Role of PDI in cellular TF activation by ATG. (A) Effect of thiol blockade by N-ethylmaleimide (NEM) on ATG-mediated (left) TF activation and (right) PS exposure. Summary statistics (mean ± SD, n = 8) and representative histograms are shown. (B) Indirect flow cytometric analysis of PDI antigen expression on THP1 cells using the monoclonal antibody RL90. A representative experiment is shown. (C) THP1 cells were treated with PBS, IgG, or ATG (100 µg/mL) or rutin (100 µM) before cell-associated insulin reductase activity was measured. Results are presented as fluorescence units (FUs) per 10⁵ cells. A representative experiment is shown. (D) IgG- or ATG-loaded THP1 cells were incubated with 100 µM rutin or vehicle for 10 minutes at RT, mixed with NHP, and analyzed for PCA as described above. Results are presented as percent of maximum PCA (mean ± SD, n = 3). (E) ATG-loaded THP1 cells received either PBS or 50 µg/mL control IgG_2a, RL90, or inhibitory anti-TF for 15 minutes at RT before being exposed to NHP and analyzed for PCA (mean ± SD, n = 9). (F) ATG-loaded THP1 cells received RL90 either before (●) or 5 minutes after mixing with NHP (○). The PDI antibody was allowed to incubate for 15 minutes. Results are presented as percent of maximum PCA. A representative experiment is shown. (G) Flow cytometric analysis of annexin V-FITC binding to ATG-loaded and NHP-exposed THP1 cells preincubated with IgG_2a or RL90. A representative experiment is shown. (H) Flow cytometric analysis of PDI antigen expression on HL60 cells using the monoclonal antibody RL90. (I) (Left) Effect of RL90 and PDI inhibitors on insulin reductase activity on HL60 cells. Results are presented as percent PDI activity of untreated cells (mean ± SD, n = 2-5). (Right) Effect of RL90 and PDI inhibitors on ATG-mediated TF activation on HL60 cells. Results are presented as percent PCA of ATG-loaded cells exposed to NHP (mean ± SD, n = 2-5). (J) Following loading of isolated monocytes with rabbit IgG or ATG (100 µg/mL), cell-associated PCA was measured in the presence of PBS, 50 µg/mL eculizumab (αC5), 100 µM rutin, or 20 µg/mL inhibitory anti-TF. Results are presented as fold increase over IgG-treated monocytes in the presence of PBS (mean ± SD, n = 5-11).