Extracellular histones mediate erythrocyte aggregation and induce erythrocyte fragility. (A) Flow cytometry detection of histone (HIS, 400 µg/mL)-induced erythrocyte (RBC) aggregation based on FSC and SSC plots (left: RBC alone, black dots, RBC-HIS, red dots) and erythrocyte autofluorescence histograms (right: FL-1 channel); autofluorescence of RBC alone is indicated by the black line and RBC-HIS by the red line. (B) SEM images of untreated (left) and histone-treated (right) human erythrocytes confirm that histones mediate erythrocyte aggregation. (C) Concentration dependence of histone-induced RBC aggregation and increased ESR, with RBC aggregation calculated as the fold increase in RBC autofluorescence relative to RBC alone. (D) Lysis of RBCs in the presence of different histone concentrations when exposed to 5 to 40 (5×, 10×, 20×, and 40×) passages through an epMotion 5070 automated 96-well microplate pipetting system at a constant flow rate of 100 mm/s or (E) a variable flow rate (25, 50, 75, and 100 mm/s) at a constant passage number (40×). Note that in the absence of shear, negligible lysis of erythrocytes occurred at all histone concentrations. (F) Erythrocyte retention in an artificial spleen in the presence of increasing histone concentrations. Data are expressed as the mean ± standard error of the mean (n = 3) and are representative of ≥3 separate experiments. *P ≤ .05; **P < .01; ***P < .001; ****P < .0001 (1-way analysis of variance with Tukey’s multiple comparisons test).