ATG induces TF activation on THP1 cells. (A) Flow cytometric analysis of TF antigen expression on monocytic THP1 in comparison with breast cancer MDA-MB231 cells using PE-conjugated TF monoclonal (αTF) and isotype-matched control antibody (IgG). (B) THP1 cells were incubated with PBS, mouse IgG, or inhibitory TF monoclonal antibody (αTF), washed twice, and analyzed for PCA both before (intact) and after repeated freeze-thawing (disrupted) (mean ± standard deviation [SD], n = 3). (C) THP1 cells were incubated with PBS, polyclonal rabbit IgG, or ATG (100 µg/mL) for 15 minutes at RT, washed, and mixed 1:1 (v:v) with NHP for 5 minutes at 37°C. Cell-associated PCA was subsequently measured by a single-stage clotting assay in the presence or absence of inhibitory anti-TF (mean ± SD, n = 4-14). (D) THP1 cells were incubated with increasing concentrations (0-200 µg/mL) of IgG or ATG. Following washing, cells were exposed to NHP and analyzed for PCA as described above (mean ± SD, n = 4). (E) THP1 cells were mixed 1:1 (v:v) with NHP. Following the addition of IgG or ATG (100 µg/mL), cell-associated PCA was measured after indicated time points. (F) THP1 cells were treated with IgG or ATG (100 µg/mL) as described above. Following exposure to NHP, cells were washed and analyzed by thrombin generation assay in the presence or absence of inhibitory anti-TF. Representative experiments are shown in panels A, E, and F.