Fig. 4: AGEs inhibit Plg binding to histone H2B and macrophages.

a Effect of AGEs on Plg binding to histone H2B in vitro. Recombinant H2B coated on ELISA plate was preincubated with either BSA or AGEs (1, 10, and 100 µg/ml), followed by treatment with Bt-Plg (1 µg/ml). Plg binding to H2B was detected with streptavidin-HRP. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Dunnett’s test (two-sided), relative to the treatment with Plg alone. b Surface plasmon resonance measurements. The interactions of Plg (10 µg/ml) with H2B immobilized on a Biacore sensor chip NTA, following the addition of either BSA or AGEs (10 or 100 µg/ml) were monitored. c Schematic representation of full length and deletion mutants of H2B (upper panel) and effects of AGEs on Plg binding to deletion mutants of H2B (lower panel). H2B mutants coated on ELISA plate were preincubated with either BSA or AGEs (100 µg/ml), followed by treatment with Bt-Plg (1 µg/ml). Plg binding to H2B mutants was detected with streptavidin-HRP. The absorbance value in the absence of competitor was defined as 100% Plg binding. Data are mean ± S.D. of triplicate samples (representative of three independent experiments). Student’s t test (two-sided), n.s. not significant (p > 0.05). d Effect of BSA or AGEs on Plg binding to J774A.1 e Effect of BSA or AGEs on Plg binding to peritoneal macrophage. Cells were preincubated with either BSA, AGEs, or tranexamic acid (a Plg inhibitor). After further incubation with Bt-Plg, Plg binding to cells was analyzed by FACS with streptavidin-APC. Plg binding (% of control) was calculated as described in the Materials and Methods section. Data are mean ± S.D. (n = 3 and 6, respectively, biologically independent experiments). Student’s t test (two-sided). f, g Effect of AGEs on plasmin formation in the presence of recombinant H2B (f) or J774A.1 cells (g). Plasmin was measured by the absorbance at 405 nm using plasmin specific substrate S-2251. The rate of plasmin generation was calculated as described in the Materials and Methods section. Data are mean ± S.D. (n = 3 and 5, respectively, biologically independent experiments). Tukey–Kramer tests (two-sided), n.s. not significant (p > 0.05).