Molecular crowding limits the role of fetal hemoglobin in therapy for sickle cell disease

The dominant assumption central to most treatments for sickle cell anemia has been that replacement of sickle hemoglobin (HbS) by fetal hemoglobin (HbF) would have major clinical benefit. Using laser photolysis, we have measured polymerization kinetics including rates of homogeneous and heterogeneous nucleation on mixtures of 20% and 30% HbF with HbS. We find that the present model for polymerization, including molecular crowding, can accurately predict the rates of such mixtures, by using the single assumption that no significant amount of HbF enters the polymer. The effects of replacing HbS by HbF on the rates of polymer formation are found to be significantly lower than previous measurements appeared to indicate because the impact of the replacement is also highly dependent on the total hemoglobin concentration. This is because the molecular crowding of non-polymerizing HbF offsets substantially the effects of decreasing the concentration of HbS concentration, an effect that increases with concentration. Most strikingly, the demonstrated benefit of hydroxyurea therapy in slowing the kinetics of intracellular polymerization cannot be primarily due to enhanced HbF, but must have some other origin, which could itself represent a promising therapeutic approach.     

Evidence for carbon monoxide binding to sickle cell polymers during melting.

The melting of sickle cell hemoglobin (HbS) polymers was induced by rapid dilution using a stopped-flow apparatus. The kinetics of polymer melting were monitored using light scattering. Polymer melting in the absence of any hemoglobin ligand was compared to melting when the diluting buffer was saturated with carbon monoxide (CO). In this way the role of CO in polymer melting could be assessed. The data were analyzed using models that assumed that melting occurs only at the ends of polymers. It was further assumed that CO could only bind to HbS in the solution phase. However, our data could not be fitted to this model, where CO cannot bind directly to the polymer. Thus, CO probably binds directly to the polymers during our melting experiments. This result is discussed in terms of oxygen induced polymer melting and polymerization processes in sickle cell disease     

Influence of sickle hemoglobin polymerization and membrane properties on deformability of sickle erythrocytes in the microcirculation.

The rheological properties of normal erythrocytes appear to be largely determined by those of the red cell membrane. In sickle cell disease, the intracellular polymerization of sickle hemoglobin upon deoxygenation leads to a marked increase in intracellular viscosity and elastic stiffness as well as having indirect effects on the cell membrane. To estimate the components of abnormal cell rheology due to the polymerization process and that due to the membrane abnormalities, we have developed a simple mathematical model of whole cell deformability in narrow vessels. This model uses hydrodynamic lubrication theory to describe the pulsatile flow in the gap between a cell and the vessel wall. The interior of the cell is modeled as a Voigt viscoelastic solid with parameters for the viscous and elastic moduli, while the membrane is assigned an elastic shear modulus. In response to an oscillatory fluid shear stress, the cell--modeled as a cylinder of constant volume and surface area--undergoes a conical deformation which may be calculated. We use published values of normal and sickle cell membrane elastic modulus and of sickle hemoglobin viscous and elastic moduli as a function of oxygen saturation, to estimate normalized tip displacement, d/ho, and relative hydrodynamic resistance, Rr, as a function of polymer fraction of hemoglobin for sickle erythrocytes. These results show the transition from membrane to internal polymer dominance of deformability as oxygen saturation is lowered. More detailed experimental data, including those at other oscillatory frequencies and for cells with higher concentrations of hemoglobin S, are needed to apply fully this approach to understanding the deformability of sickle erythrocytes in the microcirculation. The model should be useful for reconciling the vast and disparate sets of data available on the abnormal properties of sickle cell hemoglobin and sickle erythrocyte membranes, the two main factors that lead to pathology in patients with this disease.     

Metastable polymerization of sickle hemoglobin in droplets

Sickle cell disease arises from a genetic mutation of one amino acid in each of the two hemoglobin beta chains, leading to the polymerization of hemoglobin in the red cell upon deoxygenation, and is characterized by vascular crises and tissue damage due to the obstruction of small vessels by sickled cells. It has been an untested assumption that, in red cells that sickle, the growing polymer mass would consume monomers until the thermodynamically well-described monomer solubility was reached. By photolysing droplets of sickle hemoglobin suspended in oil we find that polymerization does not exhaust the available store of monomers, but stops prematurely, leaving the solutions in a supersaturated, metastable state typically 20% above solubility at 37 degrees C, though the particular values depend on the details of the experiment. We propose that polymer growth stops because the growing ends reach the droplet edge, whereas new polymer formation is thwarted by long nucleation times, since the concentration of hemoglobin is lowered by depletion of monomers into the polymers that have formed. This finding suggests a new aspect to the pathophysiology of sickle cell disease; namely, that cells deoxygenated in the microcirculation are not merely undeformable, but will actively wedge themselves tightly against the walls of the microvasculature by a ratchet-like mechanism driven by the supersaturated solution.     

Kinetics of water transport in sickle cells

This paper reports the results of stopped-flow studies on differences in the kinetics of osmotic water transport of sickle and normal erythrocytes. The kinetics of inward osmotic water permeability are similar in sickle and normal red blood cells. In contrast, the kinetics of outward water flux are significantly (approx. 38%) decreased in sickle cells. Deoxygenation does not modify the water influx kinetics in either type of cells, but accelerates considerably the rate of water efflux in sickle cells. No significant variation of water transport kinetics was observed in density-separated cell fractions of either type. The results suggest that membrane-associated hemoglobin may decrease the outward water permeability and that in deoxygenated sickle cells the fraction of hemoglobin S near the lipid bilayer is lower than in oxygenated conditions.     

Oxygen equilibrium of emulsified solutions of normal and sickle hemoglobin.

Emulsions containing microdroplets of concentrated solutions of normal or sickle hemoglobin dispersed in a continous oil phase have been prepared, and the aggregation of sickle hemoglobin within microdroplets in a deoxygenated emulsion demonstrated. The equilibrium oxygen saturation of hemoglobin in the emulsions has been measured as a function of the partial pressure of oxygen by a novel spectrophotometric technique which corrects for the scattering of light in the emulsion. The half-saturation oxygen pressure and cooperativity of oxygen binding are substantially greater in concentrated (27 g/dl) solutions of sickle hemoglobin than in solutions of non-aggregating hemoglobin. The shape of the oxygen equilibrium curve of concentrated sickle hemoglobin is qualitatively discussed in terms of a previously proposed model (1).     

Unloading oxygen in a capillary vessel under a pathological condition

In this work, we study theoretically the unloading of oxygen from a hemoglobin molecule to the wall of a typical capillary vessel, considering that the hemoglobin under pathological conditions, obeys the rheological Maxwell model. Based on recent experimental evidences in hypertension, we consider that the red blood cells (RBCs) are composed by a single continuous medium in contrast with the classical particulate or discrete RBC models, which are only valid under normal physiological conditions. The analysis considers the hemodynamic interactions between the plasma and the hemoglobin, both circulating in a long horizontal capillary. We apply numerical and analytical methods to obtain the main fluid-dynamic characteristics for both fluids in the limit of low Reynolds and Womersley numbers. A diffusion boundary layer formulation for the oxygen transport in the combined plasma-hemoglobin core region is presented. The main aspects derived are the time and spatial evolution of the membrane. The hemoglobin and plasma velocities and the pressure distributions are shown. For the oxygen unloading the results are the oxy-hemoglobin saturation, the oxygen flux and the oxygen concentration in the cell-free plasma layer. The volume fraction of red blood cells and the Strouhal number have a great influence on the hemodynamic interactions.     

The kinetics of nucleation and growth of sickle cell hemoglobin fibers

Polymerization of sickle cell hemoglobin (HbS) in deoxy state is one of the basic events in the pathophysiology of sickle cell anemia. For insight into the polymerization process, we monitor the kinetics of nucleation and growth of the HbS polymer fibers. We define a technique for the determination of the rates J and delay times theta of nucleation and the fiber growth rates R of deoxy-HbS fibers, based on photolysis of CO-HbS by laser illumination. We solve numerically time-dependent equations of heat conductance and CO transport, coupled with respective photo-chemical processes, during kinetics experiments under continuous illumination. After calibration with experimentally determined values, we define a regime of illumination ensuring uniform temperature and deoxy-HbS concentration, and fast (within <1 s) egress to steady conditions. With these procedures, data on the nucleation and growth kinetics have relative errors of <5% and are reproducible within 10% in independent experiments. The nucleation rates and delay times have steep, exponential dependencies on temperature. In contrast, the average fiber growth rates only weakly depend on temperature. The individual growth rates vary by up to 40% under identical conditions. These variations are attributed to instability of the coupled kinetics and diffusion towards the growing end of a fiber. The activation energy for incorporation of HbS molecules into a polymer is E(A)=50 kJ mol(-1), a low value indicating the significance of the hydrophobic contacts in the HbS polymer. More importantly, the contrast between the strong theta(T) and weak R(T) dependencies suggests that the homogenous nucleation of HbS polymers occurs within clusters of a precursor phase. This conclusion may have significant consequences for the understanding of the pathophysiology of sickle cell anemia and should be tested in further work.     

The structural organization of skeletal proteins influences lipid translocation across erythrocyte membrane

In order to define the influence of skeletal protein organization on transmembrane phospholipid movement in erythrocyte membranes, we measured the translocation rate of lysophosphatidylcholine in pathologic red cells. A simple method based on the differential extraction of lysophosphatidylcholine from the red cell membrane by saline and albumin solutions was used to quantitate the translocation rate. Two groups of pathologic red cells were chosen for these studies: red cells with quantitative deficiencies of the skeletal proteins, spectrin and protein 4.1, and sickle erythrocytes in which controlled reorganization of the membrane was induced by hemoglobin polymerization. Marked increase in lipid translocation rate was seen in red cells having quantitative deficiencies of spectrin and protein 4.1. The magnitude of the increase in translocation rate in spectrin-deficient red cells was related to the magnitude of protein deficiency. Translocation rate in sickle erythrocyte membranes increased by 50% upon deoxygenation as a result of sickle hemoglobin polymerization. No increase in translocation rate was seen in normal cells upon deoxygenation. By manipulating the extent of membrane reorganization that occurred following deoxygenation of sickle cells, we have been able to show that skeletal reorganization induced by hemoglobin polymerization and not hemoglobin polymerization per se is responsible for the increase in translocation rate. Together, these findings imply that the structural organization of membrane skeletal proteins plays an important role in regulating the rate of transbilayer movement of lipids across the erythrocyte membrane.     

The rate of the deoxygenation reaction limits myoglobin- and hemoglobin-facilitated O₂ diffusion in cells

A mathematical model describing facilitation of O(2) diffusion by the diffusion of myoglobin and hemoglobin is presented. The equations are solved numerically by a finite-difference method for the conditions as they prevail in cardiac and skeletal muscle and in red cells without major simplifications. It is demonstrated that, in the range of intracellular diffusion distances, the degree of facilitation is limited by the rate of the chemical reaction between myglobin or hemoglobin and O(2). The results are presented in the form of relationships between the degree of facilitation and the length of the diffusion path on the basis of the known kinetics of the oxygenation-deoxygenation reactions. It is concluded that the limitation by reaction kinetics reduces the maximally possible facilitated oxygen diffusion in cardiomyoctes by ～50% and in skeletal muscle fibers by ～ 20%. For human red blood cells, a reduction of facilitated O(2) diffusion by 36% is obtained in agreement with previous reports. This indicates that, especially in cardiomyocytes and red cells, chemical equilibrium between myoglobin or hemoglobin and O(2) is far from being established, an assumption that previously has often been made. Although the "O(2) transport function" of myoglobin in cardiac muscle cells thus is severely limited by the chemical reaction kinetics, and to a lesser extent also in skeletal muscle, it is noteworthy that the speed of release of O(2) from MbO(2), the "storage function," is not limited by the reaction kinetics under physiological conditions.     

The effects of erythrocyte membranes on the nucleation of sickle hemoglobin

Pathology in sickle cell disease begins with nucleation-dependent polymerization of deoxyhemoglobin S into stiff, rodlike fibers that deform and rigidify red cells. We have measured the effect of erythrocyte membranes on the rate of homogeneous nucleation in sickle hemoglobin, using preparations of open ghosts (OGs) with intact cytoskeletons from sickle (SS) and normal adult (AA) red cells. Nucleation rates were measured by inducing polymerization by laser photolysis of carboxy sickle hemoglobin and observing stochastic variation of replicate experiments of the time for the scattering signals to reach 10% of their respective maxima. By optical imaging of membrane fragments added to a hemoglobin solution we contrast the rate of nucleation immediately adjacent to membrane fragments with nucleation in a region of the same solution but devoid of membranes. From analysis of 29,272 kinetic curves obtained, we conclude that the effect of AA OGs is negligible (10% enhancement of nucleation rates +/-20%), whereas SS OGs caused 80% enhancement (+/-20%). In red cells, where more membrane surface is available to Hb, this implies enhancement of nucleation by a factor of 6. These experiments represent a 10-fold improvement in precision over previous approaches and are the first direct, quantitative measure of the impact of erythrocyte membranes on the homogeneous nucleation process that is responsible for polymer initiation in sickle cell disease.     

Kinetics of sickle hemoglobin polymerization. II. A double nucleation mechanism

A double nucleation mechanism for the polymerization of sickle hemoglobin is described. The mechanism accounts for all of the major kinetic observations: the appearance of a delay, the high concentration dependence of the delay time, and the stochastic behavior of slowly polymerizing samples in small volumes. The mechanism postulates that there are two pathways for polymer formation: polymerization is initiated by homogeneous nucleation in the solution phase, followed by nucleation of additional polymers on the surface of existing ones. This second pathway is called heterogeneous nucleation. Since the surface of polymers is continuously increasing with time, heterogeneous nucleation provides a mechanism for the extreme autocatalysis that is manifested as an apparent delay in the kinetic progress curves. In this mechanism, each spherulitic domain of polymers is considered to be initiated by a single homogeneous nucleation event. The mechanism explains the irreproducibility of the delay time for single domain formation as arising from stochastic fluctuations in the time at which the homogeneous nucleus for the first polymer is formed. Integration of the linearized rate equations that describe this model results in a simple kinetic form: A[cosh(Bt)-1] (Bishop & Ferrone, 1984). In the accompanying paper (Ferrone et al., 1985) it was shown that the initial 10 to 15% of progress curves, with delay times varying from a few milliseconds to over 10(5) seconds, is well fit by this equation. In this paper, we present an approximate statistical thermodynamic treatment of the equilibrium nucleation processes that shows how the nucleus sizes and nucleation equilibrium constants depend on monomer concentration. The equilibrium model results in expressions for B and B2A as a function of monomer concentration in terms of five adjustable parameters: the bimolecular addition rate of a monomer to the growing aggregate, the fraction of polymerized monomers that serve as heterogeneous nucleation sites, the free energy of intermolecular bonding within the polymer, and two parameters that describe the free energy change as a function of size for the bonding of the heterogeneous nucleus to a polymer surface. This model provides an excellent fit to the data for B and B2A as a function of concentration using physically reasonable parameters. The model also correctly predicts the time regime in which stochastic behavior is observed for polymerization in small volumes.     

Polymerization in erythrocytes containing S and non-S hemoglobins

We analyzed the effects of protein and water nonideality and of erythrocyte heterogeneity on the polymerization of hemoglobin S in cells where there were significant amounts of non-S hemoglobins, sickle trait (AS), and SC disease. For AS erythrocytes, the calculated predicted results were in good agreement with measured polymer formation as previously reported (Noguchi C.T., D.A. Torchia, and A.N. Schnechter, 1981, J. Biol. Chem. 256:4168-4171). Throughout much of the physiologically relevant oxygen saturation region, polymer was not formed in AS erythrocytes. Measurements of polymer formation in SC erythrocytes as a function of oxygen saturation using 13C NMR are reported here and also are in good agreement with the calculated predicted results. As in sickle (SS) erythrocytes, polymer can be detected in SC erythrocytes in the region above 60% oxygen saturation. The increased polymer formation in SC erythrocytes as compared with AS erythrocytes can be explained in terms of hemoglobin composition and concentration in SC erythrocytes, with the concomitant increase in the proportion of dense cells. These findings provide a basis for understanding the pathophysiology of sickle cell and of SC disease, in contrast to benign sickle trait, in terms of intracellular polymer formation.     

Hemoglobin-based O2 carrier O2 affinity and capillary inlet pO2 are important factors that influence O2 transport in a capillary

Hemopure (Biopure; Cambridge, MA) and PolyHeme (Northfield Laboratories; Evanston, IL) are two acellular hemoglobin-based O2 carriers (HBOCs) currently in phase III clinical trials for use as red blood cell substitutes. The most common adverse side effect that these HBOCs exhibit is increased vasoconstriction. Autoregulatory theory has been presented as a possible explanation for this physiological effect, where it is hypothesized that low-affinity HBOCs over-deliver O2 to tissues surrounding arterioles, thereby eliciting vasoconstriction. In this paper, we wanted to investigate HBOC oxygenation of tissue surrounding a capillary, which is the smallest element of the circulatory system. An a priori model has been developed in which the performance of mixtures of acellular HBOCs (synthesized by our group and others) and human red blood cells (hRBCs) has been simulated using a Krogh tissue cylinder model (KTCM) comprising a capillary surrounded by a capillary membrane and skeletal muscle tissue in cylindrical coordinates with specified tissue O2 consumption rates and Michaelis-Menten kinetics. In this study, the total hemoglobin (hRBCs and HBOCs) concentration was kept constant. The HBOCs studied possessed O2 affinities that were higher and lower compared to hRBCs (P50's spanned 5-55 mmHg), and the equilibrium binding/release of oxygen to/from the HBOCs was modeled using the Adair equation. At normoxic inlet pO2's, there was no correlation between O2 flux out of the capillary and the O2 affinity of the HBOC. However, a correlation was found between the average pO2 tension in the capillary and the O2 affinity of the HBOC. Additionally, we studied the change in the O2 equilibrium curve of HBOCs with different O2 affinities over a wide range of inlet pO2's and found that changing the inlet pO2 greatly affected which HBOC, having a unique O2 affinity, best delivered O2 to the surrounding tissue. The analysis of oxygen transport presented could lead to a better prediction of which acellular HBOC is best suited for a specific transfusion application that many times depends on the capillary inlet pO2 tension.     

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.     

A mathematical model of transmural transport of oxygen to the retina

A mathematical model of transmural transport of oxygen to a metabolizing retina is presented based on the equations of fluid dynamics. The equations of oxygen transfer are derived and then solved subject to the condition that the capillaries begin to transport oxygen at an initial time. The resulting transient analysis gives us insight into how diffusive and filtrative processes lead to the oxygen distributions both inside and outside capillaries. On the other hand, the steady state solution allows us to predict the cutoff intraocular pressure above which no oxygen is transferred to retinal tissue. It also gives quantitative relationships which allow us to postulate how intracapillary hypertension counterbalances elevated intraocular pressures and how low pressure glaucoma may arise from ineffective diffusive and filtrative processes of oxygen transport.     

Universal metastability of sickle hemoglobin polymerization

Sickle hemoglobin (HbS) polymerization occurs when the concentration of deoxyHbS exceeds a well-defined solubility. In experiments using sickle hemoglobin droplets suspended in oil, it has been shown that when polymerization ceases the monomer concentration is above equilibrium solubility. We find that the final concentration in uniform bulk solutions (i.e., with negligible boundaries) agrees with the droplet measurements, and both exceed the expected solubility. To measure hemoglobin in uniform solutions, we used modulated excitation of trace amounts of CO in gels of HbS. In this method, a small amount of CO is introduced to a spatially uniform deoxyHb sample, so that less than 2% of the sample is liganded. The liganded fraction is photolyzed repeatedly and the rate of recombination allows the concentration of deoxyHbS in the solution phase to be determined, even if polymers have formed. Both uniform and droplet samples exhibit the same quantitative behavior, exceeding solubility by an amount that depends on the initial concentration of the sample, as well as conditions under which the gel was formed. We hypothesize that the early termination of polymerization is due to the obstruction in polymer growth, which is consistent with the observation that pressing on slides lowers the final monomer concentration, making it closer to solubility. The thermodynamic solubility in free solution is thus achieved only in conditions with low polymer density or under external forces (such as found in sedimentation) that disrupt polymers. Since we find that only about 67% of the expected polymer mass forms, this result will impact any analysis predicated on predicting the polymer fraction in a given experiment.     

Potential of isoquercitrin as antisickling agent: a multi-spectroscopic,thermophoresis and molecular modeling approach

Abstract

Sickle cell disease is an inherited disease caused by point mutation in hemoglobin (β-globin gene). Under oxygen saturation, sickle hemoglobin form polymers, leading to rigid erythrocytes. The transition of the blood vessels is altered and initiated by the adhesion of erythrocytes, neutrophils and endothelial cells. Sickle Hemoglobin (HbS) polymerization is a major cause in red blood cells (RBC), promoting sickling and destruction of RBCs. Isoquercitrin, a medicinal bioactive compound found in various medicinal plants, has multiple health benefits. The present study examines the potential of isoquercitrin as an anti-sickle agent, showing a significant decrease in the rate of polymerization as well as sickling of RBCs. Isoquercitrin-induced graded alteration in absorbance and fluorescence of HbS, confirmed their interaction. A negative value of ΔG° strongly suggests that it is a spontaneous exothermic reaction induced by entropy. Negative ΔH° and positive ΔS° predicted that hydrogen and hydrophobic binding forces interfered with a hydrophobic microenvironment of β6Val leading to polymerization inhibition of HbS. HbS-Isoquercitrin complex exhibits helical structural changes leading to destabilization of the HbS polymer as confirmed by CD spectroscopy. MST and DSC results indicate greater changes in thermophoretic mobility and thermal stability of sickle hemoglobin in the presence of isoquercitrin, respectively. These findings were also supported by molecular simulation studies using DOCK6 and GROMACS. Hence, we can conclude that isoquercitrin interacts with HbS through hydrogen bonding, which leads to polymerization inhibition. Consequently, isoquercitrin could potentially be used as a medication for the treatment of sickle cell disease.

Communicated by Ramaswamy H. Sarma     

Differential polarization imaging. III. Theory confirmation. Patterns of polymerization of hemoglobin S in red blood sickle cells.

In this paper we test the predictions of the differential polarization imaging theory developed in the previous two papers. A characterization of the patterns of polymerization of hemoglobin in red blood cells from patients with sickle cell anemia is presented. This system was chosen because it is relatively easy to handle and because previous studies have been done on it. A differential polarization microscope designed and built in our laboratory was used to carry out this study. This microscope uses an image dissector camera, a photoelastic modulator, and a phase-lock amplifier. This design represents a substantial modification with respect to the instrumentation used in the previous results communicated on this system. Therefore, the results presented here also permit us to confirm the validity of our conclusions. On the basis of the differential polarization images obtained, models of the patterns of polymerization of the hemoglobin S inside the sickle cells are proposed and their M12 and regular images are calculated by the theory. Good agreement between those models and the experimental systems is found, as well as with the results previously reported.     

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.