Figure 1
Figure 1. Clearance of transfused ST3Gal-IV−/− platelets. (A) Average survival curves for transfused ST3Gal-IV+/+ (WT, ■), ST3Gal-IV+/− (), or ST3Gal-IV −/− (□) platelets in WT mice. CMFDA-labeled platelets were injected into WT mice, blood samples collected at the indicated time points, and the percentage of labeled platelets remaining in the circulation determined by flow cytometry. ST3Gal-IV−/− platelet recovery and platelet survival were significantly reduced compared with WT and ST3Gal-IV+/− platelets (P = .021 and P = .001, respectively). Transfusion data represent an average of 4 mice ± SD and are representative of 3 separate experiments. (B) Immunofluorescent images of cryosections of liver and spleen obtained 30 minutes after transfusion of CMFDA-labeled (green) ST3Gal-IV+/+ platelets (i,iii,v) or ST3Gal-IV−/− platelets (ii,iv,vi). Liver (i-ii) and spleen (v-vi) macrophages were stained with F4/80 antibody (red); hepatocytes were stained with anti-albumin antibody (red; iii-iv). ST3Gal-IV−/− platelets colocalized (yellow) with both liver macrophages (arrows) and hepatocytes (arrowheads). (Inset) Original magnification (×3) of the region indicated in subpanel iv. A small number of CMFDA-labeled platelets were found in splenic macrophages (v-vi). Nuclei were 4,6-diamidino-2-phenylindole–labeled (blue). Bars represent 5 μm. Platelet staining was not observed in specimens from untransfused mice (not shown). (C) Quantification of fluorescently labeled ST3Gal-IV−/− and ST3Gal-IV+/+ platelets colocalizing with liver hepatocytes or macrophages (P < .001). Data are mean ± SD of 5 randomly selected fields from stained tissue sections. (D) Effect of asialofetuin on the clearance of ST3Gal-IV−/− platelets in WT mice. Coinjection of asialofetuin (open symbols), a competitive inhibitor of the ASGPR, restored the recovery and initial circulation of ST3Gal-IV−/− platelets (triangles) to normal levels (P = .173). Circulation of ST3Gal-IV+/+ platelets (squares) was also improved by asialofetuin injection (P = .072). Fetuin coinjections (closed symbols) had no effect on ST3Gal-IV−/− platelet recovery or circulation.

Clearance of transfused ST3Gal-IV−/− platelets. (A) Average survival curves for transfused ST3Gal-IV+/+ (WT, ■), ST3Gal-IV+/− (), or ST3Gal-IV −/− (□) platelets in WT mice. CMFDA-labeled platelets were injected into WT mice, blood samples collected at the indicated time points, and the percentage of labeled platelets remaining in the circulation determined by flow cytometry. ST3Gal-IV−/− platelet recovery and platelet survival were significantly reduced compared with WT and ST3Gal-IV+/− platelets (P = .021 and P = .001, respectively). Transfusion data represent an average of 4 mice ± SD and are representative of 3 separate experiments. (B) Immunofluorescent images of cryosections of liver and spleen obtained 30 minutes after transfusion of CMFDA-labeled (green) ST3Gal-IV+/+ platelets (i,iii,v) or ST3Gal-IV−/− platelets (ii,iv,vi). Liver (i-ii) and spleen (v-vi) macrophages were stained with F4/80 antibody (red); hepatocytes were stained with anti-albumin antibody (red; iii-iv). ST3Gal-IV−/− platelets colocalized (yellow) with both liver macrophages (arrows) and hepatocytes (arrowheads). (Inset) Original magnification (×3) of the region indicated in subpanel iv. A small number of CMFDA-labeled platelets were found in splenic macrophages (v-vi). Nuclei were 4,6-diamidino-2-phenylindole–labeled (blue). Bars represent 5 μm. Platelet staining was not observed in specimens from untransfused mice (not shown). (C) Quantification of fluorescently labeled ST3Gal-IV−/− and ST3Gal-IV+/+ platelets colocalizing with liver hepatocytes or macrophages (P < .001). Data are mean ± SD of 5 randomly selected fields from stained tissue sections. (D) Effect of asialofetuin on the clearance of ST3Gal-IV−/− platelets in WT mice. Coinjection of asialofetuin (open symbols), a competitive inhibitor of the ASGPR, restored the recovery and initial circulation of ST3Gal-IV−/− platelets (triangles) to normal levels (P = .173). Circulation of ST3Gal-IV+/+ platelets (squares) was also improved by asialofetuin injection (P = .072). Fetuin coinjections (closed symbols) had no effect on ST3Gal-IV−/− platelet recovery or circulation.

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