Summary
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 moictic3 were expressed in High Five Ttichoplasiani cells, and purified
to humugciicily from the conditioned cell culture medium, with recoveries of 0.8 to
3.7 mg/1. 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
In lwo 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 pi 125I-fibrin labeled plasma clots submerged in 0.5 ml normal human plasma was obtained
with 0.7 to 0.8 μg/ml of wild-type or mutant rscu-PA-3?.k, except with LUK-5 (no significant
lysis with 16 pg/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). In hamsters with pulmonary embolism, continuous
intravenous infusion over 60 min at a dose of 1 mg/kg, resulted in 53 to 72% clot
lysis with the mutants, but only 23% with LUK-5, as compared to 36% for wild-type
rscu-PA-32k.
These data indicate that clustered charge-to-alanine mutants of rscu-PA-32k, designed
to eliminate charged regions with the highest solvent accessibility, do not have significantly
improved functional, fibrinolytic or pharmacokinetic properties.
