APC leads to a loss of the K1 domain from cell-associated TFPI. (A) Cells were treated with siRNA targeting TFPI or control for 24 or 48 hours. TFPI was detected by Western blotting in cell lysates using a K2 domain–specific antibody. (B) Cells were treated for 3 hours with the indicated agonist concentrations followed by detection of TFPI with anti-K2 or anti-K1 domain–specific antibodies. (C) Cells were treated with 20nM APC or factor Xa for the indicated time period followed by detection of TFPI with anti-K2. (D) Cells were preincubated for 15 minutes with 25 μg/mL blocking (RCR-252) or nonblocking (RCR-92) anti-EPCR followed by a 3-hour incubation with 20nM APC or factor Xa and Western blotting using anti-K2. (E) Cells and supernatant were analyzed after incubation with TNFα (5 hours) and 20nM APC (3 hours) as indicated. Growth medium was replaced with TNF-containing serum-free DMEM as in the Xa generation experiments. (F) Cells and supernatant were analyzed without replacing the growth medium for the agonist incubation. Where indicated, 25nM recombinant soluble TFPI (tTFPI) were added before APC. (G) Cells were treated for 3 hours with the indicated concentrations of thrombin in the absence or presence of protein C. (H) Cells were agonist treated as indicated in the presence of 80nM protein C. Polyclonal anti-TFPI was used for detection in panels E through H. Typical results from 2-4 experiments are shown in all panels.