Characterization of the interaction between FXIa and rTFPI. (A) Ninety-six-well plates were coated with 5 µg/mL rTFPI or BSA (♦), and increasing concentrations of FXI (◱), FXIa (○,♦), or PPACK-FXIa (●) were added to selected wells. Binding was detected with a specific antibody against FXI. (B) Ninety-six-well plates were coated with 5 µg/mL rTFPI and increasing concentrations of FXIa in the absence (♦) or presence of 25 µM Zn²⁺ (△) or 1 mM Ca²⁺ (◱). Binding was detected with a specific antibody against FXI. (C) rTFPI (10 µg) was incubated with FXIa (500 nM) for selected times at 37°C before being separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing (upper) or reducing (lower) conditions and analyzed by Coomassie blue staining. Protein bands were subjected to aminoterminal sequencing, using an automated Edman sequencer. Numbers refer to position of amino acid in full-length TFPI protein. (D) rTFPI (10 nM) was incubated with 5 nM FXIa for 10, 30, or 60 minutes in the presence or absence of 25 µM Zn²⁺ or 1 mM Ca²⁺. rTFPI was analyzed by western blotting with a polyclonal anti-TFPI antibody. (E) rTFPI (10 nM) was incubated with 5 nM FXIa for 10, 30, or 60 minutes in the presence or absence of 1A6 (20 µg/mL), 10C9 (50 µg/mL), or aprotinin (50 µM). rTFPI was analyzed by western blotting with a polyclonal anti-TFPI antibody.