Prolonged in vivo anticoagulant activity of a hirudin-albumin fusion protein secreted from Pichia pastoris

Citation
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
Citations number
35
Categorie Soggetti
Cardiovascular & Hematology Research
Journal title
BLOOD COAGULATION & FIBRINOLYSIS
ISSN journal
09575235 → ACNP
Volume
12
Issue
6
Year of publication
2001
Pages
433 - 443
Database
ISI
SICI code
0957-5235(200109)12:6<433:PIVAAO>2.0.ZU;2-X
Abstract
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.