Binding of PS to TFPI. (A) Microtiter plate assay. Wells of a 96-well plate were precoated with PS and then blocked with BSA. TFPI samples were added to the wells and incubated for 2 hours. Thereafter, wells were washed and the bound TFPI detected with a biotin-conjugated Mab2H8 as described under “TFPI immunoassay.” Specific binding of each form of TFPI to PS was obtained by subtracting the readings of wells coated with plain buffer (background) from the corresponding readings of wells coated with PS. The binding of TFPI to PS is expressed as a percentage of the binding obtained with TFPI_WT, set at 100%. Assays were done in duplicates. (B-C) SPR. A CM5 chip flow cell was coupled with TFPI to approximately 2000 RU. Cells were then perfused with increasing concentrations of PS (0, 62.5, 125, 250, 500, 1000, 2000, and 4000nM), in TBS containing 5mM CaCl₂ and 0.005% Tween 20, as described in “Binding of PS to TFPI using SPR.” The sensorgrams shown have been corrected for the signal obtained in a reference flow cell without immobilized TFPI.