Expression and characterization of clustered charge-to-alanine mutants of low M(r) single-chain urokinase-type plasminogen activator.
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)
Ueshima
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S
Center for Molecular and Vascular Biology, University of Leuven, Belgium.
Holvoet
P
P
Lijnen
H R
HR
Nelles
L
L
Seghers
V
V
Collen
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D006224:Cricetinae; D006207:Half-Life; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000493:pharmacokinetics; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis / Q000235:genetics* / Q000493:pharmacokinetics
D011993:Recombinant Fusion Proteins; D014568:Urokinase-Type Plasminogen Activator