Prolonged in vivo anticoagulant activity of a hirudin–albumin fusion protein secreted from Pichia pastoris
- Additional Document Info
- View All
Hirudin is a small, proteinaceous thrombin inhibitor that clears rapidly from the circulation. A hexahistidine-tagged hirudin-rabbit serum albumin (RSA) fusion protein, HLAH6, was characterized following secretion from Pichia pastoris. HLAH6 bound to immobilized nickel, anti-RSA, and anti-hexahistidine antibodies, and contained the expected (ITYTD) N-terminus. Its spectrometric mass was 74,490 (versus the theoretical mass of 74,410 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobility of 84 kDa). The terminal catabolic half-life in rabbits of HLAH6, recombinant Pichia-derived His-tagged RSA, or plasma-derived RSA did not differ. Injection of 2 mg/kg HLAH6 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 functional HLAH6, 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 HLAH6 and unfused hirudin inhibited clot-bound thrombin. Our results suggest that HLAH6 exhibits not only delayed clearance, but also prolonged biological activity in vivo compared with unfused hirudin.
has subject area