Figure S5.
Desialylated platelets are phagocytosed by Kupffer cells in vitro, Mac1 is dispensable for the clearance of desialylated platelets, Kupffer cells express Asgr1 and MGL1/2, but Asgr1-deficiency alone has no effect on platelet count.(A) Mice were treated with 50 mU sialidase i.v., and the amount of platelet accumulation in the liver at 30 min and 24 h after treatment was quantified as the CD49b-positive area from images obtained using SD-IVM. Data represent means ± SEM (n ≥ 3 mice per group). (B) Kupffer cells were incubated with platelets in vitro, and phagocytosis was observed using an IncuCyte incubation microscope. Kupffer cells and platelets were labeled using F4/80 antibodies and CellTracker Red dye, respectively. Representative images showing Kupffer cell platelet co-culture at 0 h (upper panel) and at 5 h (lower panel). (C) Quantification of phagocytosis (Kupffer cells with red platelet signal inside) over time. (D) Wild-type and Mac-1−/− mice were i.v. injected with 50 mU sialidase, and platelet accumulation in the liver was quantified from SD-IVM videos. n = 5 mice per group. (E) Kupffer cells were isolated from mouse liver, and ASGR1 expression was analyzed using flow cytometry. Representative flow plot showing ASGR1 signal versus fluorescence minus one (FMO). (F) Platelet count in blood from wild-type and Asgr1−/− mice was quantified using flow cytometry. Data represent means ± SEM (n = 5 mice per group). (G) Kupffer cells were isolated from mouse liver and MGL expression analyzed using flow cytometry. Representative flow plot showing MGL signal versus fluorescence minus one (FMO). (H) Washed human platelets and desialylated washed human platelets were incubated with fluorescently labeled recombinant human MGL (hMGL), and binding was quantified using flow cytometry. Representative flow plot. (I) Quantification of the binding of hMGL to control and desialylated human platelets from healthy human volunteers. Data represent means ± SEM (n = 3). Unpaired two-tailed t test (A, F, and I) was used to determine statistical significance. ***, P < 0.001; ns, not significant. Results from all experiments shown are representative of at least three independent experiments.

Desialylated platelets are phagocytosed by Kupffer cells in vitro, Mac1 is dispensable for the clearance of desialylated platelets, Kupffer cells express Asgr1 and MGL1/2, but Asgr1-deficiency alone has no effect on platelet count . (A) Mice were treated with 50 mU sialidase i.v., and the amount of platelet accumulation in the liver at 30 min and 24 h after treatment was quantified as the CD49b-positive area from images obtained using SD-IVM. Data represent means ± SEM (n ≥ 3 mice per group). (B) Kupffer cells were incubated with platelets in vitro, and phagocytosis was observed using an IncuCyte incubation microscope. Kupffer cells and platelets were labeled using F4/80 antibodies and CellTracker Red dye, respectively. Representative images showing Kupffer cell platelet co-culture at 0 h (upper panel) and at 5 h (lower panel). (C) Quantification of phagocytosis (Kupffer cells with red platelet signal inside) over time. (D) Wild-type and Mac-1−/− mice were i.v. injected with 50 mU sialidase, and platelet accumulation in the liver was quantified from SD-IVM videos. n = 5 mice per group. (E) Kupffer cells were isolated from mouse liver, and ASGR1 expression was analyzed using flow cytometry. Representative flow plot showing ASGR1 signal versus fluorescence minus one (FMO). (F) Platelet count in blood from wild-type and Asgr1−/− mice was quantified using flow cytometry. Data represent means ± SEM (n = 5 mice per group). (G) Kupffer cells were isolated from mouse liver and MGL expression analyzed using flow cytometry. Representative flow plot showing MGL signal versus fluorescence minus one (FMO). (H) Washed human platelets and desialylated washed human platelets were incubated with fluorescently labeled recombinant human MGL (hMGL), and binding was quantified using flow cytometry. Representative flow plot. (I) Quantification of the binding of hMGL to control and desialylated human platelets from healthy human volunteers. Data represent means ± SEM (n = 3). Unpaired two-tailed t test (A, F, and I) was used to determine statistical significance. ***, P < 0.001; ns, not significant. Results from all experiments shown are representative of at least three independent experiments.

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