Fibrinolysis: An International Journal of Fibrinolysis and Thrombolysis vol:10 issue:3 pages:177-182
date:INST MOL BIOTECHNOL,D-6980 JENA,GERMANY
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 evaluted. 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 less than or equal to 5% of that of wild-type SakSTAR (91+/-10 home units (HU)/mu g), whereas that of the other mutants was greater than or equal to 30% of that of the wild-type molecule. In an in vitro system consisting of a 0.06 ml radiolabeled human plasma clot submerged in 0.5 ml plasma, 50% clot lysis in 2 h (C-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 less than or equal to 3% clot lysis witin 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 mu g/kg), SakSTAR13 (32-63% lysis with doses of 81-750 mu g/kg) and SakSTAR67 (27-73% lysis with doses of 81-250 mu g/kg). 50% clot lysis was obtained at 7- to 10-fold higher dose for SakSTAR67 (175 mu g/kg) or SakSTAR13 (250 mu g/kg) than for wild-type SakSTAR (25 mu g/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.