TF activation by ATG requires lipid raft integrity, but does not involve TFPI inhibition. (A) THP1 cells were incubated with PBS, IgG, or ATG (100 µg/mL), washed, and either left intact or disrupted by repeated freeze-thawing. Intact and disrupted cells were mixed 1:1 (v:v) with NHP for 5 minutes at 37°C and analyzed for PCA by a single-stage clotting assay (mean ± SD, n = 3). THP1 cells were pretreated with (B) filipin or (C) methyl-β-cyclodextrin (MBCD) before being loaded with rabbit IgG or ATG (100 µg/mL) and analyzed for PCA as described above (mean ± SD, n = 5). (D) Binding of IgG or ATG (100 µg/mL) to THP1 cells was evaluated using indirect flow cytometry with or without (left) filipin or (right) MBCD pretreatment. Representative histograms are shown. (E) Analysis of TFPI antigen expression on THP1 cells by indirect flow cytometry using polyclonal anti-TFPI (αTFPI) in comparison with control IgG. A representative experiment is shown. (F) THP1 cells were suspended in calcium-HBS and incubated with PBS, ATG (100 µg/mL), inhibitory anti-TFPI (50 µg/mL), and/or calcium ionophore A23187 (20 µM) for 15 minutes at 37°C. Cells were washed, resuspended in PBS, and exposed to NHP before PCA was measured as described above (mean ± SD, n = 3).