Biochemical Characterization of a Purified Glycosylated β-Fibrinogenolytic Serine Protease Preparation from Gloydius ussuriensis Venom

Snake-venom serine proteases can alter hemostasis through diverse effects on fibrinogen and coagulation factors. Here, a heat-resistant protease under the tested conditions, designated TLSP, was purified from Gloydius ussuriensis venom by sequential anion-exchange and size-exclusion chromatography and characterized biochemically. TLSP migrated at approximately 35 kDa under reducing conditions and 26 kDa under non-reducing conditions. Periodic acid–Schiff staining and N-glycosidase F treatment indicated N-linked glycosylation. The N-terminal sequence, VVGGDEHNIN, showed high similarity to reported snake-venom thrombin-like serine proteases. TLSP preferentially hydrolyzed Boc-Val-Pro-Arg-pNA, with Km = 0.3 mM and kcat = 8.69 s−1, and its activity was strongly inhibited by aprotinin and moderately reduced by PMSF. TLSP preferentially cleaved the β-chain of human fibrinogen but did not induce fibrin-polymer formation or degrade preformed or factor XIIIa-cross-linked fibrin. The optimum pH was approximately 9.0, and 59% of amidolytic activity remained after heat treatment at 100 °C. N-glycosidase F treatment was accompanied by loss of detectable amidolytic activity. These findings characterize TLSP as a glycosylated β-fibrinogenolytic serine protease showing marked heat resistance under the tested conditions, with thrombin-like substrate preference but without thrombin-like fibrin-clotting activity.