TFPI blocks movement of LPS to sCD14 in the absence of LBP. (A) Time course. BODIPY-LPS (0.2 μg/mL) was incubated with sCD14 (5 μg/mL) alone (•), sCD14 plus rTFPI (20 μg/mL; ▪), or PBS alone (▴) at 37°C, and fluorescence was measured at different time points before and after adding SDS. RF(%) was calculated as described above. (B) Dose dependence. BODIPY-LPS transfer to sCD14 in the absence of LBP was performed in the presence of different doses of rTFPI. The concentration of BODIPY-LPS was 0.2 μg/mL and the concentration of sCD14 was 2.5 μg/mL. The samples were incubated at 37°C for 2 hours. (▵) A control sample with BODIPY-LPS alone without sCD14. (C) sCD14-LPS complexes formed in the presence of TFPI fail to activate PMN. The PMN adhesion assay was performed with sCD14-LPS complex prepared as follows. sCD14 and LPS (100 μg/mL and 5 μg/mL in PBS, respectively) were incubated at 37°C for 2 hours. Different amounts of rTFPI were added to the samples during (▪) or after (•) the 2 hours of incubation and the resulting mixture was added to the PMN adhesion assay. For stimulation of PMN, samples were diluted to yield a final concentration of 2 μg/mL sCD14, 0.1 μg/mL LPS, and the indicated amount of rTFPI. (▴) Control samples in which LPS alone was incubated with rTFPI without sCD14. (○) Buffer alone was added to PMN for the control of PMN adhesion assay.