Anisotropy in sickle hemoglobin fibers from variations in bending and twist

We have studied the variations of twist and bend in sickle hemoglobin fibers. We find that these variations are consistent with an origin in equilibrium thermal fluctuations, which allows us to estimate the bending and torsional rigidities and effective corresponding material moduli. We measure bending by electron microscopy of frozen hydrated fibers and find that the bending persistence length, a measure of the length of fiber required before it starts to be significantly bent due to thermal fluctuations, is 130microm, somewhat shorter than that previously reported using light microscopy. The torsional persistence length, obtained by re-analysis of previously published experiments, is found to be only 2.5microm. Strikingly this means that the corresponding torsional rigidity of the fibers is only 6x10(-27)Jm, much less than their bending rigidity of 5x10(-25)Jm. For (normal) isotropic materials, one would instead expect these to be similar. Thus, we present the first quantitative evidence of a very significant material anisotropy in sickle hemoglobin fibers, as might arise from the difference between axial and lateral contacts within the fiber. We suggest that the relative softness of the fiber with respect to twist deformation contributes to the metastability of HbS fibers: HbS double strands are twisted in the fiber but not in the equilibrium crystalline state. Our measurements inform a theoretical model of the thermodynamic stability of fibers that takes account of both bending and extension/compression of hemoglobin (double) strands within the fiber.     

Modeling sickle hemoglobin fibers as one chain of coarse-grained particles

Sickle cell disease (SCD) is caused by a single point mutation in the beta-chain hemoglobin gene, resulting in the presence of abnormal hemoglobin S (HbS) in the patients' red blood cells (RBCs). In the deoxygenated state, the defective hemoglobin tetramers polymerize forming stiff fibers which distort the cell and contribute to changes in its biomechanical properties. Because the HbS fibers are essential in the formation of the sickle RBC, their material properties draw significant research interests. Here, a solvent-free coarse-grain molecular dynamics (CGMD) model is introduced to simulate single HbS fibers as a chain of particles. First, we show that the proposed model is able to efficiently simulate the mechanical behavior of single HbS fibers. Then, the zippering process between two HbS fibers is studied and the effect of depletion forces is investigated. Simulation results illustrate that depletion forces play a role comparable to direct fiber-fiber interaction via Van der Waals forces. This proposed model can greatly facilitate studies on HbS polymerization, fiber bundle and gel formation as well as interaction between HbS fiber bundles and the RBC membrane.     

Intermolecular contacts within sickle hemoglobin fibers

By combining X-ray crystallographic co-ordinates of sickle hemoglobin (HbS) molecules with three-dimensional reconstructions of electron micrographs of HbS fibers we have synthesized a model for the structure of the clinically relevant HbS fiber. This model largely accounts for the action of 55 point mutations of HbS whose effect on fiber formation has been studied. In addition, it predicts locations at which additional point mutations are likely to affect fiber formation. The number of intermolecular axial contacts decreases with radius until, at the periphery of the fiber, there are essentially no axial contacts. We suggest that this observation accounts for the limited radial growth of the HbS fiber and that a similar mechanism may be a factor in limiting the size of other helical particles. The methodology for the synthesis of the fiber model is applicable to other systems in which X-ray crystallographic and electron microscopic data are available.     

Molecular insights into the irreversible mechanical behavior of sickle hemoglobin

Sickle cell disease is caused by the amino acid substitution of glutamic acid to valine, which leads to the polymerization of deoxygenated sickle hemoglobin (HbS) into long strands. These strands are responsible for the sickling of red blood cells (RBCs), making blood hyper-coagulable leading to an increased chance of vaso-occlusive crisis. The conformational changes in sickled RBCs traveling through narrow blood vessels in a highly viscous fluid are critical in understanding; however, there are few studies that investigate the origins of the molecular mechanical behavior of sickled RBCs. In this work, we investigate the molecular mechanical properties of HbS molecules. A mechanical model was used to estimate the directional stiffness of an HbS molecule and the results were compared to adult human hemoglobin (HbA). The comparison shows a significant difference in strength between HbS and HbA, as well as anisotropic behavior of the hemoglobin molecules. The results also indicated that the HbS molecule experienced more irreversible mechanical behavior than HbA under compression. Further, we have characterized the elastic and compressive properties of a double stranded sickle fiber using six HbS molecules, and it shows that the HbS molecules are bound to each other through strong inter-molecular forces.     

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.     

Enzyme-ligand complexes of pyridoxine 5'-phosphate synthase: implications for substrate binding and catalysis

Pathogenesis in sickle cell disease depends on polymerization of deoxyhemoglobin S into rod-like fibers, forming gels that rigidify red cells and obstruct the systemic microvasculature. Fiber structure, polymerization kinetics and equilibria are well characterized and intimately related to pathogenesis. However, data on gel rheology, the immediate cause of obstruction, are limited, and models for structure and rheology are lacking. The basis of gel rheology, micromechanics of individual fibers, has never been examined. Here, we isolate fibers by selective depolymerization of gels produced under photolytic deliganding of CO hemoglobin S. Using differential interference contrast (DIC) microscopy, we measure spontaneous, thermal fluctuations in fiber shape to obtain bending moduli (kappa) and persistence lengths (lambda(p)). Some fibers being too stiff to decompose shape accurately into Fourier modes, we measure deviations of fiber midpoints from mean positions. Serial deviations, sufficiently separated to be independent, exhibit Gaussian distributions and provide mean-squared fluctuation amplitudes from which kappa and lambda(p) can be calculated. Lambda(p) ranges from 0.24 to 13 mm for the most flexible and stiffest fibers, respectively. This large range reflects formation of fiber bundles. If the most flexible are single fibers, then lambda(p) =13 mm represents a bundle of seven single fibers. Preliminary data on the bending variations of frozen, hydrated single fibers of HbS obtained by electron microscopy indicate that the value 0.24 mm is consistent with the persistence length of single fibers. Young's modulus is 0.10 GPa, less than for structural proteins but much larger than for extensible proteins. We consider how these results, used with models for cross-linking, may apply to macroscopic rheology of hemoglobin S gels. This new technique, combining isolation of hemoglobin S fibers and measurement of micromechanical properties based on thermal fluctuations and midpoint deviations, can be used to study fibers of mutants, hemoglobin A/S, and mixtures and hybrids of hemoglobin S.     

Fiber Depolymerization: Fracture, Fragments, Vanishing Times, and Stochastics in Sickle Hemoglobin

The well-characterized rates, mechanisms, and stochastics of nucleation-dependent polymerization of deoxyhemoglobin S (HbS) are important in governing whether or not vaso-occlusive sickle cell crises will occur. The less well studied kinetics of depolymerization may also be important, for example in achieving full dissolution of polymers in the lungs, in resolution of crises and/or in minimizing gelation-induced cellular damage. We examine depolymerization by microscopic observations on depolymerizing HbS fibers, by Monte Carlo simulations and by analytical characterization of the mechanisms. We show that fibers fracture. Experimental scatter of rates is consistent with stochastic features of the analytical model and Monte Carlo results. We derive a model for the distribution of vanishing times and also show the distribution of fracture-dependent fiber fragment lengths and its time dependence. We describe differences between depolymerization of single fibers and bundles and propose models for bundle dissolution. Our basic model can be extended to dissolution of gels containing many fibers and is also applicable to other reversible linear polymers that dissolve by random fracture and end-depolymerization. Under the model, conditions in which residual HbS polymers exist and facilitate repolymerization and thus pathology can be defined; whereas for normal polymers requiring cyclic polymerization and depolymerization for function, conditions for rapid cycling due to residual aggregates can be identified.     

Free Heme and the Polymerization of Sickle Cell Hemoglobin

In search of novel control parameters for the polymerization of sickle cell hemoglobin (HbS), the primary pathogenic event of sickle cell anemia, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that the concentration of free heme in HbS solutions typically used in the laboratory is 0.02-0.04 mole heme/mole HbS. We show that dialysis of small molecules out of HbS solutions arrests HbS polymerization. The addition of 100-260 μM of free heme to dialyzed HbS solutions leads to rates of nucleation and polymer fiber growth faster by two orders of magnitude than before dialysis. Toward an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at a concentration of 66 μM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that spikes of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a significant factor in the complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell therapy.     

Modification of Axial Fiber Contact Residues Impact Sickle Hemoglobin Polymerization by Perturbing a Network of Coupled Interactions

The identity of intermolecular contact residues in sickle hemoglobin (HbS) fiber is largely known. However, our knowledge about combinatorial effects of two or more contact sites or the mechanistic basis of such effects is rather limited. Lys16, His20, and Glu23 of the α-chain occur in intra-double strand axial contacts in the sickle hemoglobin (HbS) fiber. Here we have constructed two novel double mutants, HbS (K16Q/E23Q) and (H20Q/E23Q), with a view to delineate cumulative impact of interactions emanating from the above contact sites. Far-UV and visible region CD spectra of the double mutants were similar to the native HbS indicating the presence of native-like secondary and tertiary structure in the mutants. The quaternary structures in both the mutants were also preserved as judged by the derivative UV spectra of liganded (oxy) and unliganded (deoxy) forms of the double mutants. However, the double mutants displayed interesting polymerization behavior. The polymerization behaviour of the double mutants was found to be non-additive of the individual single mutants. While HbS (H20Q/E23Q) showed inhibitory effect similar to that of HbS (E23Q), the intrinsic inhibitory propensity of the associated single mutants was totally quelled in HbS (K16Q/E23Q) double mutant. Molecular dynamics (MD) simulations studies of the isolated α-chains as well as a module of the fiber containing the double and associated single mutants suggested that these contact sites at the axial interface of the fiber impact HbS polymerization through a coupled interaction network. The overall results demonstrate a subtle role of dynamics and electrostatics in the polymer formation and provide insights about interaction-linkage in HbS fiber assembly.     

The entropically favored osmotic "compression" of sickle cell hemoglobin gels

Contrary to the accurate, hard-sphere depiction of monomeric hemoglobin in solution, sickle cell hemoglobin (HbS) polymerization/gelation requires attention to molecular interactions. From the temperature dependence of the osmotic compressibility of HbS gels, we were able to extract the entropy increase for concentrating HbS in this phase. Normalized per mole of water removed, the entropy increase from gel compression DeltaS(gel) is four times the previously measured DeltaS(trans), for the transition from monomeric HbS solution to HbS gel. The positive entropy change cannot emerge from the assembly of hard spheres but can indicate remodeling of HbS fibers driven by release of ordered water. The fourfold difference in DeltaS(gel) and DeltaS(trans) suggests that the act of initial fiber/gel formation from monomeric solution differs from the process of further polymerization due to tighter packing within the gel phase.     

Analysis of the Intermolecular Contacts within Sickle Hemoglobin Fibers: Effect of Site-Specific Substitutions, Fiber Pitch, and Double-Strand Disorder

An atomic model of the sickle hemoglobin (HbS) fiber was synthesized by combining the molecular coordinates of the fiber (obtained from electron microscopy) with atomic coordinates of the sickle hemoglobin double strand (obtained from X-ray crystallography). The model is stereochemically acceptable. The majority of polymerization-sensitive HbS mutants are located at fiber contact sites and the majority of the mutants that do not affect polymerization are not located at contact sites. The residues at intermolecular contacts in the fiber model are reported. We have searched the coordinate space in the vicinity of the EM reconstructions to find models with alternative sets of coordinates that satisfy the mutant data, contain 5-Å contacts between double strands, and are stereochemically acceptable. This involved a systematic examination over 297 different models. The alternative fiber models were generated with a range of fiber pitch, double-strand positions, and double-strand polarity. Models which had unacceptably close contacts between atoms, failed to satisfy the mutant data, or did not have 5-Å contacts between double strands were considered unacceptable. None of the acceptable alternative fiber models improved the agreement between the polymerization behavior of HbS mutants and their contact site location. However, several models could account for the polymerization data equally well. Residue locations for single-site HbS mutations that could discriminate between alternative fiber models are proposed. The twist of HbS fibers varies in an apparent random manner with an average rotation of 7.8 ± 2.5° per molecule and a maximum rotation of 16° per molecule. The number of interdouble-strand contacts as a function of fiber twist shows a broad maximum around 9° and may account for the observed range of fiber pitch. This study shows that the upper limit on the fiber twist could result from a loss of axial contacts and repulsive van der Waals interactions between residues involved in interstrand contacts. The loss of axial contacts limits the radial growth of the fiber. In the appendix we analyze the methodology used by I. Cretegny and S. J. Edelstein [(1993) J. Mol. Biol. 230, 733-738] to build a model of the fiber. Our examination reveals shortcomings in the methodology of Cretegny and Edelstein. One result of these shortcomings is that the model synthesized by Cretegny and Edelstein is not stereochemically acceptable because it gives rise to a large number of excessively close (less than 1.4 Å) atom-atom contacts, suggesting interpenetration of the molecular envelopes.     

Nitric oxide reduces sickle hemoglobin polymerization: potential role of nitric oxide-induced charge alteration in depolymerization

We previously demonstrated that inhaling nitric oxide (NO) increases the oxygen affinity of sickle red blood cells (RBCs) in patients with sickle cell disease (SCD). Our recent studies found that NO lowered the P₅₀ values of sickle hemoglobin (HbS) hemolysates but did not increase methemoglobin (metHb) levels, supporting the role of NO, but not metHb, in the oxygen affinity of HbS. Here we examine the mechanism by which NO increases HbS oxygen affinity. Because anti-sickling agents increase sickle RBC oxygen affinity, we first determined whether NO exhibits anti-sickling properties. The viscosity of HbS hemolysates, measured by falling ball assays, increased upon deoxygenation; NO treatment reduced the increment. Multiphoton microscopic analyses showed smaller HbS polymers in deoxygenated sickle RBCs and HbS hemolysates exposed to NO. These results suggest that NO inhibits HbS polymer formation and has anti-sickling properties. Furthermore, we found that HbS treated with NO exhibits an isoelectric point similar to that of HbA, suggesting that NO alters the electric charge of HbS. NO–HbS adducts had the same elution time as HbA upon high performance liquid chromatography analysis. This study demonstrates that NO may disrupt HbS polymers by abolishing the excess positive charge of HbS, resulting in increased oxygen affinity.     

Two-step mechanism of homogeneous nucleation of sickle cell hemoglobin polymers

Sickle cell anemia is a debilitating genetic disease that affects hundreds of thousands of babies born each year worldwide. Its primary pathogenic event is the polymerization of a mutant, sickle cell, hemoglobin (HbS); and this is one of a line of diseases (Alzheimer's, Huntington's, prion, etc.) in which nucleation initiates pathophysiology. We show that the homogeneous nucleation of HbS polymers follows a two-step mechanism with metastable dense liquid clusters serving as precursor to the ordered nuclei of the HbS polymer. The evidence comes from data on the rates of fiber nucleation and growth and nucleation delay times, the interaction of fibers with polarized light, and mesoscopic metastable HbS clusters in solution. The presence of a precursor in the HbS nucleation mechanism potentially allows low-concentration solution components to strongly affect the nucleation kinetics. The variations of these concentrations in patients might account for the high variability of the disease in genetically identical patients. In addition, these components can potentially be utilized for control of HbS polymerization and treatment of the disease.     

Computer models of a new deoxy-sickle cell hemoglobin fiber based on x-ray diffraction data.

A new x-ray fiber diffraction pattern from deoxygenated sickle cell erythrocytes has been observed. It displays 14 layer lines with a 109 A periodicity compared with the 64 A periodicity of the "classic" sickle cell hemoglobin (HbS) fiber. These data and association energy calculations serve as a basis for computer model building. Systematic searches over four-dimensional parameter space yielded twelve protofilament models that satisfy the following constraints: (a) two HbS molecules be related by twofold screw symmetry with a translational repeat of 109 A; (b) at least one of the substituted residues in HbS, val beta 6, should participate in intermolecular contacts; and (c) the energy of intermolecular interaction be less than -24 kcal/mol. Each of the protofilament models is a zigzag mono-strand that stands in contrast to the double-stranded protofilament of the "classic" fiber. Fiber models were constructed with each of the 12 protofilament models, pseudo-hexagonally packed. Searches of variable packing parameters showed four fiber models with minimal protofilament association energies and minimal differences between calculated transforms and observed data. The R-factor was less than 0.24 for each of these four models. In three of the fiber models the protofilament association energy is between -(93 and 130) kcal, and in a fourth, the energy is -64 kcal. One protofilament model constituted three distinct fiber models of the lower energy class, and a second protofilament model packed with a higher association energy into a fourth fiber model. The selection of a unique fiber model from among these four cannot be made because of the limited available data.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Anion transport in normal erythrocytes,sickle red cells,and ghosts in relation to hemoglobins and magnesium

"Band 3," an integral membrane protein of red blood cells, plays a relevant role in anionic transport. The C- and N-terminal portions of band 3 are cytoplasmatics, and the last is the link site for different glycolitic enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, aldolase, phosphofructokinase, and hemoglobin. All or some of these interactions on the CDB3 protein could allow a subtle modulation of anion flux. The interaction among HbA, Mg(2+), and membrane proteins has been sufficiently investigated, but not the effect of Mg(2+) on pathological hemoglobin in relation to the influx of the SO(4)(2-). The aim of this study was to evaluate the involvement of hemoglobin S in sulfate transport. This has been measured with native and increased concentrations of Mg(2+), using normal erythrocytes containing HbA, sickle red cells containing HbS, or ghosts obtained from both erythrocytes and normal erythrocytes ghosts with HbS added. The magnitude of the SO(4)(2-) rate constant measured in normal red blood cells increased markedly when measured in the presence of varied Mg(2+) concentrations. The results show that a low increase of intracellular Mg(2+) concentrations exercises a different HbA modulation on band 3 protein and consequently higher anion transport activity. The same experiments carried out in sickle red cells showed that the SO(4)(2-) rate constant measured in the presence of native concentrations of Mg(2+) was normal, compared to normal red cells, and was not affected by any increase of intracellular Mg(2+). Our suppositions with regard to the importance exercised by the hemoglobin and the Mg(2+) on the SO(4)(2-) influx were confirmed by comparison of the data obtained through measuring SO(4)(2-) influx with native and increased concentrations of Mg(2+) in both normal and sickle red cell ghosts. Both revealed the same sensitivity to Mg(2+) due to withdrawal of hemoglobins. The incorporation of HbS in normal as well as in sickle red cell ghosts reduced the Mg(2+) response to sulfate influx in both the reconstituted ghosts. Our research demonstrated that the different effects exercised on the rate constants of SO(4)(2-) influx in normal (HbA) and sickle red cells (HbS) by the increased intracellular Mg(2+) could be ascribed to the physical-chemical influence exercised either on the hemoglobins or on the intracellular contents of erythrocytes.     

Development of a 3D cell culture system for investigating cell interactions with electrospun fibers

There are many variables to be considered in studying how cells interact with 3D scaffolds used in tissue engineering. In this study we investigated the influence of the fiber diameter and interfiber spaces of 3D electrospun fiber scaffolds on the behavior of human dermal fibroblasts. Fibers of two dissimilar model materials, polystyrene and poly-L-lactic acid, with a broad range of diameters were constructed in a specifically developed 3D cell culture system. When fibroblasts were introduced to freestanding fibers, and encouraged to "walk the plank," a minimum fiber diameter of 10 microm was observed for cell adhesion and migration, irrespective of fiber material chemistry. A distance between fibers of up to 200 microm was also observed to be the maximum gap that could be bridged by cell aggregates--a behavior not seen in conventional 2D culture. This approach has identified some basic micro-architectural parameters for electrospun scaffold design and some key differences in fibroblast growth in 3D. We suggest the findings will be of value for optimizing the integration of cells in these scaffolds for skin tissue engineering.     

Analysis of the stability of hemoglobin S double strands.

The deoxyhemoglobin S (deoxy-HbS) double strand is the fundamental building block of both the crystals of deoxy-HbS and the physiologically relevant fibers present within sickle cells. To use the atomic-resolution detail of the hemoglobin-hemoglobin interaction known from the crystallography of HbS as a basis for understanding the interactions in the fibers, it is necessary to define precisely the relationship between the straight double strands in the crystal and the twisted, helical double strands in the fibers. The intermolecular contact conferring the stability of the double strand in both crystal and fiber is between the beta6 valine on one HbS molecule and residues near the EF corner of an adjacent molecule. Models for the helical double strands were constructed by a geometric transformation from crystal to fiber that preserves this critical interaction, minimizes distortion, and makes the transformation as smooth as possible. From these models, the energy of association was calculated over the range of all possible helical twists of the double strands and all possible distances of the double strands from the fiber axis. The calculated association energies reflect the fact that the axial interactions decrease as the distance between the double strand and the fiber axis increases, because of the increased length of the helical path taken by the double strand. The lateral interactions between HbS molecules in a double strand change relatively little between the crystal and possible helical double strands. If the twist of the fiber or the distance between the double strand and the fiber axis is too great, the lateral interaction is broken by intermolecular contacts in the region around the beta6 valine. Consequently, the geometry of the beta6 valine interaction and the residues surrounding it severely restricts the possible helical twist, radius, and handedness of helical aggregates constructed from the double strands. The limitations defined by this analysis establish the structural basis for the right-handed twist observed in HbS fibers and demonstrates that for a subunit twist of 8 degrees, the fiber diameter cannot be more than approximately 300 A, consistent with electron microscope observations. The energy of interaction among HbS molecules in a double strand is very slowly varying with helical pitch, explaining the variable pitch observed in HbS fibers. The analysis results in a model for the HbS double strand, for use in the analysis of interactions between double strands and for refinement of models of the HbS fibers against x-ray diffraction data.     

Fiber depolymerization

Depolymerization is, by definition, a crucial process in the reversible assembly of various biopolymers. It may also be an important factor in the pathology of sickle cell disease. If sickle hemoglobin fibers fail to depolymerize fully during passage through the lungs then they will reintroduce aggregates into the systemic circulation and eliminate or shorten the protective delay (nucleation) time for the subsequent growth of fibers. We study how depolymerization depends on the rates of end- and side-depolymerization, k(end) and k(side), which are, respectively, the rates at which fiber length is lost at each end and the rate at which new breaks appear per unit fiber length. We present both an analytic mean field theory and supporting simulations showing that the characteristic fiber depolymerization time tau= square root 1/k(end)k(side) depends on both rates, but not on the fiber length L, in a large intermediate regime 1 < k(side)L(2)/k(end) < (L/d)(2), with d the fiber diameter. We present new experimental data which confirms that both mechanisms are important and shows how the rate of side depolymerization depends strongly on the concentration of CO, acting as a proxy for oxygen. Our theory remains rather general and could be applied to the depolymerization of an entire class of linear aggregates, not just sickle hemoglobin fibers.