FIG. 4.

Inhibition by doxycycline of RgpB VPE activity production from human plasma. Normal human plasma (50 μl) supplemented with 1,10-phenanthroline (2 mM) was mixed with 25 μl of doxycycline, followed by addition of 25 μl of RgpB (40 nM) 15 s later and incubation at 25°C for 5 min. The reaction was stopped by adding 400 μl of TBS supplemented with 1,10-phenanthroline (1 mM), soybean trypsin inhibitor (20 μM), leupeptin (10 μM), or FPR-CK (10 μM). The VPE activity of each sample was measured. Activity was expressed in terms of mean micrograms of dye released in triplicate assays. Dye leakage at TBS-injected sites was used as a control and the value was subtracted from the value for each sample. Doxycycline concentrations during incubation are shown. ∗, P < 0.01 for VPE activity of RgpB-treated plasma in the absence of doxycycline. P, plasma alone; P + D, plasma plus doxycycline (1,000 μM); D, doxycycline alone (1,000 μM).