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. 2019 Aug 31;11(17):6951–6959. doi: 10.18632/aging.102229

Figure 2.

Figure 2

Determine the role of kindlin-3 in platelets and neutrophils in regulating NET release in vivo and in vitro. (A) Histological analysis of the thrombi formed in the IVC in Kindlin-3fl/flLysM-Cre mice after partial ligation for 6 hours. (B) The levels of NETs in plasma were measured using an ELISA-based assay as described in methods. Data are shown as mean ± SD; n ≥ 8 for each group. (C1–C2) Kindlin-3fl/flLysM-Cre mice were injected with DNase I, GSK484 or buffer, and the partial IVC ligation was performed. Thrombus formation in the IVC was determined 6 hours after the ligation. Dots represent individual experiments for each mouse and lines in dot plots represent mean; n ≥ 8 for each group. (D1–D3) After treatment with the indicated antibodies, Kindlin-3fl/flLysM-Cre mice were subjected to partial IVC ligation for 6 hours and then the IVC tissues were harvested and evaluated (D1–D2). Dots represent individual experiments for each mouse and lines in dot plots represent mean; n ≥ 8 for each group. Meanwhile, blood samples were collected from mice before and after the IVC stenosis and used for measuring plasma NETs by the ELISA-based assay. Data are shown as mean ± SD. (E) blood samples were collected from Kindlin-3fl/flPF4-Cre mice and Kindlin-3fl/fl mice before and after the IVC stenosis and used for measuring plasma NETs by the ELISA-based assay (D3). Data are shown as mean ± SD. (F) Mouse bone marrow neutrophils (Neuts) were incubated with TNF-α primed endothelial cell monolayers with or without the presence of washed platelets isolated from either Kindlin-3fl/fl mice (K3WT-Plts) or Kindlin-3fl/flPF4 mice (K3KO-Plts). After stimulation with either PAR4 agonist peptide (150 μM) or PMA (20 nM) for 2 hours in the presence or absence of Leo.H4 antibody, the released NETs were quantified as described in methods. Data are shown as mean ± SD. (*, p<0.05; ***, p<0.001).