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Potent antithrombin activity and delayed clearance from the circulation
characterize recombinant hirudin genetically fused to albumin
S Syed, PD Schuyler, M Kulczycky and WP Sheffield
Department of Pathology, McMaster University, Hamilton, Ontario, Canada.
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.
Volume 89,
Issue 9,
pp. 3243-3252,
05/01/1997
Copyright © 1997 by The American Society of Hematology
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