Anticoagulant Activity of UcB5 Protease

The next experiments were approved by an appropriate institution. In addition to this, all methods were performed following the relevant guidelines and regulations including ARRIVE guidelines. The in vitro anticoagulant activity was examined as the increase in the coagulation period of human blood serum in the existence of 15 µg of UcB5 protease/ml^{15 (link)}. Exactly, 100 µl of blood serum was vortexed with equivalent volumes of each thromboplastin and kaolin. After 2 min incubation at 37 °C in a water bath, exactly 100 µl of 0.3% (w/v) CaCl₂ and 100 µl of the enzyme were added. The clotting time in the presence of the enzyme was then determined in comparison with blanks containing an equivalent amount of physiological saline instead of the purified enzyme.