Prolonged in vivo anticoagulant activity of a hirudin-albumin fusion protein secreted from Pichia pastoris.
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.
Sheffield
W P
WP
Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ont., Canada. sheffiel@mcmaster.ca
Smith
I J
IJ
Syed
S
S
Bhakta
V
V
D000595:Amino Acid Sequence; D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D015870:Gene Expression; D006629:Hirudins / Q000235:genetics* / Q000494:pharmacology*; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D012709:Serum Albumin / Q000235:genetics* / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000037:antagonists & inhibitors; D014162:Transfection
D000925:Anticoagulants; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D012709:Serum Albumin; D013917:Thrombin