Fig. 9

Chelation of metal ions prevents ATP hydrolysis and promotes PMN activation. Heparinized human whole blood samples (n = 3) were incubated at 37 °C with increasing concentrations of EDTA, and plasma ATP and adenosine levels were assessed 2 h later (A). The effect of EDTA on Ca²⁺ and Mg²⁺ concentrations in blood samples was determined after EDTA treatment as described in panel A (B). The effects of EDTA on FPR-induced PMN responses were assessed by treating human blood samples (n = 7) as described in panel A, stimulating cells with 50 nM fMLP (C) or 15 nM fMLP (D), and measuring the formation of ROS and the expression of the activation marker CD63 via flow cytometry (C-D). The role of Zn²⁺ ions in maintaining the ATPase activity of mouse plasma was determined by depleting diluted plasma samples (n = 3) of divalent ions by addition of 0.1 mM EDTA, titrating back Zn²⁺ ions at the concentrations shown, and assessing ATP breakdown capacity of these samples with a luciferase assay (E). The data shown are the means ± SEMs, and the data were compared with the control values (0 mM EDTA) using one-way ANOVA and Holm-Sidak post hoc test; *p < 0.05, **p < 0.01, ***p < 0.001. MFI: mean fluorescence intensity