Prothrombotic aPLs dissociate an inhibited cell surface TF-FVIIa-FXa-TFPI complex. (A) Human monocytic MM1 cells were cultured in human serum, stained with αFVII-12C7 with a known epitope in the FVII protease domain,⁴⁶  αFX-f21-4.2C, and αTFPI-HG5 (both identified by screening for reactivity with a preassembled TF-FVIIa-FXa-TFPI complex on CHO-cells), and imaged on live cells after 15 minutes of exposure to control IgG or aPL HL5B. Nuclei were stained in blue with Hoechst 33342 or DAPI, surface membrane with cholera toxin B (CTB), and endo-lysosomes with Lysotracker or αEEA1 (red). Scale bar = 5 µm. (B) Loss of FXa surface staining on mouse CD115⁺ splenocytes grown in human serum by flow cytometry after 10 minutes of stimulation; mean ± standard deviation (SD). n = 6. *P < .0062; 1-way ANOVA, Dunnett multiple-comparison test vs unstimulated. (C) FXa clotting activity in the supernatant of aPL-stimulated monocytes; mean ± SD. n = 3. *P < .005; 1-way ANOVA, Dunnett multiple-comparison test. (D) Detection of TF-FVIIa vs TF-FVIIa-Xa-TFPI on TF-expressing CHO-cells by αVIIa-3G12⁴⁷  and 9D4 vs 12C7. (E) Coagulation inhibitory αVIIa-3G12 vs αFVII-12C7 reactivity on monocytic cells before and after stimulation with aPL HL5B Fab’2, which does not cause TF-FVIIa internalization. (F) β2GPI- and lipid-reactive HL7G, but not rJGG9, with sole β2GPI reactivity can induce FXa release from mouse CD115⁺ splenocytes; mean ± SD. n = 6.