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The vaso-occlusive crisis (VOC) is a major complication of sickle cell disease (SCD); thus, strategies to ameliorate vaso-occlusive episodes are greatly needed. We evaluated the therapeutic benefits of quercetin in a SCD transgenic sickle mouse model. This disease model exhibited very mild disease pathophysiology in the steady state. The severity of the disease in the NY1DD mouse was amplified by subjecting mice to 18 h of hypoxia followed by 3 h of reoxygenation. Quercetin (200 mg/kg body weight) administered to hypoxia challenged NY1DD mice in a single intraperitoneal (i.p.) dose at the onset of reoxygenation completely ameliorated all hypoxia reoxygenation (H/R)-induced pathophysiology. Additionally, it ameliorated the mild intrinsic steady state pathophysiology. These results are comparable with those seen with semisynthetic supra plasma expanders. In control mice, C57BL/6J, hypoxia reoxygenation-induced vaso-occlusion was at significantly lower levels than in NY1DD mice, reflecting the role of sickle hemoglobin (HbS) in inducing vaso-occlusion; however, the therapeutic benefits from quercetin were significantly muted. We suggest that these findings represent a unique genotype of the NY1DD mice, i.e., the presence of high oxygen affinity red blood cells (RBCs) with chimeric HbS, composed of mouse α-chain and human β S -chain, as well as human α-chain and mouse β-chain (besides HbS). The anti-anemia therapeutic benefits from high oxygen affinity RBCs in these mice exert disease severity modifications that synergize with the therapeutic benefits of quercetin. Combining the therapeutic benefits of high oxygen affinity RBCs generated in situ by chemical or genetic manipulation with the therapeutic benefits of antiadhesive therapies is a novel approach to treat sickle cell patients with severe pathophysiology.

In sickle cell disease, the aberrant assembly of hemoglobin fibers induces changes in red blood cell morphology and stiffness, which leads to downstream symptoms of the disease. Therefore, understanding of this assembly process will be important for the treatment of sickle cell disease. By performing the highest spatiotemporal resolution measurements (55 nm at 1 Hz) of single sickle hemoglobin fiber assembly to date and combining them with a model that accounts for the multistranded structure of the fibers, we show that the rates of sickle hemoglobin addition and loss have been underestimated in the literature by at least an order of magnitude. These results reveal that the sickle hemoglobin self-assembly process is very rapid and inefficient (4% efficient versus 96% efficient based on previous analyses), where net growth is the small difference between over a million addition-loss events occurring every second.

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.

To understand the physical basis of the wide variety of shapes of deoxygenated red cells from patients with sickle cell anemia, we have measured the formation rate and volume distribution of the birefringent domains of hemoglobin S fibers. We find that the domain formation rate depends on the approximately 80th power of the protein concentration, compared to approximately 40th power for the concentration dependence of the reciprocal of the delay time that precedes fiber formation. These remarkably high concentration dependences, as well as the exponential distribution of domain volumes, can be explained by the previously proposed double nucleation model in which homogeneous nucleation of a single fiber triggers the formation of an entire domain via heterogeneous nucleation and growth. The enormous sensitivity of the domain formation rate to intracellular hemoglobin S concentration explains the variable cell morphology and why rapid polymerization results in cells that do not appear sickled at all.

We report on observations of "zippering" that occurs when two sickle hemoglobin fibers come together side by side. A transient Y-shaped object is formed which "zips " closed. We have been able to show how the strength of the interactions that drive this may be estimated by studying the frustrated structures sometimes formed between several fibers. Our measurements, when combined with mechanical constants determined by an analysis of bending fluctuations, allow us to make the first estimate of the magnitude of these interactions, of the order of 7kBT microm(-1). Hemoglobin volume fractions of tens of %, lead to significant depletion forces. We estimate the magnitude of both the depletion and Van der Waals forces between pairs of single fibers. We study how these are effected by the helical nature of the fibers and renormalised by bending fluctuations, calculations that could have wider applications beyond sickle hemoglobin fibers. Our theoretical analysis of single fibers is in encouraging, although not fully quantitative, agreement with our measurements. We conclude that the physics and rheology of the hemoglobin gel, as well as the pathology of sickle cell anemia itself, may be influenced by depletion interactions.

We examined the depolymerization of hemoglobin (Hb) S fibers in the presence of CO by using photolysis of COHbS to create and isolate individual fibers, then removing photolysis to induce depolymerization. Depolymerization occurs at two sites, fiber ends and fiber sides, with different kinetics and by different mechanisms. At low partial pressure of CO (pCO), end-depolymerization is dominant, proceeding at approximately 1 microm s(-1), whereas at high pCO fibers vanish very rapidly, in much less than one second, by side-depolymerization. Each kind of depolymerization could occur by a ligand-independent path, in which deoxyHb depolymerizes and then is prevented from returning to the polymer by liganding with CO, or by a ligand-dependent path in which CO binds to the polymer inducing dissociation of the newly liganded molecules from it. We find that ligand-independent depolymerization is the dominant path for end-depolymerization and ligand-dependent depolymerization dominates, at least at high pCO, for side-depolymerization. On the basis of our kinetic results and electron micrographs of depolymerizing fibers, we propose a model for side-depolymerization in which a hole is nucleated by cooperative loss of a few molecules from fiber sides, followed by rapid depolymerization from the newly created fiber ends abutting the hole. Potential significance of these results for the pathophysiology of sickle cell disease is discussed.

We show that in solutions of human hemoglobin (Hb)--oxy- and deoxy-Hb A or S--of near-physiological pH, ionic strength, and Hb concentration, liquid-liquid phase separation occurs reversibly and reproducibly at temperatures between 35 and 40 degrees C. In solutions of deoxy-HbS, we demonstrate that the dense liquid droplets facilitate the nucleation of HbS polymers, whose formation is the primary pathogenic event for sickle cell anemia. In view of recent results that shifts of the liquid-liquid separation phase boundary can be achieved by nontoxic additives at molar concentrations up to 30 times lower than the protein concentrations, these findings open new avenues for the inhibition of the HbS polymerization.

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., (Copyright 2001 Academic Press.)

Deoxy-sicklecell hemoglobin (HbS) polymerizes in 0.05 M phosphate buffer to form long helical fibers. The reaction typically occurs when the concentration of HbS is about 165 mg/ml. Polymerization produces a variety of polymorphic forms. The structure of the fibers can be probed by using site-directed mutants to examine the effect of altering the residues involved in intermolecular interactions. Polymerization can also be induced in the presence of 1.5 M phosphate buffer. Under these conditions polymerization occurs at much lower concentrations (ca. 5 mg/ml), which is advantageous when site-directed mutants are being used because only small quantities of the mutants are available. We have characterized the structure of HbS polymers formed in 1.5 M phosphate to determine how their structures are related to the polymers formed under more physiological conditions. Under both sets of conditions fibers are the first species to form. At pHs between 6.7 and 7.3 fibers initially form bundles and then crystals. At lower pHs fibers form macrofibers and then crystals. Fourier transforms of micrographs of the polymers formed in 1.5 M phosphate display the 32- and 64-A(-1) periodicity characteristic of fibers formed in 0.05 M phosphate buffer. The 64-A(-1) layer line is less prominent in Fourier transforms of negatively stained fibers formed in 1.5 M phosphate possibly because salt interferes with staining of the fibers. However, micrographs and Fourier transforms of frozen hydrated fibers formed in high and low phosphate display the same periodicities. Under both sets of reaction conditions HbS polymers form crystals with the same unit cell parameters as Wishner-Love crystals (a = 64 A, b = 185 A, c = 53 A). Some of the polymerization intermediates were examined in the frozen-hydrated state in order to determine whether their structures were significantly perturbed by negative staining. We have also carried out reconstructions of the frozen-hydrated fibers in high and low phosphate to compare their molecular coordinates. The helical projection of the reconstructions in low phosphate shows the expected 14-strand structure. In high phosphate the 14-strand fibers are also formed and their molecular coordinates are the same (within experimental error) as those of fibers formed in 0.05 M phosphate. In addition, the reconstructions of high-phosphate fibers reveal a new minor variant of fiber containing 10 strands. The polymerization products in 1.5 M phosphate buffer were generally indistinguishable from those formed in 0.05 M phosphate buffer. Micrographs of frozen hydrated specimens have facilitated the interpretation of previously published micrographs using negative staining., (Copyright 2000 Academic Press.)

The polymerization properties of the fully liganded fluoromet derivative of hemoglobin S (FmetHb S) were investigated by electron microscopy and absorption spectroscopy. Polymerization progress curves, as measured by increasing sample turbidity at 700 nm, exhibit a delay time (t(d)) consistent with the double nucleation mechanism. The pattern of fiber growth, as monitored by electron microscopy, is also indicative of a heterogeneous nucleation process, and dimensions of the fibers were found to be comparable to that of deoxyHb S. The polymerization rate constant (1/t(d)) depends exponentially on Hb S concentration, and the size of the homogeneous and heterogeneous nuclei also depend on FmetHb S concentration. As for deoxyHb S, higher concentrations of protein and phosphate favor fiber formation, while lower temperatures inhibit polymerization. Solubility experiments reveal, however, that eight times more FmetHb S is required for polymerization. The current studies further show that reaction order is independent of phosphate concentration if Hb S activity and not concentration is considered. The allosteric effector, inositol hexaphosphate (IHP), promotes fiber formation, and temperature-dependent reaggregation of FmetHb S suggests that IHP stabilizes pregelation aggregates. These studies show that FmetHb S resembles deoxyHb S in many of its polymerization properties; however, IHP-bound FmetHb S potentially provides a unique avenue for future studies of the early stages of Hb S polymerization and the effect of tertiary and quaternary protein structure on the polymerization process.

Although the mean corpuscular hemoglobin concentration (MCHC) plays a dominant role in the rheologic behavior of deoxygenated density-defined sickle red blood cells (SS RBCs), previous studies have not explored the relationship between the rate of deoxygenation and the bulk viscosity of SS RBCs at a given MCHC. In the present study, we have subjected density-defined SS classes (i.e., medium-density SS4 and dense SS5 discocytes) to varying deoxygenation rates. This approach has allowed us to minimize the effects of SS RBC heterogeneity and investigate the effect of deoxygenation rates at a given MCHC. The results show that the percentages of granular cells, classic sickle cells and holly leaf forms in deoxygenated samples are significantly influenced by the rate of deoxygenation and the MCHC of a given discocyte subpopulation. Increasing the deoxygenation rate using high K+ medium (pH 6.8), results in a greater percentage of granular cells in SS4 suspensions, accompanied by a pronounced increase in the bulk viscosity of these cells compared with gradually deoxygenated samples (mainly classic sickle cells and holly leaf forms). The effect of MCHC becomes apparent when SS5 dense cells are subjected to varying deoxygenation rates. At a given deoxygenation rate, SS5 dense discocytes show a greater increase in the percentage of granular cells than that observed for SS4 RBCs. Also, at a given deoxygenation rate, SS5 suspensions exhibit a higher viscosity than SS4 suspensions with fast deoxygenation resulting in maximal increase in viscosity. Although MCHC is the main determinant of SS RBC rheologic behavior, these studies demonstrate for the first time that at a given MCHC, the rate of deoxygenation (hence HbS polymerization rates) further modulates the rheologic behavior of SS RBCs. Thus, both MCHC and the deoxygenation rate may contribute to microcirculatory flow behavior of SS RBCs.

The intracellular polymerization of deoxyhemoglobin S (HbS) into helical fibers is the primary pathological event which gives rise to sickle cell disease. The structure of these fibers has previously been studied by electron microscopy of negatively stained specimens. We are extending these studies with unstained frozen-hydrated HbS fibers (cryo-EM), which afford better visualization of the internal details of the fiber structure than can be achieved by negative staining, but have lower signal-to-noise ratio images. The pitch of the HbS fiber structure varies locally along any given particle. Because rotation about the particle axis thus is partially decoupled from translation along the axis, the pitch and angular rotation of a fiber unit cell cannot be inferred by symmetry (as is the case with constant pitch helices). Image analysis procedures are presented which are capable of explicitly identifying the pitch and angular rotation of individual HbS fiber unit cells having low signal-to-noise ratios. Fiber images are divided into segments one unit cell long (63 A) which are analyzed in two steps. First each unit cell is aligned with constant pitch electron density reference models by cross-correlation. Correlation coefficients are then used to determine angular rotation and pitch. This procedure was tested, and found to be robust, using model images corrupted to simulate experimental problems normally encountered in the analysis of cryo-electron micrographs. The effects of limited resolution, low signal-to-noise ratio, scaling errors, and rotational and axial misalignment are described.

Deoxy-sickle hemoglobin (HbS) polymerizes in vivo into long helical fibers which fill the red cell and make it rigid. This impedes red cell passage through the capillaries and is responsible for the clinical manifestations of sickle cell disease. Images of individual and laterally associated HbS fibers were obtained by electron microscopy of frozen hydrated specimens. Each fiber possesses variable pitch, having from 6 degrees to 12 degrees rotation per unit cell. Laterally associated HbS fibers display systematic inter-fiber contacts in spite of their pitch variations, and exhibit better order than isolated fibers. This suggest that inter-fiber contacts can act to couple fibers mechanically and might therefore be a factor in rigidifying red cells in vivo. Fiber variability was attributed to local torsional variations with a standard deviation of 2.5 degrees, but which are weakly coupled over a length of 2.25 unit cells. Variable pitch produces structural changes of as large as 5 A azimuthally and 6 A axially in HbS fiber unit cells.

Sickle hemoglobin macrofibers consist of rows of antiparallel double strands twisted about the particle axis. Under appropriate conditions the outer two rows can dissociate from the particle. These structures retain the helical twist of the parent macrofiber and slowly dissociate to monomers over a period of several hours. Individual double strands or pairs of parallel double strands are never observed, suggesting that only the antiparallel pairing is energetically favorable.

We have characterized new aspects of macrofiber structure and assembly which provide a mechanism for macrofiber formation from fibers. After the formation of fibers, HbS forms macrofibers by the association of small, organized bundles of partially fused fibers. These macrofibers consist of double strands, packed into antiparallel rows, and are identical to double strands found in crystalline HbS, except that the double strands in macrofibers are axially displaced from their crystalline position and are twisted about the particle axis, whereas in crystals they are linear. In lateral views, electron micrographs of macrofibers show prominent sets of "rows." We use the number of these rows to designate a particular type of macrofiber. In this study we present micrographs of macrofibers with 3 to 11 rows visible in lateral views. Such particles contain from 20 to 200 double strands. The pitch of a macrofiber is coupled to the number of rows in a manner so that the angle between the molecules in the outermost double strand is always 1.8 degrees. This observation has led us to propose that the factor limiting the extent of lateral growth of macrofibers is distortions in bonding between the hemoglobin molecules in the outermost double strands. Similar considerations have provided an explanation of the factors that limit the lateral growth of fibers. Finally, we propose a simple mechanism for the formation of macrofibers from fibers. This mechanism postulates that integral numbers of fibers form specific types of macrofibers and has the virtue of conserving the polarity of the fibers.

Electron micrographs of deoxyhemoglobin S fiber cross sections provide an end-on view of the fiber whose appearance is sensitive to small changes in orientation. We have developed a procedure to exploit this sensitivity in order to determine the hand of these particles. In a sickle hemoglobin fiber the hemoglobin molecules form long pitch helical strands which twist about the particle axis with a pitch of about 3000 A. Tilting a 400-A-thick cross section by a few degrees aligns one of the long pitch helices so that it is nearly parallel to the direction of view. When a strand of hemoglobin molecules in a fiber is aligned in this manner it appears as a strongly contrasted bright spot. It is this spot, rather than the fiber axis, which appears to be the apparent center of rotation of the cross section. The direction of the displacement of the spot from the particle axis depends upon the particle hand and tilt direction. We have used this property to determine that sickle hemoglobin fibers are right-handed particles. This method may be applicable to other particles with long pitch helices as well.

The distribution of spectrin and band 3 in deoxygenated reversibly sickled cells was visualized by immunofluorescence and immunoelectron microscopy. Antibodies against band 3, the major lipid-associated transmembrane protein, labeled the entire cell body, including the entire length of the long protruding spicule, whereas antibodies against spectrin labeled only the cell body and the base region of the spicules. The results suggest that the formation of long spicules during sickling is associated with a continuous polymerization of hemoglobin S polymers, presumably through gaps in the spectrin-actin meshwork, and a subsequent uncoupling of the lipid bilayer from the submembrane skeleton.

Electron microscopy of sickle cell hemoglobin fibers fixed at different times during gelation shows an exponential distribution of fiber lengths, with many short fibers and few long ones. The distribution does not change significantly with time as polymerization progresses. If this distribution of lengths reflects kinetic mechanism of fiber assembly, it complements information from studies of the progress of average properties of the polymers and, as has been done for other rod-like polymerizing systems, permits testing of models for the mechanism of fiber assembly. In this case, the results are consistent with the double nucleation model of Ferrone et al. or with a related alternative model based on fiber breakage. However, other possible causes of this microheterogeneity exist, including: breakage due to solution shearing of the long, rod-like, fibers; the presence of residual nuclei; equilibrium relations governing polymerization; and breakage of solid-like but weak gels that develop early and adhere to the grid. The arguments against the first three of these possibilities suggest that they are not responsible. However, breakage of entanglements or cross-links in a solid-like and adherent gel is consistent with the distributions.

In the reconstruction of helical particles, it is normally assumed that translation along the length of a particle is coupled to rotation about its axis. This assumption is not valid for particles whose pitch varies along the particle length (e.g. actin, HbS fibers), and application of the usual algorithms results in significant errors in both the shape and coordinates of subunits in the reconstructed density map. We have developed an iterative procedure for reconstructing particles with variable pitch. The goal of this procedure is to obtain an accurate estimate of the local pitch of the particle which can then be incorporated into the reconstruction algorithm. This involves synthesis of trial model structures which have constant pitch. The local pitch is derived from a cross-correlation analysis between these trial models and the variable pitch particles. The constant pitch models are constructed using coordinates measured from the reconstructed density maps. Each iteration of the procedure provides an improved estimate of the pitch which is incorporated into the succeeding iteration. The fidelity of the reconstruction is determined from cross-correlation between the original micrograph and a variable pitch model. The iterations are continued until the cross-correlation coefficient between the variable pitch model and the micrograph of the particle is maximized. The implementation of the iterative procedure is described and its behavior is evaluated using model structures which incorporate variations in pitch similar to those actually occurring in sickle hemoglobin fibers. The results indicate that the iterative reconstruction procedure considerably reduces the errors associated with constant pitch reconstructions. These tests provide a basis for applying this procedure in the structural analysis of micrographs of helical particles which display variable pitch. Application to sickle hemoglobin fibers resulted in an improvement in the accuracy with which the hemoglobin S molecules can be located in the density maps.

The spin-labeled tryptophan was used as a structural probe of hemoglobin contact sites. The ESR spectral data indicated that the probe exhibits weak binding to hemoglobin with a dissociation constant of 3.2.10(-5) and 4.0 mol bound per hemoglobin tetramer. The spectrum suggested that the bound tryptophan was 'partially immobilized' with a correlation time reflecting the environment of the tryptophan binding site of 8.2 ns. The topology of the contact sites was investigated by using dual spin-label methodology in which spin-labeled tryptophan and (2H,15N) substituted and deuterated maleimide spin label [2H-15N]MSL covalently-bound to Cys-beta 93 residue were used. The ESR spectral data suggested that the tryptophan binding sites were located within 8-10 A of the nitroxide free radical of spin-labeled hemoglobin. The environment of the contact sites is discussed.