Human α thrombin

Fibrinogen polymerization was measured based on turbidity changes at 340 nm using a Thermo Scientific Multiskan GO spectrophotometer. The final concentrations of the sample and reagents in 100 μL of HBS buffer were as follows: 0.1-NIH U/mL human α-thrombin (Hyphen BioMed) or 0.1-U/mL batroxobin (Nuokang Bio-pharmaceutical), 1mM calcium chloride (CaCl₂) and 0.5-mg/mL fibrinogen purified from the proband and normal control. This mixture was incubated at 37 °C for 110 minutes. Three important parameters were calculated based on the change in turbidity with time: lag time, maximum slope (V_max), and final turbidity.
Fibrinolysis was examined by adding 80-μg/mL human Glu-plasminogen (Haematologic Technologies), 1mM CaCl₂, and 1.2nM recombinant human tissue plasminogen activator (Alteplase; Boehringer Ingelheim) to the fibrin clots formed in the polymerization experiment mentioned above. In particular, according to the results of clottability and polymerization, the fibrinogen concentration of the proband in the thrombin group was moderately increased (∼1.1 times, Supplementary Figure S1A) to ensure the same amount of fibrin clots involved in fibrinolysis progress. The change in optical density was monitored at 340 nm for 145 minutes, and the total lysis times were measured based on the curves. All experiments were performed in triplicates.