Figure 4
Figure 4. Deficient sialylation of VWF increases its binding to platelets and enhances platelet clearance. (A) ST3Gal-IV−/− platelets have increased βGal exposure. Staining of ST3Gal-IV−/− and ST3Gal-IV+/+ platelets with the βGal recognizing lectins RCA-I and ECA revealed increased βGal exposure on ST3Gal-IV−/− platelets (red lines) compared with WT (black lines) littermates. The level of Galα1-3Gal was only marginally increased on ST3Gal-IV−/− platelets as evidenced by binding of the GS-IB4 lectin. The lectin profile was confirmed using monoclonal antibodies 1B2 (specific for Galβ1-4GlcNAcβ-R), 3C9 (specific for Galβ1-3GalNAcα-OH), and TH5 (specific for Galα1-3Galβ1-4GlcNAcβ-R). (B) Botrocetin stimulation of platelets in ST3Gal-IV−/− plasma induced significantly higher levels of anti-VWF binding to both ST3Gal-IV−/− (■) and ST3Gal-IV+/+ () platelets compared with control plasma ( and □, respectively; P < .001). ST3Gal-IV−/− platelets (■ and ) consistently bound more VWF than ST3Gal-IV+/+ platelets ( and □) independently of the plasma type used (P = .008). VWF binding was not increased in resting ST3Gal-IV−/− platelets (botrocetin = 0 U/mL). Data are representative of 9 mice. (C) WT platelet life span was significantly reduced in ST3Gal-IV−/− mice (bottom panel ▲) compared with WT mice (top panel ■; P = .014). Removal of the extracellular domain of GPIbα by O-sialoglycoprotein endopeptidase (+OSP) doubled the percentage transfused WT platelets remaining in the circulation of ST3Gal-IV−/− mice at 24 and 48 hours (P = .003 and P = .014; bottom panel △) and significantly increased platelet life span in WT mice at these times (P = .022 and P = .021; top panel □). (C inset) Flow cytometry documenting the removal of the GPIbα N-terminus. Platelets before (white) and after (gray) treatment with O-sialoglycoprotein endopeptidase (+OSP), which removes the VWF binding domain of GPIbα carrying complex type N-linked glycans. Background labeling with IgG control is also shown. (D) Transfused platelets rapidly collect ST3Gal-VI−/− VWF on their surfaces. Three-fold more VWF-positive WT platelets were detected in ST3Gal-IV−/− mice compared with WT platelets transfused into WT mice (P = .021). The data are mean ± SD of 4 mice.

Deficient sialylation of VWF increases its binding to platelets and enhances platelet clearance. (A) ST3Gal-IV−/− platelets have increased βGal exposure. Staining of ST3Gal-IV−/− and ST3Gal-IV+/+ platelets with the βGal recognizing lectins RCA-I and ECA revealed increased βGal exposure on ST3Gal-IV−/− platelets (red lines) compared with WT (black lines) littermates. The level of Galα1-3Gal was only marginally increased on ST3Gal-IV−/− platelets as evidenced by binding of the GS-IB4 lectin. The lectin profile was confirmed using monoclonal antibodies 1B2 (specific for Galβ1-4GlcNAcβ-R), 3C9 (specific for Galβ1-3GalNAcα-OH), and TH5 (specific for Galα1-3Galβ1-4GlcNAcβ-R). (B) Botrocetin stimulation of platelets in ST3Gal-IV−/− plasma induced significantly higher levels of anti-VWF binding to both ST3Gal-IV−/− (■) and ST3Gal-IV+/+ () platelets compared with control plasma ( and □, respectively; P < .001). ST3Gal-IV−/− platelets (■ and ) consistently bound more VWF than ST3Gal-IV+/+ platelets ( and □) independently of the plasma type used (P = .008). VWF binding was not increased in resting ST3Gal-IV−/− platelets (botrocetin = 0 U/mL). Data are representative of 9 mice. (C) WT platelet life span was significantly reduced in ST3Gal-IV−/− mice (bottom panel ▲) compared with WT mice (top panel ■; P = .014). Removal of the extracellular domain of GPIbα by O-sialoglycoprotein endopeptidase (+OSP) doubled the percentage transfused WT platelets remaining in the circulation of ST3Gal-IV−/− mice at 24 and 48 hours (P = .003 and P = .014; bottom panel △) and significantly increased platelet life span in WT mice at these times (P = .022 and P = .021; top panel □). (C inset) Flow cytometry documenting the removal of the GPIbα N-terminus. Platelets before (white) and after (gray) treatment with O-sialoglycoprotein endopeptidase (+OSP), which removes the VWF binding domain of GPIbα carrying complex type N-linked glycans. Background labeling with IgG control is also shown. (D) Transfused platelets rapidly collect ST3Gal-VI−/− VWF on their surfaces. Three-fold more VWF-positive WT platelets were detected in ST3Gal-IV−/− mice compared with WT platelets transfused into WT mice (P = .021). The data are mean ± SD of 4 mice.

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