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Biomedical subjects

N Mohandas

Publications and source records attributed to N Mohandas.

At least 19 recordsLinked to original sources
PubMed1992

Thrombospondin mediates adherence of CD36+ sickle reticulocytes to endothelial cells.

Initiation of vasocclusion in sickle disease pathophysiology may involve abnormal red blood cell (RBC) adhesivity to endothelium, a phenomenon influenced by both RBC and plasma factors. Using human umbilical vein endothelial cells and a gravity sedimentation adherence assay, we have examined thrombospondin (TSP) as a plasma factor in this adhesive event. The already-abnormal adherence of sickle RBCs in buffer/albumin is significantly augmented (P < .001) by the addition of TSP, with half-maximal effect at about 0.3 microgram/mL. This effect is abolished by antibodies to either TSP or glycoprotein (GP) IV (CD36), as well as peptides RGDS and CSVTCG. The even greater adherence (P < .005) of sickle RBCs in autologous platelet-rich plasma (without added TSP) is dramatically inhibited by alpha CD36 antibodies (OKM5 and alpha GPIV) and significantly diminished by alpha TSP, by peptides RGDS and CSVTCG, and by two antibodies to the vitronectin receptor (7E3 and LM609). Studies of density-separated subpopulations and of RBC adhesion to immobilized proteins, as well as analysis of sickle RBCs using fluorescence-activated cell sorting and single cell microfluorometry, show that TSP responsiveness is a feature of the immature sickle "stress" reticulocytes, which carry CD36 (and not GPIIbIIIa-like receptors) as the TSP-receptive moiety. The endothelial cell's participation in this phenomenon appears to be more complex, and the data are consistent with the notion that it involves TSP interaction with other plasma proteins and/or multiple receptor structures. Other potential adhesogenic proteins (plasma von Willebrand factor, vitronectin, fibrinogen, and fibronectin) neither exhibited an affinity for reticulocytes nor supported increased sickle RBC adherence when added to buffer/albumin in these assay systems. In aggregate, our results indicate that TSP may be the major promoter of RBC adhesivity in plasma, and they suggest that therapeutic benefit might derive from interference with sickle reticulocyte CD36, as achieved by antibodies and CSVTCG in these studies.

Anemia, Sickle Cell↗
PubMed1992

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.

Erythrocyte Deformability↗
PubMed1992

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.

Animals↗
PubMed1992

Splenic sequestration associated with sickle cell trait and hereditary spherocytosis.

Coexistence of sickle cell trait and hereditary spherocytosis (HS) is unusual, and only 16 cases have been reported in the literature. These patients have the same clinical and hematological features as individuals having HS alone. We report a serious complication, acute splenic sequestration crisis (ASSC), occurring in two patients with sickle cell trait and HS. One patient experienced four episodes of ASSC during an 11-year span, while the other had two episodes of this complication during a 4-year period. Red blood cell studies and membrane protein analysis confirmed the diagnosis of HS as a consequence of spectrin deficiency. Splenectomy resulted in marked clinical and hematological improvement in both patients. Histological examination of spleens following splenectomy confirmed that significant erythrostasis and sickling had indeed occurred. ASSC can occur in patients with coexistence of sickle cell trait and HS, and this potentially life-threatening complication should be considered in this condition.

Adolescent↗
PubMed1992

Molecular basis for red cell membrane viscoelastic properties.

An unusual combination of membrane properties allows the red cell to undergo extensive deformation without cell fragmentation, enabling it to effectively perform its function of oxygen delivery during its long life span in the circulation. These material properties are the consequence of a composite structure in which a plasma membrane envelope made up of amphiphilic surfactant molecules is anchored to a network of skeletal proteins through tethering sites (transmembrane proteins) in the bilayer. Explosive growth in our understanding of the primary structure of the various red cell membrane proteins, definition of specific mutations in various red phenotypes, and detailed biophysical characterization of membrane properties of normal and mutant red cells has enabled development of models of molecular and structural basis for red cell properties.

Anemia, Hemolytic↗
PubMed1992

Thromboerythrocytes. In vitro studies of a potential autologous, semi-artificial alternative to platelet transfusions.

In an attempt to overcome the limitations and drawbacks of using fresh platelets for transfusion therapy of thrombocytopenic patients, we have performed in vitro experiments on an autologous, semi-artificial alternative to platelet transfusions. Based on our previous studies of the interactions of unactivated and activated platelets with beads coated with peptides of various lengths, all of which contained the arginine-glycine-aspartic acid (RGD) cell recognition sequence, the peptide Ac-CGGRGDF-NH2 was chosen for covalent coupling to erythrocytes. A heterobifunctional crosslinking reagent (N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitrobenzene-4-sulfonic acid) was used to crosslink via the peptide's free sulfhydryl group and the erythrocyte's surface amino groups. Approximately 0.5-1.5 x 10(6) peptide molecules bound per erythrocyte after 2 h of incubation, and most of the peptides appeared to crosslink to glycophorin A. The resulting cells, termed thromboerythrocytes, interacted selectively with activated platelets to form mixed aggregates. Studies with fluid phase RGD peptides and monoclonal antibodies indicated that the RGD peptides on the thromboerythrocytes interacted with the GPIIb/IIIa receptors on activated platelets. Thromboerythrocytes could also bind to platelets adherent to collagen. There was minimal erythrocyte hemolysis during the formation of thromboerythrocytes and studies of thromboerythrocyte osmotic fragility and cellular deformability showed no significant changes from control erythrocytes. Whereas there is a 20:1 ratio of erythrocytes to platelets in the circulation of normal individuals, the erythrocytes from as little as 50 ml of blood could be transformed into the equivalent of 2 U of platelets by numbers (equivalent to 18 U of platelets by mass), and reinfused into the same individual within several hours. These data encourage us to proceed to in vivo studies to assess the hemostatic efficacy of thromboerythrocytes in thrombocytopenic animals.

Amino Acid Sequence↗
PubMed1992

Molecular basis for membrane rigidity of hereditary ovalocytosis. A novel mechanism involving the cytoplasmic domain of band 3.

Hereditary ovalocytic red cells are characterized by a marked increase in membrane rigidity and resistance to invasion by malarial parasites. The underlying molecular defect in ovalocytes remained a mystery until Liu and colleagues (N. Engl. J. Med. 1990. 323:1530-38) made the surprising observation that the ovalocytic phenotype was linked to a structural polymorphism in band 3, the anion transporter. We have now defined the mutation in band 3 gene and established the biophysical sequelae of this mutation. This mutation involves the deletion of amino-acids 400-408 in the boundary between the cytoplasmic and the first transmembrane domains of band 3. The biophysical consequences of this mutation are a marked decrease in lateral mobility of band 3 and an increase in membrane rigidity. Based on these findings, we propose the following model for increased membrane rigidity. The mutation induces a conformational change in the cytoplasmic domain of band 3, leading to its entanglement in the skeletal protein network. This entanglement inhibits the normal unwinding and stretching of the spectrin tetramers necessary for membrane extension, leading to increased rigidity. These findings imply that the cytoplasmic domain of an integral membrane protein can have profound effects on membrane material behavior.

Amino Acid Sequence↗
PubMed1991

Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1.

Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.

Amino Acid Sequence↗
PubMed1991

Tissue- and development-specific alternative RNA splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1.

Protein 4.1, a multifunctional structural protein originally described as an 80-kDa component of the erythroid membrane skeleton, exhibits tissue- and development-specific heterogeneity in molecular weight, subcellular localization, and primary amino acid sequence. Earlier reports suggested that some of this impressive heterogeneity is generated by alternative RNA splicing (Conboy, J. G., Chan, J., Mohandas, N., and Kan, Y. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9062-9065; Tang, T. K., Leto, T., Marchesi, V. T., and Benz, E. J. (1990) J. Cell Biol. 110, 617-624). We have now completed a systematic analysis of 4.1 mRNA isoforms expressed in erythroid cells, and have generated an "alternative splicing map" which summarizes diagrammatically a multitude of polypeptide isoforms potentially generated by combinatorial splicing of nine alternative exons. Complex 5' splicing events yield mRNA isoforms that may initiate translation at different sites and thus generate elongated or truncated NH2 termini; elongated approximately 135-kDa and prototypical approximately 80-kDa species were detected in both erythrocytes and T-lymphocytes, but in very different ratios. Among the functional domains of 4.1 responsible for interaction with other membrane skeletal elements, four variants of the 10-kDa spectrin-actin-binding region and four variants of the putative 30-kDa glycophorin-binding region are predicted. Developmentally controlled alternative RNA splicing in the spectrin-actin-binding region may help regulate remodeling of membrane architecture and mechanical properties that occur during erythropoiesis.

Amino Acid Sequence↗
PubMed1991

Contribution of the band 3-ankyrin interaction to erythrocyte membrane mechanical stability.

In an effort to evaluate the role of the band 3-ankyrin linkage in maintenance of red blood cell membrane integrity, solution conditions were sought that would selectively dissociate the band 3-ankyrin linkage, leaving other membrane skeletal interactions intact. For this purpose erythrocytes were equilibrated overnight in nutrient-containing buffers at a range of elevated pHs and then examined for changes in mechanical stability and membrane skeletal composition. Band 3 was found to be released from interaction with the membrane skeleton over a pH range (8.4 to 9.5) that was observed to dissociate the band 3-ankyrin interaction in vitro. In contrast, all other membrane skeletal associations appeared to remain intact up to pH 9.3, after which they were also seen to dissociate. Whereas hemolysis of mechanically unstressed cells did not begin until approximately pH 9.3, where the membrane skeletons began to disintegrate, enhanced fragmentation of shear stressed membranes was seen to begin near pH 8, where band 3 dissociation was first observed. Furthermore, the shear-induced fragmentation rate was found to reach a maximum at pH 9.4, ie, where band 3 dissociation was essentially complete. Based on these correlations, we hypothesize that the band 3-ankyrin linkage of the membrane skeleton to the lipid bilayer is essential for red blood cell stability in the face of mechanical distortion but not for cellular integrity in the absence of mechanical stress.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1991

Molecular characterization of erythrocyte glycophorin C variants.

Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. Immunochemical and serologic studies have identified a number of glycophorin C variants that include the Yus, Gerbich, and Webb phenotypes. We report here the molecular characterization of these variants. Amplification of glycophorin C mRNA from the Yus phenotype, using two oligonucleotide primers that span the coding domain, generated a 338-bp fragment compared with a 395-bp fragment generated by amplification of normal glycophorin C mRNA. Sequencing of the mutant 338-bp fragment identified a 57-bp deletion that corresponds to exon 2 of the glycophorin C gene. Similar analysis showed deletion of 84-bp exon 3 in the Gerbich phenotype. In contrast to the generation of shorter than normal DNA fragments from mRNA amplification in the Yus and Gerbich phenotypes, amplification of mRNA from the Webb phenotype generated a normal-sized fragment. Sequencing of this DNA fragment showed an A----G substitution at nucleotide 23 of the coding sequence, resulting in the substitution of asparagine by serine. This modification accounts for the altered glycosylation of glycophorin C seen in this phenotype. These results have enabled us to characterize glycophorin C variants in three different phenotypes that involve deletions of exons 2 and 3 of the glycophorin C gene, as well as a point mutation in exon 1 that results in altered glycosylation of this protein.

Amino Acid Sequence↗
PubMed1991

Hemoglobin aggregation and pseudosickling in vitro of hemoglobin Setif-containing erythrocytes.

Erythrocytes from individuals heterozygous for hemoglobin Setif (alpha 94 Asp----Tyr) sickle in vitro without deoxygenation when incubated in chloride buffer due to hemoglobin aggregation. We now report quantitative studies of hemoglobin polymerization and deformability in these cells. Hemoglobin polymer gradually increased in intact cells during a 24 h incubation period at 24 degrees C. After 24 hr, about 80% of the cells in 290 mOsm sodium chloride buffer contained polymer which appeared as short rods compared to greater than 99% containing polymer at 450 mOsm. Similar proportions of cells were morphologically sickled. Deformability of erythrocytes with 40% hemoglobin Setif incubated in 290 mOsm buffer at 37 degrees C decreased to 80% of normal by 210 min but in 450 mOsm decreased to 50% after only 30 min as measured by the ektacytometer. However, at 4 degrees C deformability remained normal even in 450 mOsm buffer. The solubility of gelled hemolysate containing 40% hemoglobin Setif was 24 g/dl and 21 g/dl at 290 and 459 mOsm buffer respectively. The gel persisted at 4 degrees C with a solubility of 26 g/dl, but melted when dialyzed into sodium phosphate or potassium phosphate buffer. These data suggest that hemoglobin polymerization, reduced deformability, and sickling of hemoglobin Setif-containing erythrocytes are related to reduced hemoglobin solubility. The rate and extent of intracellular polymerization in vitro are considerably reduced (as in the case of sickle trait) compared with erythrocytes from individuals with sickle cell anemia. Hence, the slower kinetics of hemoglobin aggregation in hemoglobin Setif-containing cells provide an alternate system for studying hemoglobin polymerization and abnormal rheology.

Anemia, Sickle Cell↗
PubMed1991

Reversible deformation-dependent erythrocyte cation leak. Extreme sensitivity conferred by minimal peroxidation.

To determine the threshold at which red blood cells (RBC) begin to manifest deformation-dependent leakiness to monovalent cations, we examined net passive potassium leak during elliptical deformation. Normal RBC did not begin to leak appreciable amounts of potassium until shear stress reached 204 dyn/cm2, at which point they had attained greater than 96% of their maximal deformation. In striking contrast, RBC that had undergone minimal, physiologically relevant degrees of peroxidative damage induced by t-butylhydroperoxide began to leak potassium at only 59 dyn/cm2 when they had reached only 63% of their maximal deformation. The cation leak identified in this manner is not prelytic, and it is fully reversible. Therefore, these data may be relevant to abnormal cation leaks that develop in sickle red cells that have membranes damaged by auto-oxidative stress and that manifest an exuberant but reversible leakiness to monovalent cation during sickling-induced deformation of the cell membrane.

Erythrocyte Deformability↗
PubMed1991

Detachment of agglutinin-bonded red blood cells. II. Mechanical energies to separate large contact areas.

As detailed in a companion paper (Berk, D., and E. Evans. 1991. Biophys. J. 59:861-872), a method was developed to quantitate the strength of adhesion between agglutinin-bonded membranes without ambiguity due to mechanical compliance of the cell body. The experimental method and analysis were formulated around controlled assembly and detachment of a pair of macroscopically smooth red blood cell surfaces. The approach provides precise measurement of the membrane tension applied at the perimeter of an adhesive contact and the contact angle theta c between membrane surfaces which defines the mechanical leverage factor (1-cos theta c) important in the definition of the work to separate a unit area of contact. Here, the method was applied to adhesion and detachment of red cells bound together by different monoclonal antibodies to red cell membrane glycophorin and the snail-helix pomatia-lectin. For these tests, one of the two red cells was chemically prefixed in the form of a smooth sphere then equilibrated with the agglutinin before the adhesion-detachment procedure. The other cell was not exposed to the agglutinin until it was forced into contact with the rigid cell surface by mechanical impingement. Large regions of agglutinin bonding were produced by impingement but no spontaneous spreading was observed beyond the forced contact. Measurements of suction force to detach the deformable cell yielded consistent behavior for all of the agglutinins: i.e., the strength of adhesion increased progressively with reduction in contact diameter throughout detachment. This tension-contact diameter behavior was not altered over a ten-fold range of separation rates. In special cases, contacts separated smoothly after critical tensions were reached; these were the highest values attained for tension. Based on measurements reported in another paper (Evans et al. 1991. Biophys. J. 59:838-848) of the forces required to rupture molecular-point attachments, the density of cross-bridges was estimated with the assumption that the tension was proportional to the discrete rupture force x the number of attachments per unit length. These estimates showed that only a small fraction of agglutinin formed cross-bridges at initial assembly and increased progressively with separation. When critical tension levels were reached, it appeared that nearly all local agglutinin was involved as cross-bridges. Because one cell surface was chemically fixed, receptor accumulation was unlikely; thus, microscopic "roughness" and steric repulsion probably modulated formation of cross-bridges on initial contact. To counter the steric repulsion, adhesive contacts were exposed to solutions of a high molecular weight polymer to draw the surfaces together by osmotic dehydration of the adhesion gap. These stresses exceeded initial mechanical assembly stresses by up to three orders of magnitude. As expected, the strength of adhesion was greatly enhanced by the added impingement stress.

Animals↗
PubMed1991

Hypoxia-induced in vivo sickling of transgenic mouse red cells.

To develop an animal model for sickle cell anemia, we have created transgenic mice that express a severe naturally occurring human sickling hemoglobin, Hb S Antilles. Due to its low solubility and oxygen affinity, Hb S Antilles has a greater propensity to cause red cell sickling than Hb S. To make transgenic animals that express a high level of Hb S Antilles, the erythroid-specific DNAse I hypersensitive site II from the human beta-globin cluster was linked independently to the human alpha 2-globin gene and to the beta S Antilles gene. Embryos were injected with both constructs simultaneously and seven transgenic mice were obtained, three of which contained both the human alpha and the human beta S Antilles transgene. After crossing the human transgenes into the mouse beta-thalassemic background a transgenic mouse line was derived in which approximately half the beta-globin chains in the murine red cells were human beta S Antilles. Deoxygenation of the transgenic red cells in vitro resulted in extensive sickling. An increase of in vivo sickling was achieved by placing these transgenic mice in a low oxygen environment. This murine model for red cell sickling should help to advance our understanding of sickle cell disease and may provide a model to test therapeutic interventions.

Animals↗
PubMed1991

Monozygotic twins with sickle cell anemia and discordant clinical courses: clinical and laboratory studies.

We describe a rare set of monozygotic twins with coexistent sickle cell anemia and alpha-/alpha alpha thalassemia who have asynchronous painful crises of different frequency and severity. Studies include measurements of cell deformability and other hemorheologic tests, cell density distribution, the percentage of irreversibly sickled cells, adherence of red cells to endothelial cells, membrane heme and membrane free iron, calcium containing internal vesicles and serum antioxidants. Results of these studies, including estimates of organ damage (bone, spleen, retina), were similar except for an increase in red cell membrane free iron in the patient with more frequent and severe painful crises. The study supports the concept that non-inherited factors are important contributors to the frequency and severity of painful crises in sickle cell anemia.

Adult↗
PubMed1991

Cell-cell interactions and erythropoiesis.

The erythroblastic island, a distinct anatomical unit consisting of a central macrophage surrounded by a ring of erythroblasts, is a key feature of erythropoiesis in the bone marrow. While a number of functional sequelae for the interaction between the erythroblasts and macrophage have been suggested, much remains to be learned. We suggest that this interaction may play a role in regulated assembly of membrane proteins during erythroid differentiation.

Cell Communication↗