Biochemical and Functional Characterization of LIZ, a Marine-Derived Serine Protease That Degrades Cross-Linked Fibrin and Attenuates Experimental Thrombosis

Background/Objectives: Direct fibrin-degrading proteases may provide a mechanistically distinct approach to clot proteolysis, but their development requires definition of catalytic behavior, fibrin selectivity, molecular identity, and safety. We characterized an activity-containing preparation from the marine polychaete Lepidasthenia izukai, designated LIZ.

Methods

LIZ was obtained by ammonium sulfate fractionation and sequential chromatography. N-terminal sequencing, inhibitor profiling, chromogenic-substrate screening and S-2288 kinetics, fibrinogenolysis, direct fibrin-degradation assays, a κ-carrageenan rat-tail thrombosis model, and cell viability were evaluated.

Results

The final preparation showed a predominant band at approximately 28 kDa. N-terminal sequencing yielded IMGGSEAS, providing sequence-level characterization without definitive molecular identification. PMSF abolished activity and DFP reduced it to 9.8%, supporting a serine-protease biochemical profile. For S-2288, Km was 0.029 ± 0.003 mM and apparent kcat/Km was 39.7 ± 4.3 mM−1 s−1. LIZ degraded fibrinogen Aα within 30 s and Bβ and γ within 20 min, decreased pre-formed fibrin-polymer turbidity, and degraded FXIIIa-cross-linked fibrin. In the pretreatment model, thrombotic tail length was 0.7 ± 1.0% with LIZ, 86.1 ± 3.2% with κ-carrageenan alone, and 0.5 ± 0.6% with uPA (n = 3/group).