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The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3–containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton’s physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.

The red cell membrane skeleton is a pseudohexagonal meshwork of spectrin, actin, protein 4.1R, ankyrin, and actin-associated proteins that laminates the inner membrane surface and attaches to the overlying lipid bilayer via band 3–containing multiprotein complexes at the ankyrin- and actin-binding ends of spectrin. The membrane skeleton strengthens the lipid bilayer and endows the membrane with the durability and flexibility to survive in the circulation. In the 36 years since the first primitive model of the red cell skeleton was proposed, many additional proteins have been discovered, and their structures and interactions have been defined. However, almost nothing is known of the skeleton's physiology, and myriad questions about its structure remain, including questions concerning the structure of spectrin in situ, the way spectrin and other proteins bind to actin, how the membrane is assembled, the dynamics of the skeleton when the membrane is deformed or perturbed by parasites, the role lipids play, and variations in membrane structure in unique regions like lipid rafts. This knowledge is important because the red cell membrane skeleton is the model for spectrin-based membrane skeletons in all cells, and because defects in the red cell membrane skeleton underlie multiple hemolytic anemias.

Spectrin and protein 4.1R crosslink F-actin, forming the membrane skeleton. Actin and 4.1R bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF domain, is calmodulin-like, with calcium-dependent and calcium-independent EF hands. The severely anemic sph1J/sph1J mouse has very fragile red cells and lacks the last 13 amino acids in the EF domain, implying that the domain is critical for skeletal integrity. To test this, we constructed a minispectrin heterodimer from the actin-binding domain, the EF domain, and 4 adjacent spectrin repeats in each chain. The minispectrin bound to F-actin in the presence of native human protein 4.1R. Formation of the spectrin-actin-4.1R complex was markedly attenuated when the minispectrin contained the shortened sph1J α-spectrin. The α-spectrin deletion did not interfere with spectrin heterodimer assembly or 4.1R binding but abolished the binary interaction between spectrin and F-actin. The data show that the α-spectrin EF domain greatly amplifies the function of the β-spectrin actin-binding domain (ABD) in forming the spectrin-actin-4.1R complex. A model, based on the structure of α-actinin, suggests that the EF domain modulates the function of the ABD and that the C-terminal EF hands (EF34) may bind to the linker that connects the ABD to the first spectrin repeat.

Spectrin and protein 4.1R crosslink F-actin, forming the membrane skeleton. Actin and 4.1R bind to one end of β-spectrin. The adjacent end of α-spectrin, called the EF domain, is calmodulin-like, with calcium-dependent and calcium-independent EF hands. The severely anemic sph1J/sph1Jmouse has very fragile red cells and lacks the last 13 amino acids in the EF domain, implying that the domain is critical for skeletal integrity. To test this, we constructed a minispectrin heterodimer from the actin-binding domain, the EF domain, and 4 adjacent spectrin repeats in each chain. The minispectrin bound to F-actin in the presence of native human protein 4.1R. Formation of the spectrin-actin-4.1R complex was markedly attenuated when the minispectrin contained the shortened sph1Jα-spectrin. The α-spectrin deletion did not interfere with spectrin heterodimer assembly or 4.1R binding but abolished the binary interaction between spectrin and F-actin. The data show that the α-spectrin EF domain greatly amplifies the function of the β-spectrin actin-binding domain (ABD) in forming the spectrin-actin-4.1R complex. A model, based on the structure of α-actinin, suggests that the EF domain modulates the function of the ABD and that the C-terminal EF hands (EF34) may bind to the linker that connects the ABD to the first spectrin repeat.

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 sph alleles 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 sph4J indicates 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.

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.

Defects in red blood cell (RBC) membrane skeleton components cause hereditary spherocytosis (HS). Clinically, HS varies significantly even among individuals with identical gene defects, illustrating the profound effects of genetic background on disease severity. We exploited a new spontaneous mouse model, wan, which arose on the inbred C3H/HeJ strain, to identify quantitative trait loci (QTL) that modify the HS phenotype. Homozygous wan mice have severe HS due to a complete deficiency of erythroid band 3. A QTL analysis of RBC count, hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin content (MCHC) was performed in wan/wan mice from an F2 intercross between C3H/HeJ+/wan and CAST/Ei+/+ F1 hybrids. Hematologic and survival data from C3H, CAST/Ei F2 wan homozygotes support the hypothesis that genetic modifiers significantly influence the band-3 null HS phenotype. Significant QTL were identified for the MCV trait only, suggesting that RBC membrane characteristics are a target for modifier gene action. The most significant quantitative trait locus, Hsm1 (hereditary spherocytosis modifier 1), localizes to mouse Chromosome 12 and is dominant. The peak LOD score was obtained with a marker for Spnb1 encoding erythroid β-spectrin, an obvious candidate gene. (Blood. 2004;103: 3233-3240)

Defects in red blood cell (RBC) membrane skeleton components cause hereditary spherocytosis (HS). Clinically, HS varies significantly even among individuals with identical gene defects, illustrating the profound effects of genetic background on disease severity. We exploited a new spontaneous mouse model,wan, which arose on the inbred C3H/HeJ strain, to identify quantitative trait loci (QTL) that modify the HS phenotype. Homozygouswanmice have severe HS due to a complete deficiency of erythroid band 3. A QTL analysis of RBC count, hemoglobin, hematocrit, mean corpuscular volume (MCV), and mean corpuscular hemoglobin content (MCHC) was performed inwan/wanmice from an F2 intercross between C3H/HeJ+/wanand CAST/Ei+/+F1 hybrids. Hematologic and survival data from C3H, CAST/Ei F2wanhomozygotes support the hypothesis that genetic modifiers significantly influence the band-3 null HS phenotype. Significant QTL were identified for the MCV trait only, suggesting that RBC membrane characteristics are a target for modifier gene action. The most significant quantitative trait locus,Hsm1(hereditaryspherocytosismodifier 1), localizes to mouse Chromosome 12 and is dominant. The peak LOD score was obtained with a marker forSpnb1encoding erythroid β-spectrin, an obvious candidate gene. (Blood. 2004;103: 3233-3240)

We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10–16 of β-spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length β-spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin βIV, thus encodes a full-length minor isoform (SpβIVΣ1) and a truncated major isoform (SpβIVΣ5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpβIVΣ5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpβIVΣ5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin βIV is the first β-spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds.

To begin to study the sequence variations identified in the 5' flanking genomic DNA of the ankyrin gene in ankyrin-deficient hereditary spherocytosis patients and to provide additional insight into our understanding of the regulation of genes encoding erythrocyte membrane proteins, we have identified and characterized the erythroid promoter of the human ankyrin-1 gene. This compact promoter has characteristics of a housekeeping gene promoter, including very high G+C content and enzyme restriction sites characteristic of an HTF-island, no TATA, InR, or CCAAT consensus sequences, and multiple transcription initiation sites. In vitro DNAseI footprinting analyses revealed binding sites for GATA-1, CACCC-binding, and CGCCC-binding proteins. Transfection of ankyrin promoter/reporter plasmids into tissue culture cell lines yielded expression in erythroid, but not muscle, neural, or HeLa cells. Electrophoretic mobility shift assays, including competition and antibody supershift experiments, demonstrated binding of GATA-1, BKLF, and Sp1 to core ankyrin promoter sequences. In transfection assays, mutation of the Sp1 site had no effect on reporter gene expression, mutation of the CACCC site decreased expression by half, and mutation of the GATA-1 site completely abolished activity. The ankyrin gene erythroid promoter was transactivated in heterologous cells by forced expression of GATA-1 and to a lesser degree BKLF.

Alpha‐thalassaemic erythrocytes develop a specific membrane skeletal defect that is manifest as a loss of normal spectrin‐binding sites on the inner surface of the thalassaemic membranes. To test whether this lesion could be caused by the excess free beta‐globin chains that accumulate in alpha‐thalassaemic red cells, we incubated normal red cell membranes with native, haem‐containing alpha or beta globin chains or with haemoglobin A. Spectrin‐depleted inside‐out membrane vesicles (IOVs) derived from membranes incubated with beta‐globin chains bound only 9 ± 3% as much spectrin as IOVs from control membranes incubated with bovine serum albumin. In contrast, IOVs from membranes incubated with alpha‐globin chains or haemoglobin A were nearly normal (79 ± 3% and 86 ± 5% of controls, respectively). This differential effect of globin chains was not seen when membranes were first transformed into spectrin‐depleted IOVs and then incubated with the isolated globin chains. Under these conditions, both alpha and beta globin chains reduced the spectrin‐binding capacity of the IOVs by approximately 45% (alpha 46 ± 7%, beta 43 ± 6%) whereas haemoglobin A had no effect. Unlike IOVs, spectrin isolated from membranes exposed to alpha or beta globin chains bound normally to IOVs and to actin (in the presence of protein 4.1). These studies show that isolated beta‐globin chains (but not alpha‐globin chains) can produce a spectrin‐binding defect in normal red cell membranes similar to that seen in alpha thalassaemia. The existence of similar defects in the membrane skeletons of red cells from other diseases with unstable beta globins suggests a common pathophysiology for the premature destruction of these cells.

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.

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.

Hereditary spherocytosis (HS) is the most common inherited haemolytic anaemia in Northern Europeans. The primary molecular defects reside in the red blood cell (RBC) membrane, particularly in proteins that link the membrane skeleton to the overlying lipid bilayer and its integral membrane constituents1. Ankyrin-1 is the predominant linker molecule. It attaches spectrin, the major skeletal protein, to the cytoplasmic domain of band 3, the RBC anion exchanger. Two-thirds of patients with HS have combined spectrin and ankyrin-1 deficiency2,3; deficiency of band 3 occurs in about 15 to 20% (ref. 1). These data suggest that ankyrin-1 or band 3 defects may be common in HS. To test this we screened all 42 coding exons plus the 5′ untranslated/promoter region of ankyrin-1 and the 19 coding exons of band 3 in 46 HS families. Twelve ankyrin-1 mutations and five band 3 mutations were identified. Missense mutations and a mutation in the putative ankyrin-1 promoter were common in recessive HS. In contrast, ankyrin-1 and band 3 frameshift and nonsense null mutations prevailed in dominant HS. Increased accu-mulation of the normal protein product partially compensated for the ankyrin-1 or band 3 defects in some of these null mutations. Our findings indi-cate that ankyrin-1 mutations are a major cause of dominant and recessive HS (∼35 to 65%), that band 3 mutations are less common (∼15 to 25%), and that the severity of HS is modified by factors other than the primary gene defect.

Apolipoprotein-glutamine-II (apoLP-Gln-II) is one of the two major protein constituents of human plasma high density lipoproteins (HDL). ApoLP-Gln-II contains two identical chains of 77 amino acids each, which are linked by a single disulfide bond at residue 6. Each chain contains a single methionine at position 26. In the present study we have examined the phospholipid binding properties of apoLP-Gln-II, of the NH2-terminal cyanogen bromide fragment both in the reduced form (CNBr IV, residues 1 to 26) and with the disulfide bond intact (CNBr IV)2, of the COOH-terminal cyanogen bromide fragment (CNBr III, residues 27 to 77) and of performic acid-oxidized apoLP-Gln-II. The binding of phosphatidylcholine was monitored by the inhibition of the reactivation of delipidated β-hydroxybutyrate dehydrogenase from beef heart mitochondria and by the formation of phospholipid-protein complexes which were isolated by density gradient ultracentrifugation in sucrose. The circular dichroism and immunochemical properties of these fragments and derivatives of apoLP-Gln-II were studied both in the presence and absence of phosphatidylcholine. The results may be summarized as follows. (a) ApoLP-Gln-II and its performic acid-oxidized derivative were potent inhibitors of the reactivation of β-hydroxybutyrate apodehydrogenase. One microgram of these preparations gave approximately 50% inhibition. The COOH-terminal CNBr fragment, CNBr III, also inhibited reactivation of the apodehydrogenase at low concentrations, but was less effective than the intact protein. Both CNBr IV and (CNBr IV)2failed to inhibit reactivation of the dehydrogenase. (b) ApoLP-Gln-II, performic acid-oxidized apoLP-Gln-II and CNBr III formed complexes with phosphatidylcholine which were isolated by density gradient ultracentrifugation in sucrose (d1.063 to 1.210). These isolated complexes contained, respectively, 1.16, 1.56, and 2.00 mg of phosphatidylcholine per mg of protein or peptide. When (CNBr IV)2was reconstituted with phosphatidylcholine and subjected to ultracentrifugation under identical conditions, the isolated peptide contained only 0.08 mg of phospholipid per mg of peptide. (c) Reconstitution with phosphatidylcholine resulted in an apparent increase in α helical content (as judged by an increase in θ222 nmin the circular dichroism spectrum). The magnitude of the changes were as follows: 49% to 64% for apoLP-Gln-II, 32 to 57% for performic acid-oxidized apoLP-Gln-II, and 32 to 44% for CNBr III. There was no significant change in the CD spectra of (CNBr IV)2detected in the presence of phosphatidylcholine. (d) Performic acid oxidation of apoLP-Gln-II did not alter the qualitative immunoprecipitin lines when this antigen was tested against rabbit antisera prepared against apoLP-Gln-II. The same antisera reacted with CNBr III and (CNBr IV)2, but formed lines of only partial identity between the fragments or between each fragment and apoLP-Gln-II. Rabbit antisera to CNBr III formed precipitin lines of complete identity between this fragment, apoLP-Gln-II, and performic acid-oxidized apoLP-Gln-II. No differences were noted after reconstitution with phosphatidylcholine. Anti-CNBr III did not form detectable precipitin lines with (CNBr IV)2.

Current knowledge of red cell membrane structure and function are briefly presented, and the pathophysiology, diagnosis, and treatment of hereditary spherocytosis, hereditary elliptocytosis, and hereditary pyropoikilocytosis are discussed.

Pallid is one of 12 independent murine mutations with a prolonged bleeding time that are models for human platelet storage pool deficiencies in which several intracellular organelles are abnormal. We have mapped the murine gene for protein 4.2 (Epb4.2) to chromosome 2 where it co–localizes with pallid. Southern blot analyses suggest that pallid is a mutation in the Epb4.2 gene. Northern blot analyses demonstrate a smaller than normal Epb4.2 transcript in affected pallid tissues, such as kidney and skin. This is the first gene defect to be associated with a platelet storage pool deficiency, and may allow the identification of a novel structure or biological pathway that influences granulogenesis.

The hereditary hemolytic anemias include a heterogeneous class of disorders caused by defects in the proteins that constitute the membrane skeleton of the red blood cell. The combination of classical and molecular genetics together with clinical findings is rapidly improving our understanding of the basis of these defects.

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.

In diverse cell types, ankyrin tethers a variety of ion transport and cell adhesion molecules to the spectrin-based membrane skeleton. In the whole kidney, epithelial ankyrin (Ank3) is the predominantly expressed ankyrin and is expressed as distinct spliceoforms. Antibodies against a portion of the Ank3 regulatory domain detected four major spliceoforms at 215, 200, 170, and 120 kDa. Immunoblotting of the renal cortex, which is 80% proximal tubule (PT), detected all four spliceoforms but showed significantly diminished Ank3200/215. To determine the Ank3 spliceoforms present in the mouse PT cells, PT fragments were purified to 100% from the renal cortex. Isolation was performed by incubating cortical tubule segments with fluorescein and isolating the fluorescein-laden PT fragments or fluorescein-deplete non-PT (distal) fragments under fluorescence microscopy. Distal tubule (DT) fragments displayed abundance of the Ank3200/215but no Ank3170or Ank3120. Isolated PT segments contained all four spliceoforms but dramatically diminished Ank3200/215. These larger spliceoforms bind Na-K-ATPase in diverse cell types. Densitometric analysis of Ank3200/215and Na-K-ATPase abundance measured a lower Ank3200/215-to-Na-K-ATPase ratio in the PT vs. the renal cortex. These proximal vs. distal differences in Ank3 spliceoforms were displayed in LLC-PK1cells, a proximal cell line, and MDCK cells, a distal cell line. The lower PT content of Ank3200/215suggests Na-K-ATPase in PT may be organized differently than in DT. Likely reflecting their cell-specific organization, regulation, and function, these studies indicate the different renal cell types express distinct Ank3 spliceoforms.

Ankyrin-1 (ANK-1) is an erythrocyte membrane protein that is defective in many patients with hereditary spherocytosis, a common hemolytic anemia. In the red cell, ankyrin-1 provides the primary linkage between the membrane skeleton and the plasma membrane. To gain additional insight into the structure and function of this protein and to provide the necessary tools for further genetic studies of hereditary spherocytosis patients, we cloned the human ANK-1chromosomal gene. Characterization of theANK-1gene genomic structure revealed that the erythroid transcript is composed of 42 exons distributed over ∼160 kilobase pairs of DNA. Comparison of the genomic structure with the protein domains reveals a near-absolute correlation between the tandem repeats encoding the membrane-binding domain of ankyrin with the location of the intron/exon boundaries in the corresponding part of the gene. Erythroid stage-specific, complex patterns of alternative splicing were identified in the region encoding the regulatory domain of ankyrin-1. Novel brain-specific transcripts were also identified in this region, as well as in the “hinge” region between the membrane-binding and spectrin-binding domains. Utilization of alternative polyadenylation signals was found to be the basis for the previously described, stage-specific 9.0- and 7.2-kilobase pair transcripts of theANK-1gene.

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.

ApoLp-Gln-II (or apoA-II), one of the two major apolipoprotein components of human plasma high density lipoproteins, was subjected to enzymatic and nonenzymatic digestion with trypsin and cyanogen bromide. The individual peptides were isolated to homogeneity by chromatography on DEAE-cellulose, Cm-Sephadex, Sephadex G-100, Sephadex G-75, preparative thin layer plates, and by peptide mapping. Isolated peptides were assayed for purity by amino acid analysis, thin layer chromatography, polyacrylamide gel electrophoresis, and Edman NH2-terminal analysis. Cyanogen bromide cleavage followed by reduction and S-carboxymethylation produced two peptides, C-III and C-IV, of 51 and 26 residues, respectively. The smaller peptide (C-IV) contained homoserine and homoserine lactone and had NH2-terminal pyrrolidone carboxylic acid. It was the NH2-terminal cyanogen bromide peptide. This peptide contained the single S-carboxymethylcysteine residue. C-III was the COOH-terminal peptide. It contained COOH-terminal glutamine and a penultimate threonine residue. Tryptic digestion of reduced and alkylated apoLp-Gln-II yielded eight major tryptic peptides (T-I-1, T-I-2, T-I-3, T-III, T-IV, T-VI-1, T-VII, and T-VIII) and seven additional tryptic peptides which either contained or were contained in two or more of the eight major peptides. Only single unique NH2-terminal (T-I-2), COOH-terminal (T-VII), S-carboxymethylcysteine-containing (T-VIII), methionine-containing (T-VI-1), and isoleucine-containing (T-I-3) peptides were found. The combined compositions of the tryptic or cyanogen bromide peptides were equal to the composition of S-carboxymethyl-apoLp-Gln-II. These results confirm previous observations from this laboratory on intact and reduced and alkylated apoLp-Gln-II and support the concept that the molecule is composed of two identical chains of 77 residues each linked by a disulfide bond. The data available allow a partial alignment of these peptides and indicate that the disulfide bridge is in the NH2-terminal one-third of the molecule. The first three tryptic peptides in the sequence are T-I-2, T-VIII, and T-VI-1. T-VII is the COOH-terminal tryptic peptide. These assignments have been confirmed by manual and automated Edman degradations of the isolated tryptic and cyanogen bromide peptides performed in parallel with the present studies and published elsewhere (Brewer, H. B., Jr., Lux, S. E., Ronan, R., andJohn, K. M. (1972) Proc. Nat. Acad. Sci. U.S.A.69, 1304–1308).

ApoLp-Gln-II (or apoA-II), one of the two major apolipoproteins of human plasma high density lipoprotein, was isolated from a single female volunteer by DEAE-cellulose chromatography. The purified apoprotein was shown to be homogeneous by immunoelectrophoresis, polyacrylamide gel electrophoresis, and amino acid analysis. Distinctive features of the amino acid composition were the absence of histidine, arginine, and tryptophan. Reduction and carboxymethylation yielded 2 moles of S-carboxymethylcysteine per mole of intact apoLp-Gln-II. Few carboxymethyl groups could be added to apoLp-Gln-II prior to its reduction, indicating that cystine rather than cysteine was present in the intact apoprotein. This was confirmed by identification of cystine on enzymatic digestion of the intact apoprotein. Following reduction and S-carboxymethylation of apoLp-Gln-II, its molecular weight, as determined by gel filtration in 6 mguanidine hydrochloride, decreased from 16,700 ± 300 S.E.M. to 8,950 ± 150 S.E.M., indicating that apoLp-Gln-II is composed of two polypeptide chains of similar molecular weight. The two chains appeared to be identical by polyacrylamide gel electrophoresis in urea (pH 2.9 and 9.4), immunoelectrophoresis, and DEAE-cellulose chromatography. No NH2-terminal amino acid could be demonstrated by the dansyl chloride or phenylisothiocyanate methods, but digestion of apoLp-Gln-II with pyrrolidonecarboxylyl peptidase released pyrrolidonecarboxylic acid. Carboxypeptidase A digestion released glutamine and threonine. The kinetics of release as well as the inability to demonstrate threonine on hydrazinolysis indicated that glutamine was the COOH-terminal amino acid and threonine was the penultimate residue.

The interaction of human plasma high density apolipoproteins (apoHDL) with lipids was examined by circular dichroism (CD) of the peptide and aromatic chromophores. By CD criteria native HDL contained about 70% α helical, 5 to 15% β, and 15 to 20% disordered structure. Delipidation decreased the helical content by about 20% with a corresponding increase in disordered structure. The two major apoproteins of HDL (apoA-I and apoA-II), isolated by chromatography in urea, refolded to different extents after removal of urea; apoA-I had more ordered structure than apoA-II (approximately 55% and 35% α helix, respectively). Reconstitution of apoA-I, apoA-II, and apoHDL with both phosphatidylcholine and cholesteryl ester restored, respectively, 119%, 87%, and 100% of the helical structure of the parent HDL. Phosphatidylcholine alone restored 50 to 70% of the increase produced by both phosphatidylcholine and cholesteryl oleate. With each substrate, the increase in helical content on lipid-protein recombination was accompanied by a corresponding decrease in disordered structure.

Tangier disease is an autosomal recessive disorder characterized by absence of normal high density lipoproteins (HDL), storage of cholesterol esters in foamcells, and neuropathy, Small amounts of an abnormal HDL (HDLT) having only partial immunochemical identity with normal HSL occur in Tangier plasma, HDL has recently been shown to contain two major protein subunits, one with C-terminal threonine (R-Thr), the other with C-terminal glutaminc (R-Gln). R-Thr and R-Gln are immunochemically different and occur in normal HDL, in a ratio of 2–4:1 (R-Thr:R-Gln). We now report investigations of these two moieties in HDLTisolated from a Tangier homozygote.R-Gln was identified in delipidated HDLx (apo HDLT) by polyacrylamide gel electrophoresis (PGE), DEAE cellulose chromatography and immunodiffusion with antisera to R-Gln and normal HDL (anti-HDL). An antiserum to HDLT(antiHDLT) reacted withR-Gln and absorption of antiHDL with apoHDLTremoved all reactivity to R-Gln, confirming the presence of R-Gln in HDLT. R-Gln from apoHDLTwas electrophoretically and immunochemically identical with normal R-Gln. At protein concentrations at which R-Gln was readily apparent, PGE, DEAE cellulose chromatography and immunodiffusion demonstrated R-Thr in apoHDL but not in apoHDLT. However, antiHDLTreacted weakly with R-Thr suggesting the presence of traces of R-Thr in apoHDLT. Using antiR-Thr sera and increasing the amount of apo-HDLT10 to 100-fold, an antigen was seen that migrated on PGE and eluted from DEAE the same as normal R-Thr and was immunochemically dentical with R-Thr.These results indicate a markedly disproportionate decrease in R-Thr compared to R-Gln in Tangier disease and suggest the hereditary defect is one of control of R-Thr synthesis.

The red blood cell (RBC) membrane skeleton is composed principally of short F-actin filaments crosslinked by α2β2-spectrin tetramers with the assistance of protein 4.1R. Actin and 4.1R bind to the actin-binding domain (βABD) at the N-terminus of the spectrin β-chain. The adjacent, C-terminal end of α-spectrin, contains a calmodulin-like domain (αCML, aa 2262–2418) that is also called the EF hand domain and is thought to be inert or vestigial. However, the sph1J/sph1J mouse, which has severe hereditary spherocytosis and unstable RBC membranes, makes a mutant α-spectrin that lacks the last 13 amino acids (αCMLΔC13), showing that the domain has some important function. To investigate this function we “fished” for interacting proteins using glutathione-S-transferase (GST)-fused to the CML domain—either the wildtype (αGST-CML) or sph1J (αGST-CMLΔC13). αGST-CML retrieved protein 4.2 from a 2M Tris HCl extract of spectrin-actin depleted human RBC membranes. Protein 4.2 bound αGST-CML with high affinity (Kd = 2.7 x 10−7M) but did not bind αGST-CMLΔC13. Binding was abolished by 1 mM Ca2+, which converts the CML domain to the liganded conformation. The binding site on protein 4.2 localized, at least partly, to amino acids 411–492. Because red cells lacking protein 4.2 are not as severely affected as sph1J/sph1J RBCs, we also tested the effect of the αCMLΔC13 mutation on spectrin-actin binding. A minispectrin was prepared containing the actin-binding domain plus the first four spectrin repeats of the β-chain, combined with the CML domain (±ΔC13) and the last four repeats of the α-chain. The normal and mutant minispectrins were incubated with protein 4.1R, F-actin, or both proteins. The results were striking. The minispectrin containing the normal CML domain bound actin in the presence of protein 4.1R, but the minispectrin containing the mutant CML domain did not. Similarly, the mutant minispectrin was defective in its ability to bind 125I-4.1R in the presence of a constant amount of F-actin. However, the mutation did not affect binding of the minispectrin to protein 4.1R in the absence of actin. We have not yet tested whether protein 4.2 or Ca2+ modulate the effects of the CML domain on spectrin-actin binding. In summary, these experiments clearly show that the calmodulin-like (EF hand) domain of α-spectrin, which was previously considered inert, binds protein 4.2 and also contributes to spectrin-actin binding in the presence of protein 4.1R. Further experiments will be needed to determine whether the CML domain binds actin directly or strengthens the binding of the adjacent actin-binding domain.

The red blood cell (RBC) lipid bilayer is supported by an underlying membrane skeleton. Erythroid spectrin, which is composed of flexible alpha and beta subunits, is encoded by the α (Spna1) and β (Spnb1) genes and is the major protein in the membrane skeleton. In mice, five independent autosomal recessive mutations in α-spectrin (sph, sph1J, sph2J, sph2BC, sphDem) and one in β-spectrin (ja) have been identified; all result in severe hemolytic anemia. We have identified two new mouse α-spectrin mutations, sph3J and sph4J, on the NOD.B10 and C57BL/6J background strains, respectively. Linkage analysis in F2 intercrosses localized both mutations to the distal portion of mouse chromosome 1 near Spna1, an obvious candidate gene. In both sph3J and sph4J, novel mutations distinct from the previously described five sph alleles were subsequently identified. In sph3J a cytosine to thymine transition in exon 43 causes a histidine to tyrosine substitution within the αβ nucleation site of α-spectrin (H2012Y). Spna1 message levels are significantly reduced in sph3J reticulocyte RNA. In sph4J a guanine to adenine transition in exon 52 results in a cysteine to tyrosine substitution near the C-terminus (C2384Y). Spna1 message levels are normal in sph4J reticulocytes. Both mutations cause a phenotype of severe hemolytic anemia. In homozygous adult sph3J mice, dramatic decreases in the RBC count (−67%), hemoglobin (−68%), and hematocrit (−65%) are seen. On Wright’s stained peripheral blood smears and by scanning electron microscopy, large numbers of elliptocytes and spherocytes are evident. Significantly increased spleen-to-body weight ratio (+1,200%), bilirubin (+98%), iron (+74%) and circulating reticulocytes are also present. Homozygous adult sph4J mice show similar abnormally shaped RBCs and blood profile changes. SDS-PAGE analysis of sph3J and sph4J RBC membrane skeletons revealed unique changes in membrane skeleton proteins compared to each other and to the five known sph alleles. In sph3J, α- and β-spectrin are significantly decreased but ankyrin, protein 4.1 and protein 4.2 levels are normal. Surprisingly, band 3 is reduced to ~30% of normal, and both α- and β-adducin are nearly undetectable in sph3J RBCs. The presence of normal amounts of ankyrin, which binds band 3 tetramers, suggests that band 3 dimers are absent in sph3J RBCs. These observations indicate that previously unsuspected interactions, direct or indirect, exist between spectrin and band 3 (probably dimers) and between spectrin and adducin within the RBC membrane skeleton. In contrast to sph3J, all RBC membrane skeleton proteins appear normal by SDS-PAGE and western blot analyses of sph4J RBC membranes. Coupled with the severe hemolytic anemia present in these mice, these data suggest that interactions involving the C-terminus of α-spectrin, specifically cysteine 2384, are critical to RBC membrane integrity. Together, the sph3J and sph4J mouse models provide powerful resources for identifying critical interactions within the membrane skeleton that are relevant to the pathogenesis of hereditary elliptocytosis and spherocytosis.

The red blood cell (RBC) membrane skeleton is composed principally of short F-actin filaments crosslinked by α2β2-spectrin tetramers with the assistance of protein 4.1R. Actin and 4.1R bind to the actin-binding domain (βABD) at the N-terminus of the spectrin β-chain. The adjacent, C-terminal end of α-spectrin, contains a calmodulin-like domain (αCML, aa 2262–2418) that is also called the EF hand domain and is thought to be inert or vestigial. However, the sph1J/sph1Jmouse, which has severe hereditary spherocytosis and unstable RBC membranes, makes a mutant α-spectrin that lacks the last 13 amino acids (αCMLΔC13), showing that the domain has some important function. To investigate this function we “fished” for interacting proteins using glutathione-S-transferase (GST)-fused to the CML domain—either the wildtype (αGST-CML) or sph1J(αGST-CMLΔC13). αGST-CML retrieved protein 4.2 from a 2M Tris HCl extract of spectrin-actin depleted human RBC membranes. Protein 4.2 bound αGST-CML with high affinity (Kd = 2.7 x 10−7M) but did not bind αGST-CMLΔC13. Binding was abolished by 1 mM Ca2+, which converts the CML domain to the liganded conformation. The binding site on protein 4.2 localized, at least partly, to amino acids 411–492. Because red cells lacking protein 4.2 are not as severely affected as sph1J/sph1JRBCs, we also tested the effect of the αCMLΔC13 mutation on spectrin-actin binding. A minispectrin was prepared containing the actin-binding domain plus the first four spectrin repeats of the β-chain, combined with the CML domain (±ΔC13) and the last four repeats of the α-chain. The normal and mutant minispectrins were incubated with protein 4.1R, F-actin, or both proteins. The results were striking. The minispectrin containing the normal CML domain bound actin in the presence of protein 4.1R, but the minispectrin containing the mutant CML domain did not. Similarly, the mutant minispectrin was defective in its ability to bind 125I-4.1R in the presence of a constant amount of F-actin. However, the mutation did not affect binding of the minispectrin to protein 4.1R in the absence of actin. We have not yet tested whether protein 4.2 or Ca2+modulate the effects of the CML domain on spectrin-actin binding. In summary, these experiments clearly show that the calmodulin-like (EF hand) domain of α-spectrin, which was previously considered inert, binds protein 4.2 and also contributes to spectrin-actin binding in the presence of protein 4.1R. Further experiments will be needed to determine whether the CML domain binds actin directly or strengthens the binding of the adjacent actin-binding domain.

The red blood cell (RBC) lipid bilayer is supported by an underlying membrane skeleton. Erythroid spectrin, which is composed of flexible alpha and beta subunits, is encoded by the α (Spna1) and β (Spnb1) genes and is the major protein in the membrane skeleton. In mice, five independent autosomal recessive mutations in α-spectrin (sph, sph1J, sph2J, sph2BC, sphDem) and one in β-spectrin (ja) have been identified; all result in severe hemolytic anemia. We have identified two new mouse α-spectrin mutations, sph3Jand sph4J, on the NOD.B10 and C57BL/6J background strains, respectively. Linkage analysis in F2 intercrosses localized both mutations to the distal portion of mouse chromosome 1 near Spna1, an obvious candidate gene. In both sph3Jand sph4J, novel mutations distinct from the previously described five sphalleles were subsequently identified. In sph3Ja cytosine to thymine transition in exon 43 causes a histidine to tyrosine substitution within the αβ nucleation site of α-spectrin (H2012Y). Spna1message levels are significantly reduced in sph3Jreticulocyte RNA. In sph4Ja guanine to adenine transition in exon 52 results in a cysteine to tyrosine substitution near the C-terminus (C2384Y). Spna1message levels are normal in sph4Jreticulocytes. Both mutations cause a phenotype of severe hemolytic anemia. In homozygous adult sph3Jmice, dramatic decreases in the RBC count (−67%), hemoglobin (−68%), and hematocrit (−65%) are seen. On Wright's stained peripheral blood smears and by scanning electron microscopy, large numbers of elliptocytes and spherocytes are evident. Significantly increased spleen-to-body weight ratio (+1,200%), bilirubin (+98%), iron (+74%) and circulating reticulocytes are also present. Homozygous adult sph4Jmice show similar abnormally shaped RBCs and blood profile changes. SDS-PAGE analysis of sph3Jand sph4JRBC membrane skeletons revealed unique changes in membrane skeleton proteins compared to each other and to the five known sphalleles. In sph3J, α- and β-spectrin are significantly decreased but ankyrin, protein 4.1 and protein 4.2 levels are normal. Surprisingly, band 3 is reduced to ~30% of normal, and both α- and β-adducin are nearly undetectable in sph3JRBCs. The presence of normal amounts of ankyrin, which binds band 3 tetramers, suggests that band 3 dimers are absent in sph3JRBCs. These observations indicate that previously unsuspected interactions, direct or indirect, exist between spectrin and band 3 (probably dimers) and between spectrin and adducin within the RBC membrane skeleton. In contrast to sph3J, all RBC membrane skeleton proteins appear normal by SDS-PAGE and western blot analyses of sph4JRBC membranes. Coupled with the severe hemolytic anemia present in these mice, these data suggest that interactions involving the C-terminus of α-spectrin, specifically cysteine 2384, are critical to RBC membrane integrity. Together, the sph3Jand sph4Jmouse models provide powerful resources for identifying critical interactions within the membrane skeleton that are relevant to the pathogenesis of hereditary elliptocytosis and spherocytosis.

To determine whether splenic “polishing” and “pitting” of RBC includes removal of agglomerated membrane proteins as well as membrane lipids and intracellular debris we examined the RBC membrane proteins of normal and splenectomized patients.RBC ghosts were dissolved in sodium dodecyl sulfate (SDS) and chromatographed on Sepharose 2B (exclusion limit=40×106). Membranes from splenectomized patients contained an excluded macromolecular species not present in “normosplenic” individuals. This colorless material formed insoluble fibrils on removal of SDS and was proteinaceous as judged from amino acid analysis and ultraviolet spectroscopy. It comprised 4.2±1.3% (range 2.4-6.1%) of the total membrane protein. Equal proportions were present in membranes from older (more dense) and younger (less dense) cells. When freeze-thawed ghosts were banded on a sucrose density gradient, this material remained with the membrane fraction, indicating it was not a particulars cytoplasmic contaminant. Reduction partially disaggregated the complex and revealed spectrin (a high molecular weight RBC membrane protein thought to be involved in red cell shape maintenance), and at least one other unidentified protein component on SDS-gel electrophoresis.These studies suggest the spleen normally “polishes” or “pits” from RBC membranes an aggregated complex of RBC membrane proteins. Thus RBC membrane proteins join lipids and intracellular particles as targets for splenic cleansing.

Red cell membranes from all splenectomized and some functionally asplenic persons contained a small proportion of a very high molecular weight, sodium dodecyl sulfate (SDS)-insoluble protein aggregate not found in normals. This complex averaged 3.6% ± 1.7% (SD) of the total red cell membrane protein (range 1.2%-6.3%). It was identified in the marrow reticulocytes, but not the circulating red cells, of a patient with a normal spleen. In splenectomized individuals it was present equally in young and old circulating erythrocytes. When ghosts were disrupted and banded in a sucrose density gradient, the high molecular weight protein complex remained with the membrane fraction, indicating that it was not a particulate cytoplasmic contaminant. It was accessible to iodination by lactoperoxidase only at the inner membrane surface. The complex was remarkably resistant to dissociation. It was partially disaggregated by reduction in SDS with the release of the membrane protein, spectrin. Judging from its amino acid composition, however, the complex must contain one or more additional proteins which remain to be identified. These studies suggest that reticulocyte membranes contain a high molecular weight aggregate of spectrin and other membrane proteins and that splenic “surface remodeling” includes the removal of this protein complex as well as membrane lipids. The complex may prove to be a useful tool for further study of this poorly understood splenic function and for clinical assessment of splenic hypofunction.

The activity of HMG CoA reductase, the rate-limiting enzyme in cholesterol biosynthesis, is repressed by serum in normal fibroblasts. We measured HMG CoA reductase by the conversion of [3-14C] HMG CoA to mevalonate in confluent fibroblast cultures incubated for 24 hr in Eagle's MEM with and without 10% fetal calf serum (FCS). Five normals (NLS) and 4 patients with HHLPII were studied (TP, JaP, LH, LC: Levy et al., Ann. Int. Med. 77:267, 1972). HMG CoA reductase in NLS was 1.10 ± 0.53 (avg ± SD) units (pmol/min/mg protein) with FCS and 7.70 ± 3.78 units without FCS. HMG CoA reductase in HHLPII cells was 7.83 j± 3.69 units with FCS and 10.97 ± 4.80 units without FCS. Ratios of HMG CoA reductase activity with/without FCS equals 0.16 ± 0.07 in NL cells and 0.68 ± 0.31 in all 4 HHLPII cell lines (p 0.005). An intermediate value, 0.34 ± 0.09, was observed in one HHLPII cell line (LH) which differed significantly from the other 3 HHLPII values, 0.80 ± 0.26 (p 0.05), and from NL values (p 0.01). Conclusions: (1) HMG CoA reductase levels were not inhibited by serum in cells from 3 patients with HHLPII, which confirms and extends to new patients the observation of Goldstein et al. (PNAS 70:2804, 1973). (2) Genotypic variation is suggested by one patient, LH, a phenotypic HHLPII, who exhibited partial inhibition by FCS.

A 17-year old girl with life-long T, unresponsive to splenectomy, was evaluated because of easy bruising, epistaxis, and recurrent hemarthroses. Platelets (P) were reduced in number (50,000-140,000/μl) and their size was greatly increased (3-5 μ in diameter) on peripheral blood smear. Bone marrow contained increased megakaryocytes, and 51chromium-labeled autologous P survival was normal (7.8 days), suggesting ineffective thrombopoiesis as the cause of the T. Bleeding time (BT) was consistently greater than 25 min., and P did not aggregate normally when incubated with collagen or epinephrine but did respond to ristocetin. Although rate of uptake of 3H-serotonin was reduced, P ATP/ADP ratio was normal, excluding a storage-pool defect. The patient's Factor VIII level measured by coagulation assay (VIIIAHF) or by ability of her plasma to support ristocetin-induced aggregation (VIIIVWF) varied from 35-150% and 27-160% respectively, while Factor VIII-related antigen was always normal (over 60%). Transfusions of both cryoprecipitate and P, but neither alone, transiently shortened BT (to 7-9 min.) and controlled bleeding symptoms. Post-transfusion increments in VIIIAHFwere consistent with VW disease. Family history and coagulation studies were unremarkable. This patient appears to have a unique combined defect in thrombopoiesis, in-trinsic P function, and in VIIIVWF. Effective therapy has required both normal P and Factor VIII-containing products.

Red cell (RBC) membrane shape depends, in part, on the RBC membrane skeleton; a protein meshwork of spectrin and actin which laminates the inner membrane surface. We have shown (JCI 58:955, 1976) that irreversibly sickled cells (ISCs) form ISC shaped ghosts and skeletons, which suggests the ISC shape is caused by an acquired skeletal defect. In this study we sought conditions that would alter or reverse this abnormal shape. Ghosts of ISC-rich RBCs were loaded with various agents, incubated at 37°C for 35 min and examined by phase microscopy. In isotonic media ISC ghosts retained an ISC shape. Addition of ATP, ADP, Mg++, Ca++, EDTA (all lmM), DTT (5mM) and various combinations of these agents did not alter this shape. However ISC shaped ghosts became round and indistinguishable from normal in hypertonic NaCl (>400mM), hypotonic NaCl (10-50mM) or isotonic NaCl containing small amounts of Zn++(0.1-0.5mM). ISC shape reversal was time and temperature dependent, and did not correlate with elution or proteolysis of any protein detectable on SDS gels, but did correlate with the formation, in vitro, of a high molecular weight complex of spectrin and actin. Conditions which promoted ISC ghost reversal inhibited formation of this complex.These observations indicate that the ISC shape is not maintained by covalent bonds and suggest that ISCs may be stabilized by abnormal interactions between the spectrin and/or actin components of the membrane skeleton and that disruption of these bonds may allow the skeleton to resume a normal shape.