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

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 50 ) was obtained with (mean±SD; n=3), 26±5 nM wild-type SakSTAR, whereas SakSTAR13, SakSTAR48 and SakSTAR67 at a dose of 200 nM induced ≤3% clot lysis with 2 h. The fibrin-specificity of all the active mutants was comparable to that of wild-type SakSTAR, as shown by unaltered fibrinogen levels. 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.