Summary
Chimeric 59D8-SK was designed to confer fibrin-selectivity to streptokinase by fusion
of the Fab fragment of anti-fibrin antibody 59D8 to the N-terminus of streptokinase
(SK: Ile1-Lys414). It was expressed in a mouse hybridoma cell line and purified by affinity chromatography
on a 59D8-antigen column. Chimeric 59D8-SK is a disulfide-linked heterodimer composed
of an antibody light chain (Mr 27,000) and a N-glycosylated chimeric heavy chain (Mr
90,000). The fibrin targeting by 59D8 increased plasma clot lysis by 2-fold, but connecting
59D8 to SK has provided 59D8-SK several unique properties: (i) 59D8-SK activated human
Glu-plasminogen with a significant lag period that coincided with limited proteolysis
of 59D8-SK similar to that observed for wild-type SK. In a kinetic study, both gave
very similar kinetic parameters for the activation of Glu-plasminogen even though
59D8-SK was N-glycosylated in its SK portion; (ii) 59D8-SK was relatively inactive
in human plasma, compared to SK, but it became activated in the presence of clots;
(iii) 59D8-SK lysed clots slowly but completely whereas SK lysed clots rapidly but
incompletely. Even though the mechanism behind these new properties is not fully understood,
they are characteristics of a second-generation plasminogen activator.
