Effect of PF4 on endothelial cells and microbial entrapment by NETs in vitro. Channels lined with TNF-α–stimulated HUVECs were infused with isolated human neutrophils treated with LPS (100 ng/mL) to induce NETosis with and without hPF4 (25 µg/mL). (A) Channels were incubated with the neutrophils for 16 hours, after which the number of residual adherent endothelial cells was counted using ImageJ. (Top) Representative images of remaining attached endothelial cells channels per condition. Size bar and arrows indicate direction of flow. Bottom: mean of endothelial cell counts in 3 ×10 high-powered fields per condition ± 1 SD. Statistical analysis was performed using a Mann-Whitney U test. n = 6 channels per arm. (B) Left shows representative images of NET-lined channels infused with fluorescently labeled S aureus with observed bacterial capture. Size bar and arrows indicating direction of flow are included. NETs in bottom channels compacted with PF4 (100 μg/mL). (C) Representative image of NET-lined channels previously infused with bacteria, following 30-minute infusion of heparin 100 U/mL. (D) The same as panel C, but includes images of NET-lined channels infused with S aureus bioparticles followed by a 5-minute infusion of DNase I (100 U/mL). NETs in bottom channels compacted with PF4 (100 μg/mL). (E) The same as panel C showing image of S aureus–infused channels previously treated with heparin, now following infusion with DNase 1 (100 U/mL). (F) Graph showing number of NET-adherent bacterial bioparticles in channels ± 1 SD following the initial infusion, DNase I (100 U/mL) × 15 minutes, or heparin (100 U/mL) × 30 minutes as indicated. N = 3-15 channels per condition. Analysis performed by a Kruskal-Wallis 1-way ANOVA.