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lack of annexin A7 changes red blood cell morphology and osmotic resistance\nAs annexin A7 is a component of red blood cell vesicles we studied the consequences of the loss of the protein in the anxA7-/-mouse for red blood cell morphology and osmotic resistance. In a standard 'blood smear' we did not note a deformation of the cells, however dark field microscopy revealed changes in shape and diameter of the anxA7-/-red blood cells. They had a statistically significant larger cell diameter of 6.0 μm compared to wild type with 5.7 μm (p = 0.015, n = 40), a remarkably lower emphasized central impression and a more flat shape (Fig. 4). No significant change in the mean corpuscular volume (MCV) was measured with the ADVIA 120 cell counter.\nFigure 4 Dark field microscopy of red blood cells from anxA7-/- mutant (B) and wild type mice (A). The mean cellular diameters are 6.0 μm and 5.7 μm, respectively. The size differences are statistically significant (p = 0.015; n = 40). Bar, 3 μm. Shape and MCV of a red blood cell essentially influence its function and capillary passage. The typical biconcave form depends on various influences including the membrane lipid composition and the submembranous cytoskeleton [32] where annexin A7 might play a role. The osmotic resistance, which is the resistance towards changes in the extracellular ionic strength, is a convenient assay for analysis of the red blood cell integrity. It is measured as the sodium chloride concentration at which cellular lysis starts (minimal resistance) up to a complete lysis (maximal resistance) of the cells [33]. Osmotic resistance increases with higher MCV, larger surface area and a higher degree of cellular metabolism stabilizing the intracellular ion levels. Aged red blood cells show a lowered MCV and lower osmotic resistance [33]. Similar properties can be observed in red blood cells with lowered membrane permeability. The osmotic resistance of annexin A7 deficient red blood cells is significantly increased compared to wild type cells. The minimal resistance value of knock out cells is observed in a 0.60 % NaCl solution versus 0.65 % for wild type cells. 50 % haemolysis is achieved at 0.516 % NaCl solution for knock out versus 0.564 % NaCl for wild type cells (mean values, p = 0.00066, n = 8; Fig. 5). The resistance width of the knock out cells is slightly lower (0.20 % instead of 0.25 % NaCl solution). We also performed direct measurements of the red blood cell membrane deformability, to further characterize a possible role of annexin A7 on the membrane stability. With micropipette experiments we tried to correlate mechanical characteristics of the red blood cells with their morphology. We could not observe any statistically significant difference of values describing membrane rigidity and lysis force in these experiments (M. Heil, B. Hoffmann and R. Merkel, unpublished results). However, the distinction observed in the osmotic resistance experiments was highly significant and reflects a mean value over a high number of different red blood cells.\nFigure 5 Osmotic resistance curves of red blood cells from wild type and anxA7-/- mutant mice. The osmotic resistance towards changes in the extracellular ionic strength is a convenient assay for analysis of the red blood cell integrity. It is measured as a sodium chloride concentration in which cellular lysis starts (minimal resistance) up to a complete lysis (maximal resistance) of a red blood cell. The osmotic resistance of annexin A7 deficient red blood cells is significantly increased compared to the one of wild type (p = 0.00066; n = 8). These data suggest that annexin A7 contributes to the shape of red blood cells and the osmotic resistance. As we have not observed a binding of annexin A7 to F-actin (data not shown) it could do so either by alteration of the membrane rigidity and/or by affecting the ion homeostasis."}

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lack of annexin A7 changes red blood cell morphology and osmotic resistance\nAs annexin A7 is a component of red blood cell vesicles we studied the consequences of the loss of the protein in the anxA7-/-mouse for red blood cell morphology and osmotic resistance. In a standard 'blood smear' we did not note a deformation of the cells, however dark field microscopy revealed changes in shape and diameter of the anxA7-/-red blood cells. They had a statistically significant larger cell diameter of 6.0 μm compared to wild type with 5.7 μm (p = 0.015, n = 40), a remarkably lower emphasized central impression and a more flat shape (Fig. 4). No significant change in the mean corpuscular volume (MCV) was measured with the ADVIA 120 cell counter.\nFigure 4 Dark field microscopy of red blood cells from anxA7-/- mutant (B) and wild type mice (A). The mean cellular diameters are 6.0 μm and 5.7 μm, respectively. The size differences are statistically significant (p = 0.015; n = 40). Bar, 3 μm. Shape and MCV of a red blood cell essentially influence its function and capillary passage. The typical biconcave form depends on various influences including the membrane lipid composition and the submembranous cytoskeleton [32] where annexin A7 might play a role. The osmotic resistance, which is the resistance towards changes in the extracellular ionic strength, is a convenient assay for analysis of the red blood cell integrity. It is measured as the sodium chloride concentration at which cellular lysis starts (minimal resistance) up to a complete lysis (maximal resistance) of the cells [33]. Osmotic resistance increases with higher MCV, larger surface area and a higher degree of cellular metabolism stabilizing the intracellular ion levels. Aged red blood cells show a lowered MCV and lower osmotic resistance [33]. Similar properties can be observed in red blood cells with lowered membrane permeability. The osmotic resistance of annexin A7 deficient red blood cells is significantly increased compared to wild type cells. The minimal resistance value of knock out cells is observed in a 0.60 % NaCl solution versus 0.65 % for wild type cells. 50 % haemolysis is achieved at 0.516 % NaCl solution for knock out versus 0.564 % NaCl for wild type cells (mean values, p = 0.00066, n = 8; Fig. 5). The resistance width of the knock out cells is slightly lower (0.20 % instead of 0.25 % NaCl solution). We also performed direct measurements of the red blood cell membrane deformability, to further characterize a possible role of annexin A7 on the membrane stability. With micropipette experiments we tried to correlate mechanical characteristics of the red blood cells with their morphology. We could not observe any statistically significant difference of values describing membrane rigidity and lysis force in these experiments (M. Heil, B. Hoffmann and R. Merkel, unpublished results). However, the distinction observed in the osmotic resistance experiments was highly significant and reflects a mean value over a high number of different red blood cells.\nFigure 5 Osmotic resistance curves of red blood cells from wild type and anxA7-/- mutant mice. The osmotic resistance towards changes in the extracellular ionic strength is a convenient assay for analysis of the red blood cell integrity. It is measured as a sodium chloride concentration in which cellular lysis starts (minimal resistance) up to a complete lysis (maximal resistance) of a red blood cell. The osmotic resistance of annexin A7 deficient red blood cells is significantly increased compared to the one of wild type (p = 0.00066; n = 8). These data suggest that annexin A7 contributes to the shape of red blood cells and the osmotic resistance. As we have not observed a binding of annexin A7 to F-actin (data not shown) it could do so either by alteration of the membrane rigidity and/or by affecting the ion homeostasis."}

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lack of annexin A7 changes red blood cell morphology and osmotic resistance\nAs annexin A7 is a component of red blood cell vesicles we studied the consequences of the loss of the protein in the anxA7-/-mouse for red blood cell morphology and osmotic resistance. In a standard 'blood smear' we did not note a deformation of the cells, however dark field microscopy revealed changes in shape and diameter of the anxA7-/-red blood cells. They had a statistically significant larger cell diameter of 6.0 μm compared to wild type with 5.7 μm (p = 0.015, n = 40), a remarkably lower emphasized central impression and a more flat shape (Fig. 4). No significant change in the mean corpuscular volume (MCV) was measured with the ADVIA 120 cell counter.\nFigure 4 Dark field microscopy of red blood cells from anxA7-/- mutant (B) and wild type mice (A). The mean cellular diameters are 6.0 μm and 5.7 μm, respectively. The size differences are statistically significant (p = 0.015; n = 40). Bar, 3 μm. Shape and MCV of a red blood cell essentially influence its function and capillary passage. The typical biconcave form depends on various influences including the membrane lipid composition and the submembranous cytoskeleton [32] where annexin A7 might play a role. The osmotic resistance, which is the resistance towards changes in the extracellular ionic strength, is a convenient assay for analysis of the red blood cell integrity. It is measured as the sodium chloride concentration at which cellular lysis starts (minimal resistance) up to a complete lysis (maximal resistance) of the cells [33]. Osmotic resistance increases with higher MCV, larger surface area and a higher degree of cellular metabolism stabilizing the intracellular ion levels. Aged red blood cells show a lowered MCV and lower osmotic resistance [33]. Similar properties can be observed in red blood cells with lowered membrane permeability. The osmotic resistance of annexin A7 deficient red blood cells is significantly increased compared to wild type cells. The minimal resistance value of knock out cells is observed in a 0.60 % NaCl solution versus 0.65 % for wild type cells. 50 % haemolysis is achieved at 0.516 % NaCl solution for knock out versus 0.564 % NaCl for wild type cells (mean values, p = 0.00066, n = 8; Fig. 5). The resistance width of the knock out cells is slightly lower (0.20 % instead of 0.25 % NaCl solution). We also performed direct measurements of the red blood cell membrane deformability, to further characterize a possible role of annexin A7 on the membrane stability. With micropipette experiments we tried to correlate mechanical characteristics of the red blood cells with their morphology. We could not observe any statistically significant difference of values describing membrane rigidity and lysis force in these experiments (M. Heil, B. Hoffmann and R. Merkel, unpublished results). However, the distinction observed in the osmotic resistance experiments was highly significant and reflects a mean value over a high number of different red blood cells.\nFigure 5 Osmotic resistance curves of red blood cells from wild type and anxA7-/- mutant mice. The osmotic resistance towards changes in the extracellular ionic strength is a convenient assay for analysis of the red blood cell integrity. It is measured as a sodium chloride concentration in which cellular lysis starts (minimal resistance) up to a complete lysis (maximal resistance) of a red blood cell. The osmotic resistance of annexin A7 deficient red blood cells is significantly increased compared to the one of wild type (p = 0.00066; n = 8). These data suggest that annexin A7 contributes to the shape of red blood cells and the osmotic resistance. As we have not observed a binding of annexin A7 to F-actin (data not shown) it could do so either by alteration of the membrane rigidity and/or by affecting the ion homeostasis."}