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.

SummaryHeparin cofactor II (HCII), a circulating plasma protein that inhibits thrombin, is a member of the serine proteinase (serpin) family of proteins. The extent to which HCII structure is conserved actross species lines was investigated, by obtaining cDNA clones encoding rabbit HCII. Overlapping clones corresponding to rabbit HCII were obtained by the combined use of hybridization screening of a rabbit liver cDNA library, and by rapid amplification of cDNA ends (RACE). The consensus sequence obtained spans 2178 nucleotides, and is comprised of a 5' untranslated region of 77 nucleotides, an open reading frame of 1440 nucleotides, and 3' untranslated region of 661 nucleotides that concludes with a poly A tract. The open reading frame is subdivided into a secretory signal sequence of 19 amino acids, and a mature protein of 461 amino acids. Within the region comprising the mature protein, 87% of the amino acid residues are identical to those seen in human HCII. Expression of an appropriately modified form of the rabbit HCII clone in an in vitro reticulocyte expression system yielded two major polypeptides, of 60 and 56 kD respectively, both of which were able to form SDS-stable complexes with human α-thrombin, in a reaction accelerated by dermatan sulphate. The remarkable degree of homology observed between rabbit HCII and its human conterpart, indicating a high degree of conservation of structure through evolution, suggests an important function of HCII in hemostatis.