Induction of NF-κB DNA binding activity by Xa and thrombin.

(A) Protease selectivity. Cells were incubated for 45 minutes with 10 nM of the indicated serine proteases Xa, thrombin (IIa), VIIa, activated protein C (aPC), factor IXa, or trypsin for analysis by EMSA (6% gel) using an oligonucleotide containing an NF-κB site. (B) Time dependence. Cells were incubated with Xa (50 nM), thrombin (50 nM), or TNF-α (20 ng/mL) for the indicated times, followed by nuclear extract preparation for EMSA using oligonucleotide probes specific for NF-κB (top panel) or, as a control for equal loading, for Sp1 (lower panel). (C) Effect of specific inhibitors of Xa. HeLa cells were incubated with 10 nM Xa, 10 nM thrombin, or 20 ng/mL TNF-α in the presence or absence of either 5 μM TAP33 or 1 μM NAP5.32 Nuclear extracts prepared after 45 minutes of treatment were analyzed by EMSA. (D) Effect of the thrombin inhibitor hirudin. HeLa cells were stimulated with 10 nM Xa, 10 nM thrombin, or 20 ng/mL TNF-α in the presence or absence of 100 nM hirudin for 45 minutes, followed by nuclear extract preparation for EMSA.