Gram-negative bacteria abrogate TFPI anticoagulant activity by interacting with an outer membrane component that is dependent on LPS O-antigen length. (A) Loss of TFPI activity. E coli MC4100 (10⁹ cfu/mL) were incubated with or without 1 μg/mL TFPI in plasma for 1 hour at room temperature, after which clotting times were measured. (B) Dependence on TFPI concentration. E coli MC4100 (10⁹ cfu/mL) were incubated with varying TFPI concentrations in human plasma for 30 minutes at room temperature, after which clotting times were quantified (●), compared with samples containing TFPI but no bacteria (○). (C) Requirement for physical contact with TFPI. E coli MC4100 (10⁹ cfu/mL) were incubated with 1 μg/mL TFPI for 30 minutes, then bacteria were removed by centrifugation and TFPI activity tested in clotted assays. In parallel experiments, E coli MC4100 were incubated in TFPI-free plasma for 30 minutes, removed by centrifugation, and 1 μg/mL TFPI was added to the plasma supernatants, after which clotting assays were performed. (D) TFPI inactivation by cell fractions containing the E coli envelope. E coli MC4100 (10⁹ cfu/mL) were lysed and fractionated, and the indicated cell fractions were incubated with TFPI (3 μg/mL) in HBS for 30 minutes at room temperature, then tested in clotting assays as before. (E) Only S Typhimurium strains expressing truncated O-antigen reduced clotting times. S Typhimurium (10⁹ cfu/mL) were incubated with 3 μg/mL TFPI in HBS for 30 minutes at room temperature, after which bacteria were removed by centrifugation and supernatants tested in clotting assays. The wild-type strain, 14028, expresses extended O-antigen side chains, while strains harboring a deletion (ΔgalETK) or mutation (galE496) in the UDP-4-galactose-epimerase gene express truncated O-antigen. Samples that did not clot by 600 (A,C) or 300 seconds (D,E) are indicated by asterisks. Data are mean plus SEM (n = 3).