Endogenous TFPI is required for aPL internalization. Internalization of MM1 cell surface TF (A) and FVIIa (B) after 15 minutes of live cell stimulation with fluorophore-labeled aPL HL5B (IgG or Fab’₂ fragment). Scale bar = 5 µm. (C) Schematic representation of complement-dependent and independent pathways initiated by aPL. (D) Flow cytometry detection of TF expression on CD115⁺ spleen monocytes for myeloid cell-deleted TFPI Kunitz 1 domain (TFPI^ΔK1) mice and littermate wild-type (WT) controls under basal conditions and after 3 hours of stimulation with 100 ng/mL LPS. Note that basal and LPS-induced TF expression was not affected by TFPI mutation. (E) FXa surface localization detected by flow cytometry on CD115⁺ splenocytes of TFPI^ΔK1 and littermate WT mice. (F) Live cell imaging of aPL HL5B internalization in TFPI^ΔK1 monocytes after stimulation with aPL HL5B. (G) Expression of TFPI isoforms by blood monocytes isolated from WT mice detected by RT-PCR with isoform-specific primers; mean ± SD. n = 4. (H) Dose titration of aPL HL5B binding to dry milk-blocked plates coated with 2 µg/mL soluble TF_1-218,⁸⁸  FVIIa, TFPIα⁴⁷, or FXa. (I) Effect of aPL HL5B (500 nM) on TFPIα (10 nM) inhibition of FX (100 nM) activation by 0.5 nM TF in 60% phosphatidylcholine/40 sphingomyelin⁵⁶  with 1 nM FVIIa. (J) Effect of 100 µg/mL HL5B on TFPIα (50 nM) complex formation with 1 µM soluble TFGCN4,⁸⁹  3 nM FVIIa, and 2 nM FXa measured by amidolytic assay with Spectrozyme FXa.