The specific heat and the heat of compression of human red cells,sickled red cells,and paracrystalline rat red cells

The investigation of two thermal properties of red cells throws some light on whether sickling is a process involving the crystallization of a relatively insoluble hemoglobin. These properties are the specific heat and the heat of compression, both of which would be expected to become numerically less if the hemoglobin of the red cell were to crystallize. In the case of paracrystalline rat red cells, which give spacings at 45 A and 58 A by x-ray diffraction, the specific heat is reduced to 85 per cent of that of the normal red cells, and the heat of compression is only about 75 per cent of that found for the normal red cell. In the case of the red cell sickled by a reduction of the O₂ tension, the specific heat and the heat of compression are substantially the same as found for the normal red cell. This is an argument against sickling being the result of a crystallization process, and supports the observation that sickled cells do not give x-ray spacings. The result is compatible, on the other hand, with sickling being the result of the formation of an oriented and birefringent gel.     

Viscosity and gelation studies in deer hunting hemoglobins.

Sickling, viscosity and gelling properties of the red cells and the hemoglobins of three Virginia white-tailed deer homozygous for types II and III (the sickling types) and V (the nonsickling type), respectively, have been analyzed. The sickling of erythrocytes of deer with type II or III is inhibited by urea and cyanate at concentrations which are comparable to those used in in vitro studies of red cells from patients with sickle cell anemia. No differences were observed between the viscosities of the three deer hemoglobin types at temperatures of 12 degrees C or above. High concentrations of deer hemoglobin types II and III gelled at 1 degree C and at pH values of 7.4-7.7; the minimum gelling concentration of type II was 33.5 g% and of type III was 38 g%. Gel formation was not observed at pH values between 6.7-7.1. Hemoglobin type V did not gel and prevented the formation of gels of type II and III in mixtures at pH 7.6-7.7.     

Mathematical model of red cell sickling

A mathematical model of red cell sickling without crisis was established in vitro as a function of the parameters on which it depends: the oxygen partial pressure, the abnormal hemoglobin S concentration, the temperature and the pH. This model was verified both for the homozygotic sickling SS and heterozygotic sickling SC. Such a model allows systematic investigation of patients. Moreover, it opens up the way for rational comparison in the testing of drugs which are effective in the treatment of red cell sickling.     

Water proton magnetic resonance studies of normal and sickle erythrocytes. Temperature and volume dependence.

The temperature and cell volume dependence of the NMR water proton line-width, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T2) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T1) shows no significant change upon deoxygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T1 and T2 as the cell hydration is changed indicate that these parameters are affected only slightly by a 10-20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T1 for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the "bound" water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at -15 degrees C to 500 Hz at -36 degrees C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T1 data go through a minimum at -35 degrees C for measurements at 44.4 MHz and -50 degrees C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irrotationally bound water, is altered during the sickling process.     

Water proton magnetic resonance studies of normal and sickle erythrocytes Temperature and volume dependence

The temperature and cell volume dependence of the NMR water proton linewidth, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T₂) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T₁) shows no significant change upon dexygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T₁ and T₂ as the cell hydration is changed indicate that these parameters only slightly by a 10–20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T₁ for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the “bound” water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at ?15°C to 500 Hz at ?36°C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T₁ data go through a minimum at ?35°C for measurements at 44.4 MHz and ?50°C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irratationally bound water, is altered during the sickling process.     

Covalent binding of glutathione to hemoglobin. I. Inhibition of hemoglobin S polymerization

Thiol reagents react with cysteine beta 93 of hemoglobin and as a result increase the oxygen affinity of hemoglobin. In the present studies we have used a thiol-disulfide exchange between mixed disulfides of hemoglobin and reduced glutathione to attach intracellular glutathione to hemoglobin and to study its antisickling properties. The rates of production of glutathionyl hemoglobin (G-Hb) depend on the structure of the thiol reagent linked to cysteine beta 93. Up to 25% G-Hb can be produced in normal and sickle red cells because of the high intracellular concentration of reduced glutathione. This high level of G-Hb in normal cells increases the oxygen affinity by about 35% and reduces heme-heme interactions. In sickle cells the increased oxygen affinity is associated with an inhibition of sickling of about 70% at 21 mm Hg. Inhibition of polymerization of deoxy HbS is also due to a direct inhibition of intermolecular contacts in the fibers as demonstrated by the increased solubility and the increased delay time of G-HbS compared to deoxy HbS.     

Autonomic nervous system dysfunction: Implication in sickle cell disease

Sickle cell disease is an inherited hemoglobinopathy caused by a single amino acid substitution in the β chain of hemoglobin that causes the hemoglobin to polymerize in the deoxy state. The resulting rigid, sickle-shaped red cells obstruct blood flow causing hemolytic anemia, tissue damage, and premature death. Hemolysis is continual. However, acute exacerbations of sickling called vaso-occlusive crises (VOC) resulting in severe pain occur, often requiring hospitalization. Blood rheology, adhesion of cellular elements of blood to vascular endothelium, inflammation, and activation of coagulation decrease microvascular flow and increase likelihood of VOC. What triggers the transition from steady state to VOC is unknown. This review discusses the interaction of blood rheological factors and the role that autonomic nervous system (ANS) induced vasoconstriction may have in triggering crisis as well as the mechanism of ANS dysfunction in SCD.     

Augmentation of sickling process due to turbulent blood flow.

The purpose of this study was to determine if the fluid mechanical stresses associated with turbulent blood flow can contribute to the sickling process. Blood from seven patients with sickle cell disease was subjected to intermediate and high levels of turbulent flow in vitro. Turbulence was quantitated by hot film anemometry. Control samples showed 20 +/- 3% sickled cells. Cells subjected to intermediate levels of turbulent flow showed 26 +/- 4% sickling (P less than 0.01); and blood subjected to high intensities of turbulence showed 31 +/- 4% sickling (P less than 0.01). A quantitative count by electronmicroscopy, performed in one patient, showed polymerization of the hemoglobin indicative of sickling in more cells subjected to turbulence than in the control sample. A turbulence-reducing agent, polyethylene oxide, diminished the augmentation of the sickling process as it reduced turbulence at comparable Reynolds numbers. These results support the hypothesis that a deleterious effect upon hemoglobin SS erythrocytes may occur due to the mechanical stresses of turbulent flow. The agitation associated with turbulent flow presumably modifies the stabilizing factors of the intracellular colloidal solution of hemoglobin, thereby contributing to sol-gel transformation. Such hydrodynamic stresses may supplement the previously described factors which contribute to sickle cell crises.     

Single cell microspectroscopy reveals that erythrocytes containing hemoglobin S retain a 'memory' of previous sickling cycles

Red blood cells from patients homozygotes for hemoglobin S (HbS) have been studied using a computer-controlled microspectrophotometer, which allows measurements of spectra and dynamics to be undertaken in a single erythrocyte. Complete photodissociation of HbCO results in polymerization of intracellular deoxyhemoglobin S and deformation of the cell. This is associated with a delayed optical change, which, for the same cell, was found to be highly reproducible between repeated cycles of sickling. Comparison of photographic records and absorbance time courses indicates that an erythrocyte, once having undergone a photochemically induced sickling event, always deforms along the same axis during subsequent cycles. This behaviour implies that the cell retains a 'memory' of its previous cycle(s), possibly via slow relaxations of the membrane. In addition, rebinding of CO to intracellular hemoglobin was found to be slower if measured after deformation of the cell, with possible important implications for the pathological mechanism of sickling.     

Altered amount and activity of superoxide dismutase in sickle cell anemia

The amount and activity of superoxide dismutase (SOD) (EC 1.15.1.1) were measured in red cells collected from 50 white controls, 101 black controls, 50 patients with sickle hemoglobin (SS Hb), 12 with sickle trait, and 11 with other sickling hemoglobinopathies. Red cells from normal black subjects had more SOD amount and activity than normal whites (1.77 U/mg Hb and 2.96 micrograms/mg Hb vs. 1.47 U/mg Hb and 2.64 micrograms/mg Hb, respectively) or blacks with SS Hb or other sickling hemoglobinopathies. Patients with more severe manifestations of SS Hb had lower levels of SOD activity than those with milder symptoms but had the same amount of enzyme protein. Individuals with sickle trait had amounts and activities of SOD comparable to black controls. An alteration in defense to free radical oxygen may play a role in the severity of symptoms experienced by patients with homozygous sickle cell disease.     

Biomechanics and biorheology of red blood cells in sickle cell anemia

Sickle cell anemia (SCA) is an inherited blood disorder that causes painful crises due to vaso-occlusion of small blood vessels. The primary cause of the clinical phenotype of SCA is the intracellular polymerization of sickle hemoglobin resulting in sickling of red blood cells (RBCs) in deoxygenated conditions. In this review, we discuss the biomechanical and biorheological characteristics of sickle RBCs and sickle blood as well as their implications toward a better understanding of the pathophysiology and pathogenesis of SCA. Additionally, we highlight the adhesive heterogeneity of RBCs in SCA and their specific contribution to vaso-occlusive crisis.     

Polymerization of hemoglobins in Arctic fish: Lycodes reticulatus and Gadus morhua

In vitro, and possibly in vivo, hemoglobin polymerization and red blood cell sickling appear to be widespread in codfish. In this article, we show that the hemoglobins of the two Arctic fish Lycodes reticulatus and Gadus morhua also have the tendency to polymerize, as monitored by dynamic light scattering experiments. The elucidation of the primary structure of the single hemoglobin of the zoarcid L. reticulatus shows the presence of a large number of cysteyl residues in α and β chains. Their role in eliciting the ability to produce polymers was also addressed by MALDI-TOF and TOF-TOF mass spectrometry. The G.morhua globins are also rich in Cys, but unlike in L. reticulatus, polymerization does not seem to be disulfide driven. The widespread occurrence of the polymerization phenomenon displayed by hemoglobins of Arctic fish supports the hypothesis that this feature may bea response to stressful environmental conditions.     

Detrimental effects of adenosine signaling in sickle cell disease

Hypoxia can act as an initial trigger to induce erythrocyte sickling and eventual end organ damage in sickle cell disease (SCD). Many factors and metabolites are altered in response to hypoxia and may contribute to the pathogenesis of the disease. Using metabolomic profiling, we found that the steady-state concentration of adenosine in the blood was elevated in a transgenic mouse model of SCD. Adenosine concentrations were similarly elevated in the blood of humans with SCD. Increased adenosine levels promoted sickling, hemolysis and damage to multiple tissues in SCD transgenic mice and promoted sickling of human erythrocytes. Using biochemical, genetic and pharmacological approaches, we showed that adenosine A(2B) receptor (A(2B)R)-mediated induction of 2,3-diphosphoglycerate, an erythrocyte-specific metabolite that decreases the oxygen binding affinity of hemoglobin, underlies the induction of erythrocyte sickling by excess adenosine both in cultured human red blood cells and in SCD transgenic mice. Thus, excessive adenosine signaling through the A(2B)R has a pathological role in SCD. These findings may provide new therapeutic possibilities for this disease.     

Effect of sickling on dimyristoylphosphatidylcholine-induced vesiculation in sickle red blood cells

To study the effect of sickling on dimyristoylphosphatidylcholine (DMPC)-induced vesiculation, sickle (SS) red blood cells were incubated with sonicated suspensions of DMPC under either room air or nitrogen. Like normal red cells, when sickle cells were incubated with DMPC under oxygenated conditions, incorporation of DMPC into the erythrocyte membrane occurred, followed by echinocytic shape transformation and subsequent release of membrane vesicles. On the other hand, when SS cells were induced to sickle by deoxygenation, DMPC-induced vesiculation of these cells was dramatically reduced. However, upon reoxygenation, release of vesicles from these sickle erythrocytes occurred immediately. When SS cells were incubated under hypertonic (500 mosM) and deoxygenated conditions (where hemoglobin polymerization occurs but red cells do not show the typical sickle morphology), a similar decrease in the extent of vesiculation was observed. Experiments with radiolabelled lipid vesicles indicated that incorporation of DMPC into erythrocyte membranes occurred in all cases and therefore was not the limiting factor in the reduction of vesiculation in deoxygenated SS cells. Taken together, these results indicate that cellular viscosity and membrane rigidity, both of which are influenced by hemoglobin polymerization, are two important factors in process of vesicle release from sickle erythrocytes.     

Polyamines--sickling red blood cell interaction.

The binding of polyamine as a function of concentration to normal and sickling rcc'. blood cells is analyzed by Langmuir type binding isotherms, based on the Gouy-Chapman model for an electrical double Iayer, where the zeta potential is a function of only the normal distance coordinate. For normal erythrocytes, the apparent exotropic binding constants are found to be 103, 110, and 130 dl/g at normal distance coordinates of 4, 5, and 6 Å, iezpectively. The esotropic binding constant is determined to be 420 dl/g at a distance of 7 Å. For sickling red blood cells, the apparent exotropic binding constants are 3.3, 3.8, 4.6, and 6-7 dl/g at a distance of 4 to 7 Å. The esotropic binding constant at a distance of 8 Å is found to be 12-9 dl/g. The apparent binding affinity of polyamines to the normal red blood cell. therefore, is approximately 30 times greater than to the sickling erythrocyte.The Praxis pulse nuclear magnetic resonance spectrometer is used to determine the spin-lattice relaxation time (T₁) for water in the presence of normal and sickling red blood cells. The spin-lattice relaxation time is found to be 540 ms for normal erythrocytes and 445 ms for sickling red blood cells in the oxy state. Differences in the spin-spin relaxation time (T₂) for the two types of erythrocyte are negligible, being within the range of normal experimental error.     

Relations among variations in human red cell volume,density, membrane area,hemoglobin content and cation content

It is shown how variations in different properties of red cells can be inter-related provided relations exist among these properties at the single cell level. On the basis of the cell density dependence on cell volume and hemoglobin content, and the assumed volume dependence on red cell cation and hemoglobin content, nine relations among the variations in red cell volume, density, membrane area, hemoglobin content and cation content, and their correlations are derived. Values of seven correlation coefficients are theoretically predicted and are shown to be consistent with the experiments performed by density fractionated red blood cells. The cell volume dependence on cation and hemoglobin content obtained from relations among variations is compared with the predictions obtained by the existing model about the osmotic behavior of the red blood cell. Furthermore, it is shown that data on the variations of the red cell properties indicate the existence of the relation among cation content, hemoglobin content, and membrane area at the level of a single cell.     

Plasmodium falciparum: physiological interactions with the human sickle cell.

The malaria parasite, Plasmodium falciparum, enhances the rate and extent of sickling of infected hemoglobin S heterozygous human erythrocytes. Upon sickling of the host cell, the parasite is killed. Parasite-free lysates of highly infected cells were analyzed to determine the mechanism by which sickling is enhanced. The intraerythrocytic pH of the infected cell was estimated to be 0.4 units below that of the uninfected cell, a difference which could result in a 20-fold increase in the extent of sickling under physiological conditions. Sickle-cell hemoglobin (HbS) heterozygous (AS) erythrocytes had decreased intracellular potassium after 24 hr of culture under conditions which cause sickling and parasite death. When infected AS cells were cultured in high-potassium medium under these conditions the parasites were protected. The medium did not prevent sickling but did maintain normal intracellular potassium levels. It is suggested that sequestration of trophozoite-infected AS cells in the venules leads to the sickling of the host cell, loss of erythrocytic potassium, and parasite death. The resulting attenuation of parasite multiplication would favor the survival of the HbS heterozygote and maintain the HbS gene at high frequencies in areas endemic for falciparum malaria.     

Effect of polyamines on the electrokinetic properties of red blood cells.

At the physiological pH 7.4, the zeta potential of the normal red blood cell in 1.5% glycine buffer was found to be ?52 mv, whereas that of sickling erythrocytes is ?45 mv. Addition of spermidine to normal red blood cells reduced the zeta potential by approximately 20 mv. In sickling red blood cells, where the polyamine content is determined to be 5 to 6 times greater than in the normal erythrocyte, addition of spermidine reduced the zeta potential by only 5 mv, indicating that little more polyamine binding occurs. The polyamine content of whole blood taken from 24 patients having sickle cell anemia was found to be more than ten times that of whole blood from normal donors. Binding of polyamines to the normal red blood cell was analyzed from the surface charge potential variation as a function of polyamine concentration and the apparent binding constant determined to be 130 d1/g. The difference in the electrokinetic properties of normal and sickling red blood cells in this system may be attributed, in part, to a variation in the polyamine content of the two types of erythrocytes.     

Inhibition of the in vitro formation of dense cells and of irreversibly sickled cells by charybdotoxin, a specific inhibitor of calcium-activated potassium efflux

Charybdotoxin, a specific inhibitor of the calcium-activated potassium channel, was found to inhibit the in vitro formation of irreversibly dehydrated cells and of irreversibly sickled cells, which occur as a result of repeated cycles of sickling and unsickling of sickle red blood cells. The degree of formation of dense cells was measured by Percoll-renografin density gradient centrifugation. 50% inhibition of the formation was achieved at a concentration of 30 nM of charybdotoxin. The approximate half-life of this compound in the circulation of the guinea pig was determined to be 4 h. Charybdotoxin did not inhibit the sickling of sickle cells under deoxygenation. The effects of charybdotoxin in preventing the irreversible changes of sickle cell membranes may be related to the inhibition of calcium-activated potassium efflux in sickle red blood cells.     

Hemoglobin S Travis: a sickling hemoglobin with two amino acid substitutions [beta6(A3)glutamic acid leads to valine and beta142 (h20) alanine leads to valine).

Hb S Travis is a previously undescribed sickling hemoglobin with two amino acid substitutions in the beta chain: beta6 Glu leads to Val and beta142 Ala leads to Val. The beta6 Glu leads to Val mutation imparts to Hb S Travis the characteristic properties of sickling hemoglobin, namely its association with erythrocyte sickling, the insolubility of the hemoglobin in the reduced form, and a minimum gelling concentration value identical to Hb S. Unlike Hb S, Hb S Travis exhibits an increased oxygen affinity and a decreased affinity for 2,3-bisphosphoglycerate and inositol hexakisphosphate. In addition, the variant hemoglobin's tendency to autoxidize and its mechanical precipitability suggest that there are conformational differences between Hb S and Hb S Travis.