TFPI K1 domain shedding is required for the up-regulation of procoagulant TF by APC. (A) TNFα-induced (5 hours) cells were treated for the final 3 hours with APC (20nM). Factor Xa generation was analyzed after a 30-minute pretreatment with different concentrations of blocking polyclonal anti-TFPI. (B) HEK293t cells were transfected with expression constructs for EPCR alone or in the presence of TFPIα or TFPIβ. The following day cells were incubated for 3 hours with control or APC (60nM) and TFPI in the supernatant (S) or cells (C) was analyzed by Western blotting using polyclonal anti-TFPI. (C) HEK293t cells were transfected with EPCR alone or in the presence of wild-type TFPIβ or TFPIβ K86A. The following day cells were incubated for 3 hours with control or APC (60nM) and TFPI expression in the cells was analyzed by Western blotting using polyclonal anti-TFPI. (D) HEK293t cells were triple transfected as indicated with EPCR, TFPI, and different amounts of TF expression construct. Factor Xa generation was analyzed the next day after a 3-hour incubation with control or APC (60nM). (E) HEK293t cells transfected with EPCR, TFPIβ, and TF (20 ng/well) were treated for 3 hours with APC (60nM). Xa generation was analyzed after a 30-minute pretreatment with the indicated concentrations of blocking polyclonal anti-TFPI. (F) HEK293t cells transfected with EPCR, TFPIβ, and different amounts of TF or TNFα-induced EAhy926 cells were treated for 3 hours with APC (60nM) followed by analysis of Xa generation. Means ± SEM with n = 3 are shown in panels A, D, E, and F. *P < .05; **P < .005. Typical results from 2-3 experiments are shown in panels B and C.