Plasmin

The purified Fbg and citrullinated Fbg were diluted to give a final concentration of 1 mg/mL in PBS (20 μL). Plasmin (34 units/μL, Fujifilm Wako) was diluted in PBS, and the diluted Plasmin solution was added to the Fbg and citrullinated Fbg solutions. The reaction mixtures were incubated at 37 °C for 5 min to 8 h. The reactions were terminated by adding aprotinin (10,000 U/mL, Fujifilm Wako). The samples were centrifuged at 600×g for 10 min. The supernatants were analyzed by SDS-PAGE and western blot.

Purified fibrinogen (Wako Pure Chemical Industries, Tokyo, Japan; 70 µg) in PBS buffer was incubated with thrombin (Wako; final conc. 2 U/mL [17 (link)]), plasmin (Wako; 1 U/mL [18 (link)]) or neutrophil elastase (Sigma-Aldrich, St. Louis, MO, USA; 1 U/mL [19 (link)]) for 2 h at 25 °C. The reaction was stopped by adding EDTA (pH 8.0, 10 mM) and aprotinin (Wako; 1 U/mL). Under these conditions, purified fibrinogen is extensively degraded by thrombin, plasmin and neutrophil elastase [17 (link)–19 (link)].

Serum collection tubes (tubes are evacuated; Insepack II, Sekisui Medical Co.) with added thrombin (Wako; 20 U/mL), hirudin (Thermo Fisher; 1 U/mL [17 (link)]), plasmin (Wako; 0.8 U/mL), tranexamic acid (Wako; 10 mM [18 (link)]), sivelestat sodium (Cosmo Bio Co.; Tokyo Japan; 80 µM [19 (link)]) or the same volume of saline were used to collect blood samples from eleven healthy volunteers with an evacuated by Safetouch™ Blood Collection system (NIPRO Co., Osaka Japan). The collected blood was clotted for 0, 5, 30, 60, 90 min at 25 °C. After blood clotting, serum was obtained by centrifugation at 1500g for 10 min at 4 °C. The level of FIC5.9 was measured using a FIC5.9 ELISA kit described in [6 (link)]. Written informed consent was obtained prior to the sample collection, and the study was approved by the research ethics committee of the graduate school of medicine, Chiba University (Approval no. 677).