The Gardos effect drives vesiculation-dependent sialic acid loss and adhesion molecule activation. (A) Control, aged, valinomycin-treated (Val.), stored, propranolol-treated (Prop.), sickle, and neuraminidase-treated (N’ase) erythrocytes (1 × 10⁸) were incubated with Arachis hypogea lectin. Exposure of the T-antigen that follows removal of terminal sialic acid residues will allow A hypogea lectin to agglutinate these cells. Neuraminidase treatment was used as a positive control. (B) Micrograph of erythrocytes treated with propranolol. Although a minority of erythrocytes remained round and biconcave, a large fraction of erythrocytes had already assumed stomatocyte-like appearance with shrinking and loss of biconcavity. The width of the exerpt is 50 μm. (C) ARCA quantification of erythrocyte surface area under a sheer stress of 10 dynes/cm². (D) Imagestream analysis was performed to identify erythrocytes and vesicles in response to propranolol treatment. (E) Shedding of relatively large vesicles (>0.3 µm) in response to propranolol treatment was quantified by flow cytometry. (F) Effect of propranolol and inhibition of serine proteases by DFP on expression of α2,3-linked membrane sialic acid. (G) A total of 10⁷ control, propranolol-treated, and DFP-pretreated erythrocytes were flown over laminin-α5 and HA at 0.2 dynes/cm², and adhesion frequency was assessed by microscopy. (H) Erythrocytes were stained with the Ca²⁺ dye Fluo-4 and treated with propranolol or with DFP before treatment with propranolol. Flow cytometry was performed to quantify Ca²⁺ influx in response to these stimuli (n = 3; 1-way ANOVA). (I) Relative intracellular potassium levels were assessed by potassium-binding benzofuran isophthalate acetoxymethyl ester (PBFI) staining and flow cytometry. PBFI was used instead of ion-specific electrodes because this allowed us to work with fewer erythrocytes, which enabled us to detect the effect of the various protease inhibitors (n = 3-4; 1-way ANOVA). (J) Micrograph of erythrocyte morphology upon treatment with various protease inhibitors and propranolol. The induction of stomatocytosis by propranolol was quantified by flow cytometry based on an increase in side scatter. This was also used to measure the degree of inhibition of stomatocytosis by various protease inhibitors (n = 3; 1-way ANOVA). *P < .05; **P < .01; ***P < .001. FSC, forward scatter; inh, inhibitor; SSC, side scatter; ZVAD, Z-VAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp[O-Me] fluoromethyl ketone).