Construction and characterization of novel staphylokinase variants with antiplatelet aggregation activity and reduced immunogenecity.

To develop target thrombolytic agents with fibrinolytic activity, antiplatelet aggregation activity and reduced immunogenicity, two staphylokinase variants containing Arg-Gly-Asp (RGD) motif were constructed. Gene expression was induced in E. coli JF1125 and the variants, designated DGR and RL1, were purified with gel filtration and ion-exchange chromatography and the purity was over 95%. The fibrinolytic activity and kinetic constants of the two variants were comparable to those of recombinant wild-type staphylokinase. Both the variants can inhibit the platelet aggregation at a final concentration of 2 microM. The titers of antibodies against variants were much lower than those against recombinant staphylokinase in guinea pigs, which indicated that the immunogenicity of the variants was greatly reduced. These results confirm that it is possible to design and produce a bifunctional protein that possesses fibrinolytic and antiplatelet aggregation activities.