMed  Google Scholar 

  45. Zwiebel JA et al. High-level recombinant gene expression inrabbit endothelial cells transduced by retroviral vectors Science 1989 243: 220–222

    Article  CAS  PubMed  Google Scholar 

  46. Connelly S et al. In vivo gene delivery and expression of physiological levels of functional human factor VIII in mice Hum Gene Ther 1995 6: 185–193

    Article  CAS  PubMed  Google Scholar 

  47. Kahn ML, Lee SW, Dichek DA . Optimization of retroviral vector–mediated gene transfer into endothelial cells in vitro Circ Res 1992 71: 1508–1517

    Article  CAS  PubMed  Google Scholar 

  48. Ory DS, Neugeboren BA, Mulligan RC . A stable human-derived packaging cell line for production of high titer retrovirus/vesicular stomatitis virus G pseudotypes Proc Natl Acad Sci USA 1996 93: 11400–11406

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Harker L et al. Reduction in vascular lesion formation (VLF) by hirudin secreted from retroviral-transduced confluent endothelial cells on vascular grafts in baboons Thromb Haemost 1997 77 (Suppl.): 585

    Google Scholar 

  50. Dichek DA, Nussbaum O, Degen SJF, Anderson WF . Enhancement of the fibrinolytic activity of sheep endothelial cells by retroviral vector-mediated gene transfer Blood 1991 77: 533–541

    CAS  PubMed  Google Scholar 

  51. Dichek DA, Lee SW, Nguyen NH . Characterization of recombinant plasminogen activator production by primate endothelial cells transduced with retroviral vectors Blood 1994 84: 504–516

    CAS  PubMed  Google Scholar 

  52. Dichek DA et al. Enhanced in vivo antithrombotic effects of endothelial cells expressing recombinant plasminogen activators transduced with retroviral vectors Circulation 1996 93: 301–309

    Article  CAS  PubMed  Google Scholar 

  53. Dunn PF et al. Seeding of vascular grafts with genetically modified endothelial cells. Secretion of recombinant T-PA results in decreased seeded cell retention in vitro and in vivo Circulation 1996 93: 1439–1446

    Article  CAS  PubMed  Google Scholar 

  54. Shayani V, Newman KD, Dichek DA . Optimization of recombinant t-PA secretion from seeded vascular grafts J Surg Res 1994 57: 495–504

    Article  CAS  PubMed  Google Scholar 

  55. Berg DT, Grinnell BW . Signal and propeptide processing of human tissue plasminogen activator: activity of a pro-t-PA derivative Biochem Biophys Res Commun 1991 179: 1289–1296

    Article  CAS  PubMed  Google Scholar 

  56. McLachlin JR et al. Factors affecting retroviral vector function and structural integrity Virology 1993 195: 1–5

    Article  CAS  PubMed  Google Scholar 

  57. Krall WJ et al. Increased levels of spliced RNA account for augmented expression from the MFG retroviral vector in hematopoietic cells Gene Therapy 1996 3: 37–48

    CAS  PubMed  Google Scholar 

  58. Markowitz D, Goff S, Bank A . A safe packaging line for gene transfer: separating viral genes on two different plasmids J Virol 1988 62: 1120–1124

    CAS  PubMed  PubMed Central  Google Scholar 

  59. Miller AD, Buttimore C . Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production Mol Cell Biol 1986 6: 2895–2902

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Spannagl M, Bichler H, Lill H, Schramm W . A fast photometric assay for the determination of hirudin Haemostasis 1991 21 (Suppl. 1): 36–40

    Google Scholar 

  61. Laemmli UK . Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Nature 1970 227: 680–685

    Article  CAS  PubMed  Google Scholar 

  62. Towbin H, Staehelin T, Gordon J . Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications Proc Natl Acad Sci USA 1979 76: 4350–4354

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Johnson PH et al. Structure-function and refolding studies of the thrombin-specific inhibitor hirudin Haemostasis 1991 21 (Suppl. 1): 41–48

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD, USA

    J J Rade, M Cheung, S Miyamoto & D A Dichek

  2. Division of Cardiology, Johns Hopkins School of Medicine, Baltimore, MD, USA

    J J Rade

  3. Gladstone Institute of Cardiovascular Disease and Department of Medicine, University of California, San Francisco, CA, USA

    D A Dichek

Authors
  1. J J Rade
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. M Cheung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Miyamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D A Dichek
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rade, J., Cheung, M., Miyamoto, S. et al. Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins. Gene Ther 6, 385–392 (1999). https://doi.org/10.1038/sj.gt.3300824

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3300824

Keywords

This article is cited by

Search

Advanced search

Quick links