Active caspase-1 is present in both thrombi and blood cells in murine DVT and can be induced by histones and NETotic neutrophils in human platelets. (A) FLICA incorporation (active caspase-1) in platelets (from thrombi and blood) and leukocytes 48 hours after IVC stenosis. (B) Quantification of FLICA-positive platelets and leukocytes in both thrombi and blood (n = 5, mean ± standard deviation). (C) Immunofluorescence staining of thrombi sections for FLICA (green), SYTOX Orange (red), and CD41 (blue). Arrows indicate areas of colocalization of FLICA and SYTOX Orange. Scale bars: left = 100 μm, middle = 50 μm, right = 10 μm. (D) Total and cleaved (activated) caspase-1 content in thrombus lysates at either 48 or 6 hours after IVC stenosis (representative gel, n = 4-5). (E) MFI for FLICA incorporation (caspase-1 activation) by washed human platelets stimulated with ATP, nigericin, PAF, and calf histones, at indicated concentrations, or left untreated (n = 3, mean ± standard deviation). One-way analysis of variance, Dunnett’s multiple comparison test, *P ≤ .05. (F) Representative plot of FLICA incorporation by human platelets treated with individual human histones and evaluated by flow cytometry. (G) Immunofluorescent staining of histone-stimulated platelets with FLICA (green) and CD41 (red). Scale bar = 10 µm, n = 3. (H) MFI quantification for FLICA from autologous platelets seeded on NETotic neutrophils after treatment with PMA for 2 hours (n = 3 biological replicates, data points indicate technical replicates). One-way analysis of variance, Tukey’s multiple comparison test, *P ≤ .05.