Fibrinolytic properties of staphylokinase mutants obtained by ‘clustered charge-to-alanine’ mutagenesis

Abstract

Summary The fibrinolytic properties of 18 mutants of recombinant staphylokinase (SakSTAR) in which clusters of two or three charged residues were converted to alanine (‘clustered charge-to-alanine scan’) were evaluated. The specific fibrinolytic activity, determined in a clot lysis time assay, of SakSTAR13 (with K11, D13 and D14 to A), SakSTAR48 (with E46 and K50 to A), and SakSTAR67 (with E65 and D69 to A) was Evaluation of the thrombolytic properties of selected SakSTAR moieties in hamsters with pulmonary embolism revealed dose-dependent clot lysis with wild-type SakSTAR (30–82% lysis with doses of 9–81 μg/kg), SakSTAR13 (32–63% lysis with doses of 81–750 μg/kg) and SakSTAR67 (27–73% lysis with doses of 81–250 μg/kg). 50% clot lysis was obtained at 7- to 10-fold higher dose for SakSTAR67 (175 μg/kg) or SakSTAR13 (250 μg/kg) than for wild-type SakSTAR (25 μg/kg). Thus, three clusters of charged amino acids were identified in SakSTAR (regions 11–14, 46–50 and 65–69) which are important for its fibrinolytic potency.