Figure 4.
Shedding of GPC+vesicles lift the restriction on Lu/BCAM and CD44 activity. (A) Expression of anti-GPA/GPC on control, propranolol, and valinomycin-treated erythrocytes. MFI was normalized to control cells and expressed as fold change. (B) Imagestream analysis was performed to identify erythrocyte and vesicle populations (see Figure 3B) in response to propranolol treatment. The distribution of GPA and GPC on erythrocytes and their vesicles was determined (lower panel). (C) CD44 immunoprecipitation (IP) was performed on control and neuraminidase-treated erythrocytes and blotted for GPC using BRIC10 monoclonal antibody. Band height difference between control and neuraminidase-treated erythrocytes indicate successful desialylation of GPC. Immunoglobulin G (IgG) control and lysates were taken along as controls. (D) We quantified expression of a sialylated epitope of GPC using BRIC10 on control erythrocytes and on erythrocytes lacking exon 3 of GPC known as the Gerbich (Ge+2–3) phenotype. Neuraminidase-treated erythrocytes were taken along as control (n = 3-7; 1-way ANOVA). (E-F) A total of 107 control, Ge–2–3 and neuraminidase-treated erythrocytes were flowed over laminin-α5 and HA at 0.2 dynes/cm2, and adhesion/rolling frequency was assessed by microscopy (n = 3-5; 1-way ANOVA). (G) Flow cytometry staining of CD44-FITC and GPC-allophycocyanin (APC) on wild-type (WT) K562 and GPC knockout (KO) K562 cells. IgG controls were used for gating. (H) Adhesion of WT and GPC KO K562 cells to HA. (I) Schematic representation (created with Biorender.com) of senescence of RBCs as a consequence of Gardos activation. First, calcium enters the cell, either through a stimulus such as propranolol or as a consequence of transient leakage of calcium such as that observed during erythrocyte ageing. The increased intracellular calcium levels activate the calcium-dependent potassium efflux channel known as the Gardos channel, causing RBC dehydration. In response to dehydration, erythrocytes shed vesicles that contain GPC, causing loss of membrane sialic acid which directly results in Lu/BCAM and CD44 activation.2,3 *P < .05; **P < .01; ***P < .001; ****P < .0001. FITC, fluorescein isothiocyanate. 

Shedding of GPC+vesicles lift the restriction on Lu/BCAM and CD44 activity. (A) Expression of anti-GPA/GPC on control, propranolol, and valinomycin-treated erythrocytes. MFI was normalized to control cells and expressed as fold change. (B) Imagestream analysis was performed to identify erythrocyte and vesicle populations (see Figure 3B) in response to propranolol treatment. The distribution of GPA and GPC on erythrocytes and their vesicles was determined (lower panel). (C) CD44 immunoprecipitation (IP) was performed on control and neuraminidase-treated erythrocytes and blotted for GPC using BRIC10 monoclonal antibody. Band height difference between control and neuraminidase-treated erythrocytes indicate successful desialylation of GPC. Immunoglobulin G (IgG) control and lysates were taken along as controls. (D) We quantified expression of a sialylated epitope of GPC using BRIC10 on control erythrocytes and on erythrocytes lacking exon 3 of GPC known as the Gerbich (Ge+2–3) phenotype. Neuraminidase-treated erythrocytes were taken along as control (n = 3-7; 1-way ANOVA). (E-F) A total of 107 control, Ge–2–3 and neuraminidase-treated erythrocytes were flowed over laminin-α5 and HA at 0.2 dynes/cm2, and adhesion/rolling frequency was assessed by microscopy (n = 3-5; 1-way ANOVA). (G) Flow cytometry staining of CD44-FITC and GPC-allophycocyanin (APC) on wild-type (WT) K562 and GPC knockout (KO) K562 cells. IgG controls were used for gating. (H) Adhesion of WT and GPC KO K562 cells to HA. (I) Schematic representation (created with Biorender.com) of senescence of RBCs as a consequence of Gardos activation. First, calcium enters the cell, either through a stimulus such as propranolol or as a consequence of transient leakage of calcium such as that observed during erythrocyte ageing. The increased intracellular calcium levels activate the calcium-dependent potassium efflux channel known as the Gardos channel, causing RBC dehydration. In response to dehydration, erythrocytes shed vesicles that contain GPC, causing loss of membrane sialic acid which directly results in Lu/BCAM and CD44 activation.2,3  *P < .05; **P < .01; ***P < .001; ****P < .0001. FITC, fluorescein isothiocyanate. 

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