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Title: Expression and characterization of clustered charge-to-alanine mutants of low Mr single-chain urokinase-type plasminogen activator
Authors: Ueshima, S ×
Holvoet, Paul
Lijnen, Roger
Nelles, Lucien
Seghers, V
Collen, Desire #
Issue Date: Jan-1994
Series Title: Thrombosis and haemostasis vol:71 issue:1 pages:134-40
Abstract: In an effort to modify the fibrinolytic and/or pharmacokinetic properties of recombinant low M(r) single-chain urokinase-type plasminogen activator (rscu-PA-32k), mutants were prepared by site-directed mutagenesis of clusters of charged amino acids with the highest solvent accessibility. The following mutants of rscu-PA-32k were prepared: LUK-2 (Lys 212, Glu 213 and Asp 214 to Ala), LUK-3 (Lys 243 and Asp 244 to Ala), LUK-4 (Arg 262, Lys 264, Glu 265 and Arg 267 to Ala), LUK-5 (Lys 300, Glu 301 and Asp 305 to Ala) and LUK-6 (Arg 400, Lys 404, Glu 405 and Glu 406 to Ala). The rscu-PA-32k moieties were expressed in High Five Trichoplasiani cells, and purified to homogeneity from the conditioned cell culture medium, with recoveries of 0.8 to 3.7 mg/l. The specific fibrinolytic activities (220,000 to 300,000 IU/mg), the rates of plasminogen activation by the single-chain moieties and the rates of conversion to two-chain moieties by plasmin were comparable for mutant and wild-type rscu-PA-32k moieties, with the exception of LUK-5 which was virtually inactive. Equi-effective lysis (50% in 2 h) of 60 microliters 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained with 0.7 to 0.8 microgram/ml of wild-type or mutant rscu-PA-32k, except with LUK-5 (no significant lysis with 16 micrograms/ml). Following bolus injection in hamsters, all rscu-PA-32k moieties had a comparably rapid plasma clearance (1.3 to 2.7 ml/min), as a result of a short initial half-life (1.4 to 2.5 min).(ABSTRACT TRUNCATED AT 250 WORDS)
ISSN: 0340-6245
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Atherosclerosis and Metabolism (-)
Molecular and Vascular Biology
× corresponding author
# (joint) last author

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