This is of interest for diseases, such as systemic infections, rheumatoid arthritis and osteoarthritis, which are associated with an increased activation of coagulation and the presence of physiological concentrations

of coagulation proteases, which may contribute to pro- or anti-inflammatory responses in a PAR-dependent manner. Therefore, in this study, it was investigated whether coagulation proteases (FVIIa, the binary TF-FVIIa complex, the binary TF-FVIIa complex with free FX, free FX, free FXa and thrombin) in physiological concentrations can elicit pro- or anti-inflammatory responses in a PAR-dependent manner in naïve (non-preactivated) human monocytes and PBMCs. Ficoll-Paque was purchased from Pharmacia (Uppsala, Sweden) and CD14 microbeads from Miltenyi Biotec (Bergisch Gladbach, Autophagy Compound Library cell line Germany). Dulbecco’s modified Eagle’s medium (DMEM) was obtained from Invitrogen (Carlsbad, CA, USA). Heat-inactivated human male AB serum was from

Sigma-Aldrich (St. Louis, MO, USA). Allophycocyanin (APC)-conjugated monoclonal mouse anti-human CD14 antibody and APC-conjugated isotype control antibody were from BD Biosciences (Franklin Lakes, NJ, USA). Phycoerythrin (PE)-conjugated monoclonal mouse anti-human PAR-1 (ATAP2) antibody, FITC-conjugated monoclonal mouse anti-human PAR-2 (SAM11) antibody, buy LY294002 PE-conjugated monoclonal mouse anti-human PAR-3 (8E8) antibody, and APC-, PE- and FITC-conjugated isotype control antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). FITC-conjugated polyclonal rabbit anti-human PAR-4 (APR-034-F) antibody was obtained from Alomone Labs (Jerusalem, Israel). PE-conjugated monoclonal mouse anti-human TF (HTF-1) antibody and PE-conjugated isotype control antibody were from BD Biosciences. Recombinant human FVIIa was kindly provided by Novo Nordisk A/S (Maaloev, Denmark). Recombinant human tissue factor (4500L), human factor X (527) and human activated factor X (526) were purchased from American Diagnostica

Inc. (Stamford, CT, USA). Human alpha thrombin factor IIa (IHT; activity ≥2700 NIH units/mg) was obtained from Innovative Research (Novi, USA). The HSP90 activity of the purchased coagulation proteases was tested positive in coagulation assays before use. Purified LPS was purchased from Sigma-Aldrich. PAR-1 antagonist FR171113 was obtained from Tocris Bioscience (Bristol, UK). FR171113 is a highly purified (>98%) specific PAR-1 antagonist which is able to inhibit thrombin-induced platelet aggregation. Interleukin-1β (IL-1β), Interleukin-10 (IL-10) and tumour necrosis factor-alpha (TNF-α) enzyme-linked immunosorbent assay (ELISA) kits were from Invitrogen. Interleukin-6 and IL-8 ELISA kits were obtained from eBioscience (San Diego, CA, USA). All other chemicals were from Sigma-Aldrich. Peripheral blood was obtained from five different healthy donors after informed consent (age 37.2 ± 4.9 years; 2 males and 3 females). PBMCs were isolated by Ficoll-Paque (Pharmacia) according to standard procedures.