Role of the intact catalytic center and lysine binding sites for the plasmin-induced expression of IL-1α, IL-1β, TNF-α, and TF mRNA in human monocytes.

(A) Effect of active site-blocked plasmin. The catalytic center of plasmin was blocked by preincubation with the irreversible inhibitor VPLCK. Monocytes were cultured in the absence of any stimulus (lane 1), VPLCK 25 μM (lane 2), LPS 1 μg/mL (lane 3), LPS 1 μg/mL, and VPLCK 25 μM (lane 4), in the presence of plasmin 0.43 CTA U/mL (lane 5) or with 0.43 CTA U/mL active site-blocked plasmin (VPLCK-plasmin, lane 6). (B) Effects of t-AMCA. Monocytes were cultured in the absence of any stimulus (lane 1), t-AMCA 3 mM (lane 2), LPS 1 μg/mL (lane 3), LPS 1 μg/mL, and t-AMCA 3.0 mM (lane 4), in the presence of plasmin 0.43 CTA U/mL (lane 5) and with plasmin 0.43 CTA U/mL and t-AMCA 0.3 mM (lane 6) or 3.0 mM (lane 7), respectively. In both experiments, cells were harvested after 4 hours, and mRNA was extracted and subjected to RT-PCR. HLA(B) was used for normalization. Results of 1 of 3 experiments are shown in each case.