stimulated wild-type TF promoter. Discussion Emerging evidence has suggested a potential role for HMGB1 in the pathogenesis of atherosclerosis [14]. Moreover, HMGB1 increased expression of Egr-1 and nuclear translocation of NF-B (c-Rel/p65) in ECs. Taken together, our data suggest that HMGB1 induces TF expression in vascular endothelial cells via cell surface receptors (TLR4, TLR2, and RAGE), and through activation of transcription factors (NF-B and Egr-1). amebocyte lysate assay (Endochrome, Charles River), and contained 500 pg endotoxin per microgram of rHMGB1. Cell culture Primary human coronary artery endothelial cells (HCAECs) (Clonetics) were cultured in EGM-2MV medium (Clonetics) with full supplements and 5% FBS as described [25]. The purity of EC cultures was 99% as determined by immunostaining with the anti-von Willebrand factor monoclonal antibody (Dako, Carpinteria, CA). Twenty-four hours prior to the experiment, ECs were cultured in M199 supplemented with 0.1% human serum albumin. Human umbilical vein endothelial cell line (HUVEC-CS) was purchased from American Type Culture Collection (ATCC) and maintained at 37C under 5% CO2 in RPMI 1640 medium (Gibco) supplemented with 10% fetal calf serum (Gibco). Primary peritoneal macrophages were isolated from Balb/C mice as previously described [26]. Murine macrophages were pre-cultured in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco) and 2 mmol/L glutamine. All experiments with recombinant HMGB1 were performed in the presence of 1 g/mL polymyxin Ro 31-8220 mesylate B (PMB) to neutralize activities of contaminating LPS. Murine macrophage-like RAW264.7 cells, obtained from the American Type Culture Collection (ATCC, MD), were cultured in RPMI medium 1640 (Life Technologies, NY) supplemented with 10% heatinactivated FBS (GIBCO/BRL), 2mM glutamine (GIBCO/BRL). Western blot analysis Cells were lysed in 2 sodium dodecyl sulfate (SDS) buffer, and resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were transferred to a PVDF membrane (Millipore) by semidry transfer, and equal loading was confirmed by Ponceau S staining. Antibodies to human TF and TF pathway inhibitor (TFPI) (both from American Diagnostica Inc.) were used at 1:1000 dilution. Blots were normalized to GAPDH expression (1:2000 dilution; Sigma). Total RNA extraction and real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted using the TRIzol reagent (Invitrogen Corp., Carlsbad, CA), according to the manufacturers instructions. Reverse transcription was performed as described earlier [27]. Total RNA (1 g) was incubated with DNaseI, and real-time quantitative PCR Hoxa10 was performed as described previously [27]. TF activity analysis The actichrome TF activity assay kit (American Diagnostica Inc.) was used to determine the TF activity of cell lysates of ECs following HMGB1 (1 g/mL) stimulaiton. Protein samples (30 g) were assayed for the cellular TF activity in accordance with the manufacturers instructions. Ro 31-8220 mesylate For measuring the cell surface TF activity, ECs at the Ro 31-8220 mesylate same density of 104 cells/well were seeded onto 96-well plates. Following overnight incubation, cells were treated with 1 g/mL HMGB1 for the indicated time periods. Cell monolayers were washed twice with PBS, and the reagents were directly added to measure TF activity. Procoagulant activity assay Murine macrophages were solubilized with 15 mmol/L octyl-D-glucopyranoside (Sigma) at 37C for 15 min, and the procoagulant activities were evaluated using a one-step clotting assay, as described previously [11]. Blocking of TLR4, TLR2, or RAGE Primary ECs were pretreated for 30 min at 37C with mouse anti-human TLR4 (eBioscience, Catalog Number: 16C9917; 20 g/mL), mouse anti-human TLR2 (eBioscience, Catalog Number: 16C9922; 20 g/mL), mouse anti-human RAGE (R&D Systems, Catalog Number: MAB11451; 20 g/mL), or control IgG2a antibody (eBioscience, Catalog Number: 16C4724; 60 g/mL). The cells were harvested at 5 h after stimulation for obtaining cell lysates and at 3 h after stimulation for RNA extraction. Effects of HMGB1 on NF-B (c-Rel/p65 heterodimer) nuclear translocation and Egr-1 expression Nuclear protein was isolated using the Nuclear Extraction Kit (Chemicon). NF-B (c-Rel/p65 heterodimer) nuclear translocation and Egr-1 expression were measured by western blotting. Antibodies against human c-Rel, p65, and Egr-1 (all from Cell Signaling) were used at 1:1000 dilutions. Blots were normalized to PCNA Ro 31-8220 mesylate expression (1:10000 dilution; Sigma). Plasmids and transient transfection The wild-type human TF promoter (?227) and a construct containing a mutation of the NF-B (NF-Bm) or Egr-1 (Egr-1m) site have been described elsewhere [6]. For transfection, the HUVEC-CS cell line (ATCC) was seeded at a density of 105 cells/well in a 24-well culture plate one day before the experiment. The cells were transfected with 400 ng DNA/well for 5 h using the Lipofectamine reagent (Gibco), according to the manufacturers protocol. All samples were co-transfected with equal amounts of a pRL-TK construct encoding luciferase (Promega) to compensate for variations in transfection efficiencies. Twenty-four hours after transfection, the cells were incubated with HMGB1 (10 g/mL) for 12 h. The cells were harvested, and luciferase assays were performed using the Ro 31-8220 mesylate dual luciferase reporter system (Promega) according to the manufacturers protocol. Statistical analysis.