Wp. Sheffield et al., Prolonged in vivo anticoagulant activity of a hirudin-albumin fusion protein secreted from Pichia pastoris, BL COAG FIB, 12(6), 2001, pp. 433-443
Hirudin is a small, proteinaceous thrombin inhibitor that clears rapidly fr
om the circulation. A hexahistidine-tagged hirudin-rabbit serum albumin (RS
A) fusion protein, HLAH(6), was characterized following secretion from Pich
ia pastoris. HLAH(6) bound to immobilized nickel, anti-RSA, and anti-hexahi
stidine antibodies, and contained the expected (ITYTD) N-terminus. Its spec
trometric mass was 74490 (versus the theoretical mass of 74410 and sodium d
odecyl sulfate-polyacrylamide gel electrophoresis mobility of 84 kDa). The
terminal catabolic half-life in rabbits of HLAH(6), recombinant Pichia-deri
ved His-tagged RSA, or plasma-derived RSA did not differ. Injection of 2 mg
/kg HLAH(6) into rabbits raised the activated partial thromboplastin time (
aPTT) above initial values for 4-24 h, while the equimolar dose of unfused
hirudin was without significant effect. A higher dose of HLAH6 (3 mg/kg fun
ctional HLAH(6), equivalent to 37.6 thrombin-inhibitory units/g) raised the
aPTT by 2.0- to 2.5-fold; the elevation persisted for > 48 h. Importantly,
both HLAH(6) and unfused hirudin inhibited clot-bound thrombin. Our result
s suggest that HLAH(6) exhibits not only delayed clearance, but also prolon
ged biological activity in vivo compared with unfused hirudin. (C) 2001 Lip
pincott Williams & Wilkins.