Figure 3.
Desialylated platelets rapidly accumulate on Kupffer cells.(A) Ex vivo desialylated (desial. plts) and untreated control platelets were labeled with CellTracker Green and Red dye, respectively, and transfused into control and Kupffer cell–depleted mice. Representative still images from videos obtained using SD-IVM of the mouse liver. Scale bars are 70 µm. (B) Quantification of the accumulation of ex vivo desialylated platelets and untreated (untxt) control platelets in wild-type and Kupffer cell–depleted mice. n = 3 mice per group. (C) SD-IVM of the liver of mice treated with 50 mU sialidase. Representative still images obtained at the indicated time points. Kupffer cells, endothelial cells, and endogenous platelets were stained using fluorescently labeled anti-F4/80, anti-CD31, and anti-CD49b antibody, respectively. Scale bars are 33 µm. (D) Quantification of the accumulation of platelets within the first 30 min after treatment with 50 mU sialidase in control (Ctrl) and Kupffer cell (KC)–depleted mice. n = 5 mice per group. (E) Colocalization of platelets with Kupffer cells, endothelial cells (ECs), and hepatocytes (Hep) was quantified at the 20-min time point after sialidase treatment using Imaris software. Data represent means ± SEM (n = 4 mice per group). (F) Platelet count was measured using flow cytometric analysis of blood from control mice as well as mice treated with 250 parts-per-billion (ppb) arsenic in drinking water for 5 wk to specifically cause defenestration of the liver sinusoidal endothelial cells. Data represent means ± SEM (n = 5 mice per group). (G) Platelet count was measured using flow cytometric analysis of blood from control mice, control mice treated with sialidase, and Kupffer cell–depleted (using clodronate liposomes) mice treated with sialidase. Measurement was performed 24 h after sialidase application. Data represent means ± SEM (n = 4 mice per group). (H) Galactose exposure on platelets of untreated control mice and Kupffer cell–depleted (using clodronate liposomes) mice treated with sialidase was measured using flow cytometry. Data represent means ± SEM (n = 3 mice per group). (I) SD-IVM of the liver of control and ST3Gal-IVΔ/Δ mice shows massive accumulation of platelets on Kupffer cells of ST3Gal-IVΔ/Δ mice already under steady-state conditions. Scale bars are 70 µm. (J) Platelet count was quantified using flow cytometry of blood from ST3Gal-IVΔ/Δ mice before and 5 d after Kupffer cell depletion using clodronate liposomes. Platelet count is expressed relative to untreated wild-type controls. Data represent means ± SEM (n = 4 mice per group). One-way ANOVA with post-hoc testing (E and G) and unpaired two-tailed t test (F, H, and J) were used to determine statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Results from all experiments shown are representative of at least three independent experiments.

Desialylated platelets rapidly accumulate on Kupffer cells. (A) Ex vivo desialylated (desial. plts) and untreated control platelets were labeled with CellTracker Green and Red dye, respectively, and transfused into control and Kupffer cell–depleted mice. Representative still images from videos obtained using SD-IVM of the mouse liver. Scale bars are 70 µm. (B) Quantification of the accumulation of ex vivo desialylated platelets and untreated (untxt) control platelets in wild-type and Kupffer cell–depleted mice. n = 3 mice per group. (C) SD-IVM of the liver of mice treated with 50 mU sialidase. Representative still images obtained at the indicated time points. Kupffer cells, endothelial cells, and endogenous platelets were stained using fluorescently labeled anti-F4/80, anti-CD31, and anti-CD49b antibody, respectively. Scale bars are 33 µm. (D) Quantification of the accumulation of platelets within the first 30 min after treatment with 50 mU sialidase in control (Ctrl) and Kupffer cell (KC)–depleted mice. n = 5 mice per group. (E) Colocalization of platelets with Kupffer cells, endothelial cells (ECs), and hepatocytes (Hep) was quantified at the 20-min time point after sialidase treatment using Imaris software. Data represent means ± SEM (n = 4 mice per group). (F) Platelet count was measured using flow cytometric analysis of blood from control mice as well as mice treated with 250 parts-per-billion (ppb) arsenic in drinking water for 5 wk to specifically cause defenestration of the liver sinusoidal endothelial cells. Data represent means ± SEM (n = 5 mice per group). (G) Platelet count was measured using flow cytometric analysis of blood from control mice, control mice treated with sialidase, and Kupffer cell–depleted (using clodronate liposomes) mice treated with sialidase. Measurement was performed 24 h after sialidase application. Data represent means ± SEM (n = 4 mice per group). (H) Galactose exposure on platelets of untreated control mice and Kupffer cell–depleted (using clodronate liposomes) mice treated with sialidase was measured using flow cytometry. Data represent means ± SEM (n = 3 mice per group). (I) SD-IVM of the liver of control and ST3Gal-IVΔ/Δ mice shows massive accumulation of platelets on Kupffer cells of ST3Gal-IVΔ/Δ mice already under steady-state conditions. Scale bars are 70 µm. (J) Platelet count was quantified using flow cytometry of blood from ST3Gal-IVΔ/Δ mice before and 5 d after Kupffer cell depletion using clodronate liposomes. Platelet count is expressed relative to untreated wild-type controls. Data represent means ± SEM (n = 4 mice per group). One-way ANOVA with post-hoc testing (E and G) and unpaired two-tailed t test (F, H, and J) were used to determine statistical significance. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Results from all experiments shown are representative of at least three independent experiments.

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