Fig. 4.

Functional inhibition of histones by MBP-p33. a The bactericidal activity of histones is neutralized by p33. Histone H1 (500 nM), histone H2A (500 nM), histone H2B (500 nM), histone H3.1 (500 nM) and histone H4 (250 nM) were incubated with 2 × 10⁶ CFU/ml bacteria (E. coli strain ATCC 25922) in the presence or absence of 2.5 μM MBP-p33. Samples were plated on Todd-Hewitt agar plates and colonies were counted the following day. Data are mean and SE from 3 separate experiments. *** p < 0.001, ** p < 0.01, * p < 0.05. b MBP-p33 blocks the cytolytic effect of histones. Histones (10 μM) were incubated with washed human erythrocytes in the presence or absence of 10 μM MBP-p33. Samples were centrifuged and hemolysis was measured at 540 nm in the supernatant. Hemolysis was calculated as percentage of cells treated with Tox-7 lysis buffer. Data are mean and SE from 3 separate experiments. *** p < 0.001, ** p < 0.01, * p < 0.05. c MBP-p33 inhibits histone-induced LDH release from endothelial cells. Confluent EA.hy926 cells were treated overnight with histone H1 (4 μM), histone H2A (4 μM), histone H2B (4 μM), histone H3.1 (4 μM), histone H4 (2 μM) or CTH (50 μg/ml) in the presence or absence of 10 μM MBP-p33. LDH release was measured in the supernatant. Cell lysis was calculated as the percentage of cells treated with Tox-7 lysis buffer. Data are mean and SE from 3 separate experiments. *** p < 0.001, ** p < 0.01, * p < 0.05. d MBP-p33 prevents histone-triggered platelet aggregation. CTH (300 μg/ml) were added to platelet-rich plasma in the presence or absence of increasing concentrations of MBP-p33 (0.65-6.2 μM). Platelet aggregation was monitored for 12 min and was calculated as the percentage of light transmission through each sample with platelet-poor plasma in a reference channel. One representative experiment out of 3 is shown.