ITA-LNPs alleviate venous thrombosis in IVC ligation model. (A) Schematic of the experiments. WT mice IV pretreated (50 mg/kg, bolus) with ITA- or Ctrl-LNPs were subjected to IVC ligation surgery followed by the isolation of the blood clots; n = 6 for Ctrl-LNP and n = 8 for ITA-LNP. (B) Gross pathology with clot weight quantification. (C) Hematoxylin and eosin staining of blood clot sections from these experiments. (D) Immunofluorescence staining of the same sections using Ly6G/C mAb (Red) and nuclei (blue). (E) MPO (top, alkaline phosphatase immunohistochemistry) and IL-1β (bottom, immunofluorescence) staining in the same clots. (F) Quantification of the staining in panel E. For each biological replicate (n = 6 for Ctrl-LNP and n = 8 for ITA = LNP), 3 to 4 histopathological locations at different depths (100 μm apart) were analyzed. (G) NET staining in the same sections using triple-positive identification through colocalization of the signals from MPO (green), H4Cit3 (red), and DNA (DAPI; blue). (H) Quantification of the staining in panel G. Quantification was accomplished using 2 to 3 different histopathological locations and the same aforementioned biological replicates. Statistical analysis is through a 2-tailed t test. DAPI, 4′,6-diamidino-2-phenylindole; H4Cit3, citrullinated histone H4; MPO, myeloperoxidase; WT, wild type.