Genetic deletion of P-selectin attenuates pulmonary vaso-occlusion in SCD mice. (A) SS and SS-Selp^−/− mice were challenged IV with 0.1 µg/kg LPS, and qFILM was used 2 to 2.5 hours later to visualize the pulmonary microcirculation. (B) IV LPS (0.1 µg/kg) triggered occlusion of pulmonary arteriolar bottlenecks (large dotted ovals) in SS mice by large aggregates of neutrophils (red) and platelets (green). The same FOV is shown over 3 time points. A neutrophil (red; small dashed circles) flows toward the occlusion but cannot pass through it and is forced to flow toward another open vessel to the side of the occlusion. Supplemental Video 1 shows the complete time series for the FOV in panel B. (C) In contrast to SS mice, the majority of FOVs in SS-Selp^−/− mice were free of pulmonary vaso-occlusions. The same FOV is shown over 3 time points. A neutrophil (red; small dashed circles) is seen trafficking up the pulmonary arteriole that has no aggregates present. Supplemental Video 2 shows the complete time series for the FOV in panel C. The pulmonary microcirculation was labeled with FITC-Dextran and pseudo-colored purple. Neutrophils and platelets were labeled by IV administration of AF546-conjugated anti-Ly6G mAb and V450-conjugated anti-CD49b mAb, respectively. Neutrophils are shown in red, and platelets are pseudo-colored green. The arrows denote the direction of blood flow, and the asterisks (*) denote alveoli. Scale bars, 20 μm. The diameters of the vessels are 34 μm and 40 μm in panels B-C, respectively. (D-F) The neutrophil-platelet aggregates blocking pulmonary arterioles were quantified as described in Methods. After IV LPS administration, SS-Selp^−/− mice had a significantly decreased average number of pulmonary vaso-occlusions per FOV (D), percentage of FOVs with pulmonary vaso-occlusions (E), and large pulmonary vaso-occlusions (area >1000 μm²) (F) compared with SS mice. The average number of pulmonary vaso-occlusions per FOV and large pulmonary vaso-occlusions (area >1000 µm²) were compared between groups using the unpaired Student t test, and the percentage of FOVs with pulmonary vaso-occlusions were compared between groups using fourfold table with χ² analyses. SS mice: n = 1 male mice and 2 female mice, FOV = 45. SS-Selp^−/− mice: n = 1 male mice and 2 female mice, FOV = 46. Error bars are mean ± SE. *P < .05.