Fibrinogen

Recombinant osteopontin was purchased from R&D Systems (Minneapolis, MN, USA, #1433-OP-050/CF), ACROBiosystems (Newark, DE, #OPN-H5227), and Abcam (Cambridge, UK, #ab92964, #ab281819). Fibronectin, tenascin-C, bone sialoprotein, and vimentin were purchased from R&D Systems (Minneapolis, MN, USA). a-enolase and type II collagen were purchased from Abcam (Cambridge, UK). Fibrinogen was purchased from the Fujifilm Wako Pure Chemical Corporation (Osaka, Japan). Antibodies against total focal adhesion kinase (FAK) and phosphorylated FAK were purchased from Cell Signaling Technology (Danvers, MA, USA, #3285 and #8556). Recombinant human tumor necrosis factor (TNF) was purchased from Peprotech (Cranbury, NJ, USA, #300-01A). The secondary antibody used for immunoblot analysis was peroxidase-conjugated anti-rabbit IgG (Agilent, Santa Clara, CA, USA; #P0399).

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)].