The importance of erythroid expansion in determining the extent of apoptosis in erythroid precursors in patients with beta-thalassemia major.

Beta-thalassemia major is characterized by ineffective erythropoiesis leading to severe anemia and extensive erythroid expansion. The ineffective erythropoiesis is in part due to accelerated apoptosis of the thalassemic erythroid precursors; however, the extent of apoptosis is surprisingly variable. To understand this variability as well as the fact that some patients undergoing allogeneic marrow transplantation are resistant to the myeloablative program, we attempted more quantitative analyses. Two groups of patients totaling 44 were studied, along with 25 healthy controls, and 7 patients with hemolysis and/or ineffective erythropoeisis. By 2 flow cytometric methods, thalassemic erythroid precursors underwent apoptosis at a rate that was 3 to 4 times normal. Because thalassemic marrow has between 5- to 6-fold more erythroid precursors than healthy marrow, this translated into an absolute increase in erythroid precursor apoptosis of about 15-fold above our healthy controls. In searching for the causes of the variability in thalassemic erythroid precursor apoptosis, we discovered tight direct correlations between the relative and absolute extent of apoptosis and the extent of erythroid expansion as measured either by the absolute number of marrow erythroid precursors or by serum soluble transferrin receptor levels. These results could mean that the most extreme rates of erythroid proliferation lend themselves to cellular errors that turn on apoptotic programs. Alternatively, extreme rates of erythroid hyperplasia and apoptosis might be characteristic of more severely affected patients. Lastly, extreme erythroid hyperplasia could generate such numbers of apoptotic erythroid precursors that marrow macrophages are overwhelmed, leaving more apoptotic cells in the sample.

A correlation of erythrokinetics, ineffective erythropoiesis, and erythroid precursor apoptosis in thai patients with thalassemia.

The variety of patients with thalassemia in Thailand offers an opportunity to fully characterize the kinetic causes of the anemia and to study apoptosis of marrow erythroid precursors as a possible factor contributing to its severity. Kinetic studies showed that in hemoglobin H (HbH) disease, the extent of hemolysis, as well as the minimally ineffective erythropoiesis, usually falls within the compensatory capacity of normal erythropoiesis; therefore, anemia in patients with HbH partly represents a failure to expand erythropoiesis adequately. Hemoglobin Constant Spring (HbCS), a common variant of alpha thalassemia in Bangkok, causes more severe hemolysis and a distinct increase in ineffective erythropoiesis. Ineffective erythropoiesis plays a much more prominent role in beta thalassemia/hemoglobin E (beta-thal/HbE) disease, in which the variability of the anemia is puzzling. We compared mild and severe cases and found that patients with severe disease had a maximal marrow erythropoietic response that failed to compensate for very short survival of red blood cells and a marked quantitative increase in ineffective erythropoiesis. Analysis of apoptosis of marrow erythroid precursors done both on shipped samples and in Bangkok showed a moderate increase in HbH disease, consistent with the small increase in ineffective erythropoiesis. In patients with homozygous HbCS, there was a further increase in apoptosis, consistent with the additional increase in ineffective erythropoiesis. Patients with beta-thal/HbE disease had the most ineffective erythropoiesis and the most erythroid apoptosis. Thus, it appears that alpha-chain deposition in erythroid precursors, either alpha(A) or alpha(cs), leads to accelerated apoptosis and ineffective erythropoiesis.

Membrane phospholipid asymmetry in human thalassemia.

Phospholipid asymmetry in the red blood cell (RBC) lipid bilayer is well maintained during the life of the cell, with phosphatidylserine (PS) virtually exclusively located in the inner monolayer. Loss of phospholipid asymmetry, and consequently exposure of PS, is thought to play an important role in red cell pathology. The anemia in the human thalassemias is caused by a combination of ineffective erythropoiesis (intramedullary hemolysis) and a decreased survival of adult RBCs in the peripheral blood. This premature destruction of the thalassemic RBC could in part be due to a loss of phospholipid asymmetry, because cells that expose PS are recognized and removed by macrophages. In addition, PS exposure can play a role in the hypercoagulable state reported to exist in severe beta-thalassemia intermedia. We describe PS exposure in RBCs of 56 comparably anemic patients with different genetic backgrounds of the alpha- or beta-thalassemia phenotype. The use of fluorescently labeled annexin V allowed us to determine loss of phospholipid asymmetry in individual cells. Our data indicate that in a number of thalassemic patients, subpopulations of red cells circulate that expose PS on their outer surface. The number of such cells can vary dramatically from patient to patient, from as low as that found in normal controls (less than 0.2%) up to 20%. Analysis by fluorescent microscopy of beta-thalassemic RBCs indicates that PS on the outer leaflet is distributed either over the entire membrane or localized in areas possibly related to regions rich in membrane-bound alpha-globin chains. We hypothesize that these membrane sites in which iron carrying globin chains accumulate and cause oxidative damage, could be important in the loss of membrane lipid organization. In conclusion, we report the presence of PS-exposing subpopulations of thalassemic RBC that are most likely physiologically important, because they could provide a surface for enhancing hemostasis as recently reported, and because such exposure may mediate the rapid removal of these RBCs from the circulation, thereby contributing to the anemia.

Removal of erythrocyte membrane iron in vivo ameliorates the pathobiology of murine thalassemia.

Abnormal deposits of free iron are found on the cytoplasmic surface of red blood cell (RBC) membranes in beta-thalassemia. To test the hypothesis that this is of importance to RBC pathobiology, we administered the iron chelator deferiprone (L1) intraperitoneally to beta-thalassemic mice for 4 wk and then studied RBC survival and membrane characteristics. L1 therapy decreased membrane free iron by 50% (P = 0.04) and concomitantly improved oxidation of membrane proteins (P = 0.007), the proportion of RBC gilded with immunoglobulin (P = 0.001), RBC potassium content (P < 0.001), and mean corpuscular volume (P < 0.001). Osmotic gradient ektacytometry confirmed a trend toward improvement of RBC hydration status. As determined by clearance of RBC biotinylated in vivo, RBC survival also was significantly improved in L1-treated mice compared with controls (P = 0.007). Thus, in vivo therapy with L1 removes pathologic free iron deposits from RBC membranes in murine thalassemia, and causes improvement in membrane function and RBC survival. This result provides in vivo confirmation that abnormal membrane free iron deposits contribute to the pathobiology of thalassemic RBC.

Pathophysiology of the thalassemias. The Albion Walter Hewlett Award presentation.

The Albion Walter Hewlett Award (named for Professor of Medicine and Chair of the Stanford Department of Medicine 1916-1925) recognizes a role model, accomplished in discovery of the biological sciences and at the same time a consummate and compassionate physician. In introductory remarks, Dr. Stanley L. Schrier, Professor of Medicine (Hematology), the tenth recipient of the Award, indicated that the person so identified is no longer a viable model in academic medicine. The loss of this sort of person is serious because this appropriately trained physician-investigator was uniquely positioned to study pathophysiology, defined as the processes by which disordered biology produces disease. He used his own studies on the clinical manifestations of the thalassemias to clarify what he meant by pathophysiology. Thus he and his colleagues first defined membrane material properties of alpha and beta thalassemic RBC membranes and the states of hydration of alpha and beta thalassemic RBC and found them to be strikingly divergent. The biochemical counterparts of these alterations proved to be the accumulation of the excess unmatched partially oxidized globin chains on the membrane skeleton. In vitro studies with chemical oxidants selectively oxidized alpha and beta globin chains which then attached to the RBC membrane skeleton and reproduced the membrane material properties characteristic of beta and alpha thalassemia respectively. Many of these alterations had occurred prior to the reticulocyte stage so that pursuit of pathophysiology shifted to studies of marrow erythroid precursors, and it was shown that in beta thalassemia major there was accelerated programmed cell death as well as defective assembly of the membrane skeleton.

Improved perfusion with rt-PA and hirulog in a rabbit model of embolic stroke.

We conducted a study using diffusion-weighted (DWI) and perfusion-weighted (PWI) magnetic resonance imaging (MRI) to evaluate the efficacy of thrombolysis in an embolic stroke model with recombinant tissue plasminogen activator (rt-PA) and hirulog, a novel direct-acting antithrombin. DWI can identify areas of ischemia minutes from stroke onset, while PWI identifies regions of impaired blood flow. Right internal carotid arteries of 36 rabbits were embolized using aged heterologous thrombi. Baseline DWI and PWI scans were obtained to confirm successful embolization. Four animals with no observable DWI lesion on the initial scan were excluded; therefore, a total of 32 animals were randomized to one of three treatment groups: rt-PA (n = 11), rt-PA plus hirulog (n = 11), or placebo (n = 10). Treatment was begun 1 h after stroke induction. Intravenous doses were as follows: rt-PA, 5 mg/kg over 0.5 h with 20% of the total dose given as a bolus; hirulog, 1 mg/kg bolus followed by 5 mg/kg over 1 h. MRI was performed at 2, 3, and 5 h following embolization. Six hours after embolization, brains were harvested, examined for hemorrhage, then prepared for histologic analysis. The rt-PA decreased fibrinogen levels by 73%, and hirulog prolonged the aPTT to four times the control value. Posttreatment areas of diffusion abnormality and perfusion delay were expressed as a ratio of baseline values. Significantly improved perfusion was seen in the rt-PA plus hirulog group compared with placebo (normalized ratios of the perfusion delay areas were as follows: placebo, 1.58, 0.47-3.59; rt-PA, 1.12, 0.04-3.95; rt-PA and hirulog, 0.40, 0.02-1.08; p < 0.05). Comparison of diffusion abnormality ratios measured at 5 h showed trends favoring reduced lesion size in both groups given rt-PA (normalized ratios of diffusion abnormality areas were as follows: placebo, 3.69, 0.39-15.71; rt-PA, 2.57, 0.74-5.00; rt-PA and hirulog, 1.95, 0.33-6.80; p = 0.32). Significant cerebral hemorrhage was observed in one placebo, two rt-PA, and three rt-PA plus hirulog treated animals. One fatal systemic hemorrhage was observed in each of the rt-PA groups. We conclude that rt-PA plus hirulog improves cerebral perfusion but does not necessarily reduce cerebral injury. DWI and PWI are useful methods for monitoring thrombolysis.

Pathobiology of thalassemic erythrocytes.

Although study of the thalassemias has focussed on possible cure or amelioration by genetic techniques, considerable progress has been made in understanding the underlying pathobiology of these diseases. Better control of childhood infectious diseases has led to a clearer understanding of the frequency and clinical severity of some of these disorders. The striking differences between alpha- and beta-thalassemia are now well documented and the role of oxidant attack in the pathobiology is becoming clearer. Some authors believe that severe beta-thalassemia induces a hypercoagulable state that could be partially caused by scrambling of the phospholipid bilayer of affected erythrocytes. There is growing appreciation that double heterozygosity for hemoglobin E/beta-thalassemia, while causing variable anemia, can produce a clinical condition as severe as Cooley's anemia (beta-thalassemia major). Anemia severity may be related to the extent of oxidant attack on the unstable hemoglobin E. Studies of the hemoglobin Constant Spring variants demonstrate the consequences of accumulating excess unmatched beta globin as well as the unique alpha CS. Studies on marrow erythroid precursors in the beta-thalassemias have already shown accelerated programmed cell death and abnormal assembly of membrane proteins. Such studies in the future will likely further delineate the underlying differences between alpha- and beta-thalassemias.

The unusual pathobiology of hemoglobin constant spring red blood cells.

Hemoglobin Constant Spring (HbCS) is the most common nondeletional alpha-thalassemic mutation and is an important cause of HbH-like disease in Southeast Asia. HbCS variants have an almost normal mean cell volume (MCV) and the anemia is more severe when compared with other alpha-thalassemic variants. We explored the pathobiology of HbCS red blood cells (RBCs) because the underlying cause(s) of this MCV "normalizing" effect of HbCS and the more severe anemia are not fully explained. HbCS containing RBCs are distinctly overhydrated relative to deletional alpha-thalassemia variants, and the derangement of volume regulation and cell hydration occurs early in erythroid maturation and is fully expressed at the reticulocyte stage. Furthermore, the membrane rigidity and membrane mechanical stability of HbCS containing RBCs is increased when compared with HbH and alpha-thalassemia-1 trait RBCs. In seeking the cause(s) underlying these cellular alterations we analyzed membranes from HbCS and deletional alpha-thalassemic variants and found that in addition to oxidized beta-globin chains, oxidized alpha cs-globin chains are also associated with the membranes and their skeletons in HbCS containing RBCs. We propose that the membrane pathology of HbCS variants is caused by combination of the deleterious effects induced by membrane-bound oxidized alpha cs- and beta-globin chains. The membrane alterations induced by alpha cs chains are more akin to those induced by beta A-globin chains than those induced by the alpha A-globin chains that accumulate in the beta-thalassemias. Thus, each globin chain, alpha cs, alpha A, beta A, appears to produce its own form of membrane perturbation.

Abnormal assembly of membrane proteins in erythroid progenitors of patients with beta-thalassemia major.

The life threatening anemia in beta-thalassemia major (Cooley's anemia) is characterized by profound intramedullary lysis, the cause of which is incompletely understood. Using marrow obtained from beta thalassemia major patients undergoing allogeneic bone marrow transplantation in Pesaro Italy, it became possible to directly study the mechanism of the intramedullary hemolysis. Based on our previous studies, we hypothesized that the unmatched alpha globin chains would interfere with normal assembly of erythroid precursor membrane proteins. Patient and control erythroid precursors were reacted with monospecific polyclonal rabbit antibodies directed against spectrin, band 3, and band 4.1 and with a monoclonal anti-alpha globin chain antibody. Using laser confocal fluorescence microscopy, normal erythroid precursors show no alpha globin chain accumulation and exhibited uniformly smooth rim fluorescence of the three membrane proteins. In some thalassemic precursors, spectrin appeared to interact with large alpha globin accumulations, and in many of these cells the spectin appeared clumped and discontinuous. Band 4.1 interacted strongly with accumulations of alpha globin in thalassemic precursors to produce bizarrely clumped zones of abnormal band 4.1 distribution. Band 3 was incorporated smoothly into thalassemic erythroblast membranes. However, the proerythroblasts and basophilic erythroblasts were significantly deficient in band 3. Thus, accumulations of alpha globin in beta-thalassemia major colocalized with and disrupt band 4.1 and spectrin assembly into the membrane. The cause of deficient band 3 incorporation into thalassemic proerythroblast membranes remains unknown. These profound membrane alterations would likely contribute to the intramedullary lysis seen in Cooley's anemia.

The instability of the membrane skeleton in thalassemic red blood cells.

The thalassemias are a heterogeneous group of disorders characterized by accumulation either of unmatched alpha or beta globin chains. These in turn cause the intramedullary and peripheral hemolysis that leads to varying anemia. A partial explanation for the hemolysis came our of our studies on material properties that showed that beta-thalassemia (beta-thal) intermedia ghosts were very rigid but unstable. A clue to this instability came from the observation that the spectrin/band 3 ratio was low in red blood cells (RBCs) of splenectomized beta-thal intermedia patients. The possible explanations for the apparent decrease in spectrin content included deficient or defective spectrin synthesis in thalassemic erythroid precursors or globin chain-induced membrane changes that lead to spectrin dissociation from the membrane during ghost preparation. To explore the latter alternative, samples from different thalassemic variants were obtained, ie, beta-thal intermedia, HbE/beta-thal, HbH (alpha-thal-1/alpha-thal-2), HbH/Constant Spring (CS), and homozygous HbCS/CS. We searched for the presence of spectrin in the first lysate of the standard ghost preparation. Normal individuals and patients with autoimmune hemolytic anemia, sickle cell anemia, and anemia due to chemotherapy served as controls. Using gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, no spectrin was detected in identical aliquots of the supernatants of normals and these control samples. Varying amounts of spectrin were detected in the first lysate supernatants of almost all thalassemic patients. The identification of spectrin was confirmed by Western blotting using an affinity-purified, monospecific, rabbit polyclonal antispectrin antibody. Relative amounts of spectrin detected were as follows in decreasing order: splenectomized beta-thal intermedia including HbE/beta-thal; HbCS/CS; nonsplenectomized beta-thal intermedia, HbH/CS; and, lastly, HbH. These findings were generally confirmed when we used an enzyme-linked immunosorbent assay technique to measure spectrin in the first lysate. Subsequent analyses showed that small amounts of actin and band 4.1 also appeared in lysates of thalassemic RBCs. Therefore, the three major membrane skeletal proteins are, to a varying degree, unstably attached in severe thalassemia. From these studies we could postulate that membrane association of abnormal or partially oxidized alpha-globin chains has a more deleterious effect on the membrane skeleton than do beta-globin chains.

Defective assembly of membrane proteins in erythroid precursors of beta-thalassemic mice.

beta-Thalassemic mice provide a useful model for studying the pathophysiology of human beta-thalassemia in that one can perform experiments that are difficult to perform in humans. The ease of access to beta-thalassemic mouse marrow provided the opportunity to explore the cause of the ineffective erythropoiesis that characterizes severe beta-thalassemia in mouse and man. We hypothesized that the accumulation of excess alpha-globin might interfere with the normal assembly of red blood cell (RBC) membrane proteins, thus contributing to the severe intramedullary lysis. Femoral marrow was obtained from normal and beta-thalassemic mice, and RBC precursors were purified (> 90%) by panning and harvesting CD45- cells. The assembly of RBC membrane proteins was assessed by observing immunofluorescence patterns obtained on fixed permeabilized precursors using rabbit polyclonal antibodies directed against human spectrin, and band 4.1, and murine band 3. The distribution of the proteins was shown with a fluorescein-tagged goat antirabbit antibody. In contrast to normal mice, about 30% of intermediate and late stage erythroblasts in beta-thalassemic mice appear abnormal. Neither spectrin nor band 4.1 formed crisp rim fluorescence in these erythroid precursors of thalassemic mice, whereas assembly of band 3 appeared normal. Therefore, the assembly of membrane skeletal proteins is abnormal in murine beta-thalassemic erythroid precursors perhaps because of the deposition of unmatched alpha-globin chains.

Thalassemia: pathophysiology of red cell changes.

The thalassemias are extremely heterogeneous in terms of their clinical severity, and their underlying pathophysiology relates directly to the extent of accumulation of excess unmatched globin chains: alpha in beta thalassemia and beta in the alpha thalassemias. However, the accumulation of each separate globin chain affects red cell membrane material properties and the state of red cell hydration very differently. These observations presumably account for the varying extent of ineffective erythropoiesis and peripheral blood hemolysis in the major variants of thalassemia. The thalassemias are a worldwide group of inherited disorders of globin-chain synthesis that developed in multiple geographic regions, probably because they provided partial protection against malaria. In normal assembly of adult hemoglobin (HbA-alpha 2 beta 2), alpha and beta globin are synthesized by genes on different chromosomes, whereas heme is synthesized primarily on mitochondria. The synthesis of these chains is very tightly coordinated so that the ratio of alpha globin to beta globin (beta in this case including the beta-like globins delta and gamma) is normally 1 +/- 0.05. Furthermore, specific erythroid proteases are designed to attack and destroy excess alpha or beta globin chains, demonstrating the deleterious impact of the accumulation of excess unmatched globin chains. In beta thalassemia, production of beta globin decreases and excess alpha globin accumulates. In alpha thalassemia, on the other hand, this process occurs in reverse. Perhaps in these disorders more than any others, molecular biologists have documented the deletional and transcriptional events leading to diminished synthesis of specific classes of globin chains.(ABSTRACT TRUNCATED AT 250 WORDS)

Accelerated programmed cell death (apoptosis) in erythroid precursors of patients with severe beta-thalassemia (Cooley's anemia)

The profound and life-threatening anemia in patients with Cooley's anemia is ascribed primarily to intramedullary hemolysis (ineffective erythropoiesis), the cause of which is obscure. Based on prior morphologic data showing nuclear abnormalities, we hypothesized that accelerated apoptosis could occur in these erythroid precursors. The highly successful bone marrow (BM) transplantation program for patients with Cooley's anemia provided us with a unique opportunity to test this hypothesis. We obtained pretransplantation BM aspiration samples from patients undergoing BM transplantation in Pesaro, Italy and from their allogeneic donors. The erythroid precursors were isolated using ficoll sedimentation and then panning selecting fro CD45- cells. Cytospin and Giemsa staining showed that the separation provided greater than 90% erythroblasts. Five million of these erythroblasts were lysed and their DNA was isolated. There were obvious ladder patterns of DNA breakdown products in beta-thalassemia major samples, with less occurring in beta-thalassemia trait. Normal individuals showed only a slight smear of breakdown of DNA. These results indicate there is enhanced apoptosis in the erythroblasts in the BMs of Cooley's anemia patients. This finding might partially explain why most of these erythroblasts never survive to become mature erythrocytes.

Transmembrane redistribution of phospholipids of the human red cell membrane during hypotonic hemolysis.

The transmembrane distribution of spin-labeled phospholipids was measured in human erythrocytes before and after hypotonic hemolysis by electron paramagnetic resonance. With a first series of partially water soluble probes a complete randomization of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and sphingomyelin analogues was achieved when cells were resealed in the absence of Mg-ATP or when the aminophospholipid translocase was inhibited by vanadate or calcium. If the ghosts were resealed with Mg-ATP inside, the transmembrane asymmetry of the aminophospholipids was reestablished. With long chain insoluble spin-labeled lipids complete randomization was obtained with the phosphatidylcholine analogue but even in the presence of vanadate only a small percentage (approx. 15%) of the spin-labeled phosphatidylserine flopped to the outer monolayer and comparable percentage of the spin-labeled sphingomyelin flipped to the inner monolayer, indicating a hierarchy in the phospholipid redistribution for these water insoluble lipids during hemolysis. The mechanism by which a selective randomization takes place is not known. It may involve phosphatidylserine-protein interactions in the inner leaflet and sphingomyelin-cholesterol or sphingomyelin-sphingomyelin interaction in the outer leaflet.

Globin-chain specificity of oxidation-induced changes in red blood cell membrane properties.

We have previously shown that excess unpaired alpha- and beta-globin chains in severe alpha- and beta-thalassemia interacting with the membrane skeleton induce different changes in membrane properties of red blood cells (RBCs) in these two phenotypes. We suggest that these differences in membrane material behavior may reflect the specificity of the membrane damage induced by alpha- and beta-globin chains. To further explore this hypothesis, we sought in vitro models that induce similar membrane alterations in normal RBCs. We found that treatment of normal RBCs with phenylhydrazine produced rigid and mechanically unstable membranes in conjunction with selective association of oxidized alpha-globin chains with the membrane skeleton, features characteristic of RBCs in severe beta-thalassemia. Methylhydrazine, in contrast, induced selective association of oxidized beta-globin chains with the membrane skeleton and produced rigid but hyperstable membranes, features that mimicked those of RBCs in severe alpha-thalassemia. These findings suggest that consequences of oxidation induced by globin chains are quite specific in that those agents that cause alpha-globin chain accumulation at the membrane produce rigid but mechanically unstable membranes, whereas membrane accumulation of beta-globin chains results in rigid but mechanically stable membranes. These in vitro experiments lend further support to the hypothesis that membrane-associated alpha- and beta-chains induce oxidative damage to highly specific different skeletal components and that the specificity of this skeletal damage accounts for the differences in material membrane properties of these oxidatively attacked RBCs and perhaps of alpha- and beta-thalassemic RBCs as well.

Mechanisms of amphipath-induced stomatocytosis in human erythrocytes.

We studied stomatocytosis induced in human red blood cells (RBC) by vinblastine and chlorpromazine, monitoring the movements of spin-labeled phosphatidylcholine (PC*) and sphingomyelin (SM*) by electron spin resonance (ESR) spectroscopy. This technique allows determination of the fraction of labeled lipids, respectively, on the external leaflet, on the cytosol face, or trapped in endocytic vacuoles. Both vinblastine and chlorpromazine produce a time- and concentration-dependent stomatocytic shape change, which is paralleled by a shift of approximately 10% to 33% of outer leaflet SM* and PC* inward. Of this amount, 8% to 12% was trapped in endocytic vacuoles and 8% to 19% had flipped to the inner leaflet. Vanadate, while inhibiting the stomatocytosis, did not block the flip of either SM* or PC* to the inner leaflet. To explain the inhibiting effect of vanadate, as well as the adenosine triphosphate (ATP) requirement for drug-induced stomatocytosis, we propose the following model: (1) addition of amphipath partially scrambles the bilayer; and (2) the flop of phosphatidylserine (PS) and phosphatidylethanolamine (PE) to the outer leaflet provides substrate for the aminophospholipid translocase (APLT), which flips back PS and PE inward faster than PC or SM can diffuse outward--thereby producing inner layer expansion or stomatocytosis. This role of APLT accounts for the vanadate inhibition of amphipath stomatocytosis. However, the vanadate effect can be overcome by increasing the amphipath concentration, which at such levels probably passively expands the inner leaflet.

Characterization and comparison of the red blood cell membrane damage in severe human alpha- and beta-thalassemia.

The aim of the present work was to understand the pathophysiology of the severe human thalassemias as represented by beta-thalassemia intermedia and hemoglobin (Hb) H (alpha-thalassemia) disease. We have previously shown that the material properties of the red blood cell (RBC) and its membrane differ in severe alpha- and beta-thalassemia, and we now show that this difference is probably caused by accumulation of alpha-globin chains at the cytoskeleton in beta-thalassemia, whereas beta-globin chains are associated with the cytoskeleton in alpha-thalassemia. In both alpha- and beta-thalassemia, some of these globin chains have become oxidized as evidenced by loss of the free thiols. Furthermore, there is similar evidence of oxidation of protein 4.1 in beta-thalassemia, whereas beta-spectrin appears to be subject to oxidation in alpha-thalassemia. These observations support the idea that the association of partly oxidized globin chains with the cytoskeleton results in oxidation of adjacent skeletal proteins. The abnormality of protein 4.1 in beta-thalassemia is consistent with a prior observation, and is also in accord with the known importance of protein 4.1 in maintenance of membrane stability, a property that is abnormal in beta-thalassemic membranes.

Oxidative red blood cell membrane injury in the pathophysiology of severe mouse beta-thalassemia.

In severe human beta-thalassemia, the pathophysiology relates to accumulation of excess alpha-globin chains at the membrane. One hypothesis is that membrane-associated alpha-globin by virtue of it's iron or hemichromes produces oxidation of adjacent membrane proteins. The availability of a mouse model of severe beta-thalassemia, as well as a transgenic (thalassemic-sickle) mouse that expresses 12% of human beta s-chain, has allowed us to study the effect of graded accumulation of alpha-chains at the red blood cell (RBC) membrane on the clinical status of the animal and on the material properties of its RBCs. Proteins from control, beta-thalassemic, and transgenic mouse RBC membranes were analyzed for evidence of oxidation, as measured by thiol-disulfide exchange chromatography, which detects intramolecular sulfhydryl oxidation. Ratios of oxidized globin to protein 7 were calculated and increased amounts were seen in thalassemic mice as compared with control mice and transgenic mice. Furthermore, there were increased amounts of thiol-free protein 4.1 in the thalassemic mice, compared with very small amounts in the control mice and intermediate amounts in the transgenic mice. Membrane mechanical stability as assessed by ektacytometry showed that the thalassemic mouse RBCs were markedly unstable. Transgenic mouse RBCs showed intermediate levels of membrane instability compared with the controls. We propose that this oxidized globin, in conjunction with oxidized protein 4.1, accounts (at least in part) for membrane instability. A 12% increase in beta s-globin chain synthesis (by decreasing excess globin available) confers considerable protection against both oxidative damage and the consequent membrane instability.

Myosin content and distribution in human neonatal erythrocytes are different from adult erythrocytes.

Neonatal erythrocytes (N-RBC) are different from adult erythrocytes (A-RBC). N-RBC are larger, less deformable, and undergo enhanced spontaneous and drug-induced endocytosis. The reticulocyte population of N-RBC is also different, consisting primarily of the youngest (R1) reticulocytes that are motile and capable of receptor-mediated endocytosis. Processes such as motility could require a contractile system. Myosin, a contractile protein, was identified in both A-RBC and N-RBC. We proposed to compare myosin content and distribution in A-RBC and N-RBC by immunofluorescence and enzyme-linked immunosorbent assay (ELISA) using a monospecific polyclonal rabbit antimyosin. There was bright immunofluorescence on 44% of N-RBC with some heterogeneity contrasting with a barely detectable fluorescence on A-RBC. ELISA measurements showed that A-RBC had 4,315 myosin copies/RBC, whereas N-RBC had 10,855 copies/RBC (or 2.5 times as much). ELISA measurements of white ghosts showed that A-ghosts contained 1,250 copies of myosin/RBC (29% of total) whereas N-ghosts contained 3.4 times as much at 4,320 copies/RBC (39% of total). Therefore, N-RBC not only have more myosin, but the amount that is membrane-associated is disproportionately increased. It is proposed that such differences in amount and distribution of myosin could account for some of the unusual properties of neonatal RBC indicated.