Biochemical and Immunological Properties of Engineered Low-Immunogenic Staphylokinases for Next-Generation Thrombolytic Therapy

Abstract Staphylokinase (SAK) is a highly fibrin-specific plasminogen activator with significant potential as a safe and affordable thrombolytic. Yet, its clinical translation can be limited by potential immunogenicity. To accelerate the development of improved thrombolytics, a critical step is identifying the most suitable molecular template. Therefore, we performed a comparative analysis of biochemical and immunological properties of three engineered low-immunogenic variants (SAK SY155, SAK THR174, and SAK STAR FRIDA) and two wild-types (SAK STAR and SAK 42D), using a newly established panel of assays. All variants retained potent thrombolytic activity, with SAK SY155 displaying the highest catalytic efficiency and fibrin-clot permeability. However, this advantage did not fully translate into improved clot reduction under flow conditions. Comprehensive immunological profiling, including T lymphocyte proliferation, dendritic cell maturation, mouse immunization models, and human serum reactivity tests, confirmed decreased immunogenicity for two low-immunogenic variants. Overall, low-immunogenic SAK SY155 emerged as the most promising template for rational engineering of next-generation thrombolytics.