PGN induces monocyte TF expression and extrinsic pathway activation in vivo and in vitro. (A-C) PGN challenge induces monocyte TF expression in vivo and priming of the extrinsic tenase protease. (A) Serial blood smears were methanol fixed, stained overnight at 4°C with biotinylated HTF-1 monoclonal anti-human TF, followed by detection with Cy3-labeled streptavidin (Jackson ImmunoResearch, West Grove, PA). Monocytes were identified morphologically. Confocal images were acquired on a Nikon Eclipse TE2000-U inverted microscope equipped with a Nikon C1 scanning head using EZ-C1 software. Representative micrographs of TF immunostaining in time-paired PGN-challenged baboons are shown (scale bar represents 20 µm). TF staining is stronger in high-dose challenged baboons (right panel) as opposed to low-dose challenge (middle panel). For comparison, no monocyte TF staining is observed on blood smears processed before the high-dose PGN challenge (left panel). (B) Time-course quantitation of TF immunostaining on serial blood smears depicted in panel A. Monocyte TF staining was quantified in at least 5 individual fields from each time point. (C) Time-course evaluation of FVIIa-AT complexes, as a marker of in vivo FVII activation, in PGN-challenged primates. Statistical analysis and representation are consistent with Figure 1. (D-F) PGN stimulation induces de novo TF expression in primary human monocytes in vitro. Time-course analysis of TF mRNA (D), surface antigen levels (E), and TF procoagulant activity (F) on monocytes stimulated with either 10 µg/mL PGN or 1 µg/mL LPS. Data are represented as mean ± SEM from 3 experiments using independent donors. Multiple comparisons were performed by 2-way repeated measures ANOVA followed by Holm-Sidak post hoc test and statistically significant changes from unstimulated (T0) controls are depicted: *P < .05; ** P < .01; ***P < .001.