Omptin gene deletion prevents E coli and S Typhimurium from inactivating TFPI. Incubation of TFPI with omptin-positive bacterial strains resulted in time-dependent reductions of clotting times (bar graphs), which coincided with reductions in band intensity of full-length TFPI on Western blots (photographs). TFPI (1 μg/mL) was incubated with (A) E coli MC4100 or ΔompT (107 cfu/mL) or (B) Y pestis KIM5 or Δpla (108 cfu/mL) in HBS at room temperature for varying times, then tested in clotting assays. Parallel samples were subjected to Western blot analysis and detected with an anti-TFPI monoclonal antibody reactive with the Kunitz-2 domain. (C) This procedure was repeated using S Typhimurium ΔgalETK or ΔgalETK ΔpgtE (109 cfu/mL) and 3 μg/mL TFPI (3 μg/mL) and an anti-TFPI monoclonal antibody reactive with the Kunitz-1 domain. Samples that did not clot by 300 seconds are indicated by an asterisk. Clotting data are mean plus SEM (n = 3).
