title: Retroviral vector-mediated expression of hirudin by human vascular endothelial cells: implications for the design of retroviral vectors expressing biologically active proteins.
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/10(6) 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/microgram), compared with specific activities of approximately 12 ATU/microgram 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/microgram. 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.
authors.affiliation: Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD, USA.
authors.forename: J J
authors.initials: JJ
authors.lastname: Rade
authors.forename: M
authors.initials: M
authors.lastname: Cheung
authors.forename: S
authors.initials: S
authors.lastname: Miyamoto
authors.forename: D A
authors.initials: DA
authors.lastname: Dichek
mesh_terms: D000991:Antithrombins / Q000235:genetics*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D013006:Growth Hormone / Q000378:metabolism; D006629:Hirudins / Q000235:genetics*; D006801:Humans; D013058:Mass Spectrometry; D021382:Protein Sorting Signals / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D010959:Tissue Plasminogen Activator / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D000991:Antithrombins; D006629:Hirudins; D021382:Protein Sorting Signals; D013006:Growth Hormone; D010959:Tissue Plasminogen Activator