Both thrombocytopenia and anticoagulation aggravate inflammatory bleeding. (A) Experimental scheme of subacute lung injury model with or without antibody-mediated platelet depletion. The red arrow indicates antibody administration; the blue arrow indicates intranasal LPS administration. (B) Representative macroscopic images of lungs from control (C301) and thrombocytopenic animals (R300 treatment) 24 hours after LPS challenge. (C) Flow cytometric analysis of peripheral blood (platelet and RBC count) and BALF (RBC and leukocyte count/µL BALF). Student’s t test, two-tailed, unpaired. (D) Representative micrographs of immunofluorescence stainings of alveolar hemorrhage in control (C301) and thrombocytopenic animals (R300). Bar represents 100 µm. (E) Magnified excerpts of representative micrographs, corresponding to white rectangles in Figure 1D. Bar represents 50 µm. (F) Representative images of immunofluorescence stainings of lungs from control (Ctrl) or septic animals (E. coli–derived LPS 1 mg/kg BW intraperitoneally). Bar represents 100 µm (left and mid panel) and 20 µm (right panel). White arrowheads indicate fibrin(ogen)-positive platelets. Red: anti-PS antibody (Merck). (G) Quantification of fibrinogen deposition and overlap of fibrinogen/platelet positive areas. n = 3 to 4 mice per group. Student’s t test, two-tailed, unpaired. (H) Quantification of fibrinogen deposition in lungs from septic control (C301) and platelet-depleted animals (R300) per field of view (FOV). n = 4 mice per group. Student’s t test, two-tailed, unpaired. (I) Representative micrographs of control (C301) and platelet-depleted mice (R300), referring to (H). Bar represents 200 µm (left and right panels). Magnified excerpt (middle; bar represents 20 µm) corresponds to the white rectangle of the left panel. Red: anti-PS antibody (Merck). (J) Experimental scheme of (sub)acute lung injury. Bl6 mice were treated with 20 µg of LPS intranasally (blue arrow) and intravenously injected with vehicle or rivaroxaban (3 mg/kg BW) right before and 8 hours after challenge or argatroban (5 mg/kg BW) right before and 4 and 8 hours after challenge; rivaroxaban-treated animals received vehicle injections at 4 hours after challenge (red arrows indicate timing of intravenous injections). (K) Representative macroscopic image of BALF derived from 1 simultaneously performed set of experimental groups, collected in 2 mL Eppendorf tubes. The right tube corresponds to the maximum bleeding observed in argatroban-treated animals. (L) Flow-cytometric assessment of BALF (RBC, polymorphonuclear, and PLT counts/µL BALF). One-way analysis of variance (ANOVA) with Holm-Šídák's multiple comparisons test compared with control groups. (M) Representative images of immunofluorescence stainings of lung slices from different experimental groups. Bar represents 100 µm. (N) Quantification of alveolar hemorrhage (TER119⁺ area) and neutrophil recruitment. n = 3 to 4 mice/group. One-way ANOVA with Holm-Šídák's multiple comparisons test compared with control groups. (O) Magnified excerpts of representative micrographs, corresponding to white rectangles in Figure 1M. Bar represents 50 µm.