The HIT Ab complex induces TF expression in PBMCs and monocytes. (A) PBMCs were incubated for 6 hours with the different reagents, and the number of TF⁺/CD14⁺ double-positive events was measured with flow cytometry. Results are expressed as MFI (of the anti-TF Ab on CD14⁺ cells). (B) Measurement of cellular TF activity. An additional control consisted of RTO (isotype control Ab for KKO, 100 μg/mL) + heparin + PF4. TF activity was measured with a 1-stage clotting assay. (C) Monocytes were incubated in media alone (control), KKO (100 μg/mL), KKO (100 μg/mL) + heparin (1 U/mL) + PF4 (10 μg/mL), and LPS (1 μg/mL) for 2 hours. Total RNA was isolated from the cells, and TF mRNA levels were determined by real-time PCR. Results are presented as relative TF mRNA levels compared with HPRT. Results are from 5 independent experiments. ***P < .001, **P < .01, and *P < .05 compared with control.