FXIa cleaves the K1 domain of TFPI on endothelial cells. (A) HUVECs were pretreated with increasing concentrations of FXIa for 2 hours. Cell lysates were used for western blotting with a polyclonal anti-TFPI antibody under nonreducing conditions. (B) HUVECs were pretreated with increasing concentrations of FXIa for 2 hours, followed by cell surface detection of TFPI, using an anti-TFPI K1 antibody (●) or a polyclonal anti-TFPI antibody (○). (C) HUVECs were pretreated with 30 nM FXIa (black bars) or vehicle (white bars) for 2 hours in the presence or absence of 25 µM Zn²⁺, 50 nM HK, or 50 µM aprotinin, followed by cell surface detection of TFPI, using an anti-TFPI K1 antibody. P < .05 with respect to FXIa in the presence of Zn²⁺. (D) HUVECs were pretreated with increasing concentrations (0-50 nM) of FXIa (●), elastase (▪), plasmin (▲), kallikrein (◱), or FXIIa (○) for 2 hours, followed by cell surface detection of TFPI, using an anti-TFPI K1 antibody. Data are mean ± standard error (n = 3). Mann-Whitney U test was used for statistical comparisons.