Analysis of TFPI cleavage by whole bacteria. (A) Omptin-positive bacterial strains, E coli MC4100, S Typhimurium ΔgalETK, or Y pestis KIM5 (10⁹ cfu/mL) were incubated with TFPI (100 μg/mL) in HBS without BSA for 24 hours at room temperature, after which mixtures were resolved on SDS-PAGE and stained with Coomassie blue. (B) TFPI (3 μg/mL) was incubated with omptin-positive bacterial strains (10⁹ cfu/mL) in HBS for varying times, after which the mixtures were Western blot analyzed and detected with an anti-TFPI monoclonal antibody reactive with the Kunitz-2 domain. Discrete fragments are indicated by arrows and numbered. (C) S Typhimurium, (D) E coli, or (E) Y pestis at 10⁹ cfu/mL were incubated with TFPI (3 μg/mL) for 30 minutes at room temperature in HBS after which bacteria were removed by centrifugation at 16 000g for 4 minutes. Supernatants were transferred to separate tubes while the bacterial pellets were washed once with HBS, pelleted, and resuspended in HBS. Western blot analysis of the resulting supernatant and bacterial pellet fractions were probed with monoclonal antibodies reactive with the Kunitz-2 domain of TFPI. (F) Amino acid sequence following the C-terminal ends of the Kunitz-3 domains of human (Homo sapiens), rabbit (Oryctolagus cuniculus), mouse (Mus musculus), and rat (Rattus norvegicus) TFPI. Residues identical in all 4 species are highlighted in gray.