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Five spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sphalleles reveals band 3 and adducin deficiency as well. In sph4J, a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, β-adducin. The severity of anemia in sph4Jindicates that the highly conserved cysteine residue at the C-terminus of α-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin, adducin, and band 3.

Cellular cation homeostasis in mouse erythrocytes with defective membrane skeletons was examined in three mouse mutants, hemolytic anemia (sphha/sphha), spherocytosis (sph/sph), and normoblastosis (nb/nb), and compared with reticulocytes produced by repetitive bleeding of congenic normal mice. To assess reticulocyte maturity, nucleic acid and transferrin receptor contents were measured by fluorescence flow cytometry; mutant cells were somewhat more mature than normal reticulocytes by these criteria. Red blood cell (RBC) sodium contents (Na+c) in homozygous sphha/sphha, sph/sph, and nb/nb animals were 30.1 ± 0.9, 28.9 ± 0.3, and 26.9 ± 1.5 mmol/L cell, respectively, whereas cellular potassium (K+c) was 102 ± 2.6, 101 ± 7.8, and 97.4 ± 3.0. Na+cand K+cin normal reticulocyte preparations were 11.3 ± 0.7 and 123 ± 10, respectively. Net Na+and K+fluxes in the presence of ouabain were markedly increased in mutant RBCs. Sodium uptake was 14.8 ± 1.6, 15.4 ± 3.3, and 14.7 ± 3.1 mmol/L cell/h in sphha/sphha, sph/sph, and nb/nb mutants, respectively, whereas K+loss was 17.0 ± 4.0, 15.0 ± 3.8, and 14.1 ± 2.6. Normal mouse reticulocytes gained Na+at a rate of 3.9 ± 1.0 mmol/L cell/h and lost K+at 6.0 ± 2.1, rates indistinguishable from those in mature mouse RBCs. Potassium loss from sphha/sphhaand nb/nb cells was not dependent on the presence of a Na+gradient, and net cation movements were insensitive to bumetanide (sphha/sphhaand nb/nb RBCs) and to chloride replacement with sulfamate (nb/nb cells). We conclude that mutant mouse RBCs with dysfunctional membrane skeletons have increased passive permeability to monovalent cations. These findings support a role of the membrane skeleton in the maintenance of the membrane permeability barrier and suggest that the abnormal permeability associated with human hereditary spherocytosis and elliptocytosis may be a consequence of the membrane skeleton defects reported in these disorders.

A method has been developed for determining the relative tendency of red cell membranes to fragment under shear stress. For these measurements, resealed ghosts were subjected to constant, high fluid shear stress in an ektacy-tometer. The deformability signal, which this instrument provides, progressively declined as the deformable cells were transformed into less deformable, more spherical cell fragments. The deformability signal for normal ghost preparations decayed over a narrowly defined time period; this decay was accompanied by the production of cell fragments, as shown by microscopic examination and analysis of particle volume distribution. The method was used to study cells from patients with various hemolytic disorders. Membrane preparations from patients with various types of nonhemolytic congenital elliptocytoses showed a reduction in the time required for fragmentation to about half the normal value. Membrane preparations from patients with hemolytic elliptocytosis associated with a severe deficiency of the membrane skeletal protein band 4.1 showed an even greater reduction in fragmentation time, distinct from the values for nonhemolytic samples. In elliptocytoses secondary to myelofibrosis, the fragmentation times were within the normal range. In hereditary spherocytosis (HS), heterogeneity in fragmentation times was observed; marginally decreased or normal fragmentation times were seen for cells from 8 patients, but a pronounced shortening in fragmentation time was found for one other HS patient. A defect was previously found in spectrin-band 4.1 binding for this same patient. These observations suggest that this membrane fragmentation technique may be useful for the identification of certain specific disorders or their clinical variants in which the integrity of membrane skeletal interactions has been perturbed in such a way that the mechanical stability of the membrane is reduced.

To understand more clearly the hemolytic anemia associated with administration of certain oxidant drugs, the mechanism by which H2O2 causes hemolysis in rat red cells, deficient in vitamin E was investigated. It was demonstrated that the locus of attack by H2O2 was the red cell membrane, in which one phospholipid, i.e., phosphatidyl ethanolamine, was specifically destroyed prior to the onset of hemolysis. No perturbation of intracellular components or metabolism was noted during peroxidative hemolysis. E-deficient red cells incorporated 14C-labelled fatty acids into this phosphatide at nearly twice the rate that in E-supplemented cells, reflecting the continual tendency of phosphatidyl ethanolamine to be destroyed. Young red cells were especially active in this regard and concomitantly were less vulnerable to damage by H2O2 both in vitro and when circulating in rats exposed to hyperbaric oxygenation. If, however, replacement of fatty acids in phosphatidyl ethanolamine was prevented by inhibition of metabolism or if fatty acids were enzymatically removed by a phospholipase-A, H2O2 hemolysis was potentiated. Hemolysis was also associated with, and potentiated by, loss of membrane sulfhydryl activity. It is suggested that hemolytic anemia may occur in patients with vitamin E deficiency (i.e., with steatorrhea) if oxidant drugs capable of generating H2O2 and oxidizing membrane thiols are administered. Two such cases are under investigation.