Binding of DG-KKO to PF4/NET complexes in vitro. (A) Graphs quantifying binding of increasing concentrations of KKO (gray) and DG-KKO (red) to PF4-heparin (PF4-H) using fluorescent plate assay. (B) The same as panel A but using flow cytometry to quantify antibody binding to the platelet surface. (C) Mean ± 1 SD of P-selectin MFI in human whole blood samples incubated with the indicated concentration of antibody, reflecting the degree of platelet activation. N = 3. (D) Mean ± 1 SD of the % decrease in platelets counts in HIT mice injected with 400 µg of the indicated antibody, measured every 12 hours for 3 days. N = 10. (E) Representative confocal images of released NETs as in Figure 2 exposed to no PF4 or 6.5 µg/mL of PF4, labeled with the nucleic acid stain SYOTX green (green) demonstrating change in morphology. The indicated channels were then infused with fluorophore-labeled DG-KKO (white). Size bar and arrows indicating direction of flow are included. Image were obtained at ×10 magnification. (F) Representative widefield images of adherent NETs as in panel A, but in the presence of 100 U/mL DNase I and 6.5 µg/mL PF4 ± 25 µg/mL of DG-KKO. (G) Mean ± 1 SD of the relative area of NETs compacted with PF4 (6.5 µg/mL) alone or with PF4 plus DG-KKO or a polyclonal anti-PF4 antibody control (Ctl) (each, 25 µg/mL) after an infusion of DNase I (100 U/mL, 3 minutes) compared with preinfusion area. N = 7-10 channels per condition. Comparative statistical analysis performed by Kruskal-Wallis 1-way ANOVA.