title: Potent antithrombin activity and delayed clearance from the circulation characterize recombinant hirudin genetically fused to albumin.
abstract: In this study we sought to extend the plasma half-life while maintaining the potent antithrombin activity of hirudin. We hypothesized that gene fusion of hirudin to albumin would result in the expression of a slowly cleared hirudin molecule. A hirudin variant 3 (HV3) cDNA was obtained by gene synthesis, while a 1,996-bp full-length rabbit serum albumin (RSA) cDNA was selected from a rabbit liver cDNA library. Expression of the former in COS-1 cells conferred antithrombin activity on media conditioned by the cells, while expression of the latter resulted in the secretion of a 67-kD protein that reacted with mono-specific anti-RSA antibodies. Having shown independent expression of the two proteins, we next expressed two fusion proteins: HV3 linked via its C-terminus to albumin (HLA), and HV3 linked via its N-terminus to albumin (ALH). The former, but not the latter, inhibited both the amidolytic and fibrinogenolytic activities of thrombin. HLA also retained the dye-binding characteristics of RSA, as judged by Affi-Gel Blue chromatography. Highly similar concentrations of either commercial HV1 (40 nmol/L) or HLA (30 nmol/L) were required to halve the initial rate of thrombin reaction with chromogenic substrate S2238, suggesting the retention of high-affinity inhibition of thrombin by the fusion protein. An His-tagged form of HLA was purified by Ni2+-chelate affinity and heparin-Sepharose chromatography. The purified, radioiodinated protein was injected into rabbits, and demonstrated a catabolic half-life of 4.60 +/- 0.16 days. This represents an extension of hirudin half-life in vivo of greater than two orders of magnitude; gel analysis of HLA(H)6 recovered from rabbits showed that it circulated in intact form. Our results provide a rationale for future testing of the biological effects of HLA, and support our initial hypothesis.
authors.affiliation: Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
authors.forename: S
authors.initials: S
authors.lastname: Syed
authors.forename: P D
authors.initials: PD
authors.lastname: Schuyler
authors.forename: M
authors.initials: M
authors.lastname: Kulczycky
authors.forename: W P
authors.initials: WP
authors.lastname: Sheffield
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000493:pharmacokinetics*; D019556:COS Cells; D017931:DNA Primers; D015723:Gene Library; D005813:Genes, Synthetic; D014644:Genetic Variation; D006207:Half-Life; D006629:Hirudins / Q000096:biosynthesis / Q000097:blood / Q000493:pharmacokinetics*; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000097:blood / Q000493:pharmacokinetics*; D012709:Serum Albumin / Q000096:biosynthesis / Q000737:chemistry / Q000493:pharmacokinetics*; D014162:Transfection
chemical_list: D000991:Antithrombins; D017931:DNA Primers; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin