Dcf300

To extract the total protein, THP-1 cells were lysed in RIPA buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) containing 20 mM Tris–HCl, 150 mM NaCl, 1 mM Na₂-EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 µg/mL leupeptin, and a protease inhibitor cocktail (Sigma-Aldrich). The cell extracts were centrifuged at 10,000 rpm for 5 min at 4 °C. The supernatants were stored at − 80 °C until further use. The levels of human TF protein in THP-1 cells were measured using an ELISA kit according to the manufacturer’s instructions (#DCF300, R&D Systems, Minneapolis, MN, USA).

Plasma levels of TF (range: 7.8–500 pg/mL), TFPI (range 31.3–2000 pg/mL) and TM (range 62.5–4000 pg/mL) were measured using ELISA kits, in accordance with the manufacturer’s instructions: TF (cat. DCF300, R&D Systems); TFPI (cat. DTFP10, R&D Systems) and TM (cat. DTHBD0, R&D Systems). Standard curves of known concentrations of recombinant human proteins (TF, TFPI and TM) were used to convert optical density (OD) into concentration units (pg/mL). The plates were read in a SpectraMax Paradigm^® machine (Molecular Devices, California, USA). Plasma levels of D-dimer were measured using HemosIL D-Dimer HS 500 kit (cat. 0020500100) on an automated coagulation analyzer ACL TOP 300 CTS (Instrumentation Laboratory, Bedford, MA, USA).