Macrofiber structure and the dynamics of sickle cell hemoglobin crystallization

Fibers of deoxyhemoglobin S undergo spontaneous crystallization by a mechanism involving a variety of intermediate structures. These intermediate structures, in common with the fiber and crystal, consist of Wishner-Love double strands of hemoglobin S molecules arranged in different configurations. The structure of one of the key intermediates linking the fiber and crystal, called a macrofiber, has been studied by a variety of analytical procedures. The results of the analysis indicate that the intermediates involved in the fiber to crystal transition have many common structural features. Fourier analysis of electron micrographs of macrofibers confirms that they are composed of Wishner-Love double strands of hemoglobin molecules. Electron micrographs of macrofiber cross-sections reveal that the arrangement of the double strands in macrofibers resembles that seen in micrographs of the a axis projection of the crystal. This orientation provides an end-on view of the double strands which appear as paired dumb-bell-like masses. The structural detail becomes progressively less distinct towards the edge of the particle due to twisting of the double strands about the particle axis. Serial sections of macrofibers confirm that these particles do indeed rotate about their axes. The twist of the particle is right handed and its average pitch is 10,000 Å. The effect of rotation on the appearance of macrofiber cross-sections 300 to 400 Å thick can be simulated by a 15 ° rotation of an a axis crystal projection. The relative polarity of the double strands in macrofibers and crystals can be determined easily by direct inspection of the micrographs. In both macrofibers and crystals they are in an anti-parallel array.On the basis of these observations we conclude that crystallization of macrofibers involves untwisting and alignment of the double strands.     

Determination of the transition-state entropy for aggregation suggests how the growth of sickle cell hemoglobin polymers can be slowed

Sickle cell anemia is associated with the mutant hemoglobin HbS, which forms polymers in red blood cells of patients. The growth rate of the polymers is several micrometers per second, ensuring that a polymer fiber reaches the walls of an erythrocyte (which has a 7-μm diameter) within a few seconds after its nucleation. To understand the factors that determine this unusually fast rate, we analyze data on the growth rate of the polymer fibers. We show that the fiber growth follows a first-order Kramers-type kinetics model. The entropy of the transition state for incorporation into a fiber is 95 J mol^− 1 K^− 1, very close to the known entropy of polymerization. This agrees with a recent theoretical estimate for the hydrophobic interaction and suggests that the gain of entropy in the transition state is due to the release of the last layer of water molecules structured around contact sites on the surface of the HbS molecules. As a result of this entropy gain, the free-energy barrier for incorporation of HbS molecules into a fiber is negligible and fiber growth is unprecedentedly fast. This finding suggests that fiber growth can be slowed by components of the red cell cytosol, native or intentionally introduced, which restructure the hydration layer around the HbS molecules and thus lower the transition state entropy for incorporation of an incoming molecule into the growing fiber.     

Ligand binding and the gelation of sickle cell hemoglobin.

The solubility equilibrium between monomer and polymer which has been shown to exist in deoxyhemoglobin S solutions is examined in solutions partially saturated with carbon monoxide. The total solubility is found to increase monotonically with increasing fractional saturation. At low fractional saturations the increase is nearly linear, amounting roughly to an increase of 0.01 g cm^?3 in solubility for each 10% increase in fractional saturation. Linear dichroism measurements on the spontaneously aligned polymer phase are used to examine the composition of the polymer as a function of the fractional saturation of the corresponding solution phase. The dichroism experiments show that the polymer phase contains less than 5% of CO-liganded hemes even at supernatant fractional saturations in excess of 70%. The polymer selects against totally liganded hemoglobin molecules by a minimum factor of 65 and against singly liganded molecules by a factor of at least 2.5. Consequently, polymerized hemoglobin S has a ligand affinity which is significantly lower than that of monomeric hemoglobin S in the deoxy quaternary structure.The kinetics of the polymerization reaction in the presence of CO are similar to those observed in pure deoxyhemoglobin S solutions. The polymerization is preceded by a pronounced delay, the duration of which, t_d, is proportional roughly to the 30th power of the solubility. At low fractional saturations, this amounts to a tenfold increase in t_d for each 10% increase in the fractional saturation.These results show that the polymerization reaction is nearly specific for deoxyhemoglobin. Models for the dependence of the solubility and the polymer saturation on ligand partial pressure demonstrate the importance of solution phase non-ideality in determining the solubility of mixtures. The results require selection against partially liganded species which is significantly greater than is predicted by the two-state allosteric model. The data are compatible with either sequential or allosteric models in which the major polymerized component is the unliganded hemoglobin molecule.     

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.     

Mixed gelation theory. Kinetics, equilibrium and gel incorporation in sickle hemoglobin mixtures.

This paper outlines a theoretical formalism for describing the gelling behavior of sickle cell hemoglobin in mixtures with other hemoglobin and non-hemoglobin proteins. Experimental applications are reported for hybridized and unhybridized mixtures of HbS (sickle hemoglobin), HbA (adult hemoglobin), HbF (fetal hemoglobin), and HbC Harlem. The theory is a general one based on a modification of the sol—gel phase equilibrium equation to take into account the varying tendencies of different hemoglobin species to promote gelation, and specific hemoglobin interactions are encoded in gelling coefficients which quantify gelling capability. Gelling coefficients for the hemoglobin species dealt with here are evaluated by measuring incorporation into the polymer phase in S-A, S-F, and S-C_H mixtures. Given this information, the theory is found to provide accurate prodictions for the equilibrium gelling behavior of the calibrating pairs themselves when they are hybridized or unhybridized, for gelation kinetics in diverse mixtures of these species taken two, three and four at a time, for the anomalous equilibrium and kinetic gelling behavior of A- C_H mixtures, and it also accounts for a variety of results previously published by others. Apparently, given the gelling coefficients for any mutant hemoglobin, one can compute gelling behavior (equilibrium, kinetics, incorporation, etc.) in any specified mixture with any other known hemoglobin(s). The gelling coefficients for any mutant hemoglobin depend upon, and therefore provide information about, gel interactions at the mutant site. From the gelling coefficients one can also obtain the change in free energy of interaction in the gel due to the altered residue. Experimental approaches are described which allow an analysis for the gelling coefficients of any mutant hemoglobin to be performed in a few hours.     

X-ray diffraction studies of 14-filament models of deoxygenated sickle cell hemoglobin fibers. Models based on electron micrograph reconstructions

The transforms of a large number of models of deoxygenated sickle hemoglobin fibers, related to that derived from image reconstruction of electron micrographs, have been calculated and compared with X-ray diffraction data of 15 A resolution. The model of the fiber, determined from the reconstructed image, is a helix consisting of 14 filaments that associate in a specific mode to form seven pairs, or protofilaments. Pairs were identified through the pattern of filament loss in partially disassembled fibers and by the separation between molecules, in adjacent filaments, of half a molecular diameter, along the fiber axis. An alternative mode of filament association can be derived also from the surface lattice of the reconstruction, which meets these criteria for the pairing of molecular filaments. Both pairing modes have been used in the search for structures whose transforms show the best agreement with the diffraction data. Models were generated by the systematic translation of six protofilaments, taken in symmetry related pairs, in steps of 3.5 A along the fiber axis relative to a fixed central protofilament. Each translation of a protofilament corresponds to a different fiber model, whose transform was compared with observed data. In all, over 11,000 transforms were calculated. Of all the models considered, three have been found whose residuals are minimal. At 30 A resolution, similar to that of electron micrographs, the model derived from image reconstruction and the three found through our search procedure are indistinguishable. At 15 A, however, the transforms of these models show better agreement with the observed data than the transform of the reconstructed image. Comparison of residuals shows that the model derived from the reconstructed image can be rejected with 99.5% probability relative to the model, with the same pairing scheme, found by our search procedures. The two other models, derived from the alternative pairing scheme, are also more credible than the reconstructed image, but at a lower confidence level. Each of our three models is equally acceptable. Their existence may reflect structural polymorphism of the fiber.     

RNA aptamer therapy for vaso-occlusion in sickle cell disease

Patients with sickle cell disease (SCD) often suffer painful vaso-occlusive episodes caused in part by the adhesion of sickle erythrocytes (SS-RBC) to the vascular endothelium. To investigate inhibition of SS-RBC adhesion as a possible treatment for vaso-occlusion, 2 adhesion molecules, α(v)β(3) and P-selectin, were targeted by high-affinity RNA aptamers. An in vitro flow chamber assay was used to test the antiadhesion activity of α(v)β(3) aptamer clone 17.16. Human SS-RBC were passed across a confluent monolayer of thrombin-stimulated human umbilical vein endothelial cells (HUVEC) at a constant rate. α(v)β(3) aptamer reduced SS-RBC adhesion to activated endothelial cells to the level seen with untreated HUVEC. An aptamer reactive with complement component 8 was used as a negative control and exerted no inhibition, confirming the specificity of α(v)β(3) aptamer (P=0.04). At 2?dyn/cm(2) shear stress, 30?nM α(v)β(3) aptamer showed maximal effect in decreasing SS-RBC adhesion to HUVEC. The antiadhesive activity of the P-selectin aptamer clone PF377 was also tested using HUVEC pretreated with IL-13 to upregulate expression of P-selectin as seen in activated endothelial cells. At 1?dyn/cm(2) shear stress, 60?nM of P-selectin aptamer had antiadhesion activity similar to heparin, a known inhibitor of SS-RBC adhesion to P-selectin. A negative control did not prevent adhesion (P=0.05). These data show the potential utility of aptamers to block endothelial adhesion molecules to prevent or treat vaso-occlusion in SCD.     

Frequency and origin of haplotypes associated with the beta-globin gene cluster in individuals with trait and sickle cell anemia in the Atlantic and Pacific coastal regions of Colombia

Sickle cell anemia is a genetic disease with high prevalence in people of African descent. There are five typical haplotypes associated with this disease and the haplotypes associated with the beta-globin gene cluster have been used to establish the origin of African-descendant people in America. In this work, we determined the frequency and the origin of haplotypes associated with hemoglobin S in a sample of individuals with sickle cell anemia (HbSS) and sickle cell hemoglobin trait (HbAS) in coastal regions of Colombia. Blood samples from 71 HbAS and 79 HbSS individuals were obtained. Haplotypes were determined based on the presence of variable restriction sites within the β-globin gene cluster. On the Pacific coast of Colombia the most frequent haplotype was Benin, while on the Atlantic coast Bantu was marginally higher than Benin. Eight atypical haplotypes were observed on both coasts, being more diverse in the Atlantic than in the Pacific region. These results suggest a differential settlement of the coasts, dependent on where slaves were brought from, either from the Gulf of Guinea or from Angola, where the haplotype distributions are similar. Atypical haplotypes probably originated from point mutations that lost or gained a restriction site and/or by recombination events.     

Gelation of sickle cell hemoglobin in mixtures with normal adult and fetal hemoglobins.

We report the results of thermodynamic and kinetic studies on the gelation of mixtures of sickle cell (S) deoxyhemoglobin with normal human adult (A) and fetal (F) deoxyhemoglobins. The delay time of thermally induced gelation was monitored by the increase in turbidity. At the completion of gelation the solubility was determined by sedimenting the polymers and measuring the supernatant concentration spectrophotometrically. Addition of hemoglobins A or F, at mole fractions from 0 to 0.6, resulted in large increases in both the solubility and the delay time. For a 50:50 mixture of deoxyhemoglobin F with deoxyhemoglobin S, the solubility increased by a factor of 1.8 and the delay time by a factor of 10⁷ relative to pure deoxyhemoglobin S at the same total concentration, while for a 50:50 mixture of deoxyhemoglobins A and S the solubility increased by a factor of 1.4 and the delay time by a factor of 10⁴. The relative delay times were independent of both temperature and total hemoglobin concentration. The data have been analyzed according to theoretical models which treat the effects of temperature, concentration, non-ideality and solution composition on the thermodynamics and kinetics of gelation. The increased solubility in mixtures with deoxyhemoglobin F is fully explained by a model in which only deoxyhemoglobin S molecules polymerize. The effect of fetal hemoglobin (α₂γ₂) and hybrid α₂γβ^S molecules is to increase the solution non-ideality through the contribution of their excluded volume. The smaller increase in the solubility observed in comparable mixtures with deoxyhemoglobin A requires that the hybrid α₂β^Aβ^S molecules copolymerize with the deoxyhemoglobin S. The kinetic results for the mixtures can be quantitatively accounted for using a nucleation model in which the equilibrium properties of the polymer are used to describe the critical nucleus. The very large increases in delay time observed for the $\text{S}\text{F}$ mixtures can be explained by assuming that only α₂β₂^S molecules participate in the formation of a nucleus containing about 25 monomers. As in the thermodynamic analysis, the smaller effect of adding deoxyhemoglobin A can be attributed to the contribution of the hybrid molecules in forming the critical nucleus. Thus the difference between the polymerization properties of mixtures of deoxyhemoglobin S with deoxyhemoglobins A and F can be attributed solely to the copolymerization of the α₂β^Aβ^S hybrid molecule and the absence of any significant copolymerization of the α₂γβ^S hybrid.     

The role of pH on instability and aggregation of sickle hemoglobin solutions

Understanding the physical basis of protein aggregation covers strong physical and biomedical interests. Sickle hemoglobin (HbS) is a point-mutant form of normal human adult hemoglobin (HbA). It is responsible for the first identified "molecular disease," as its propensity to aggregation is responsible for sickle cell disease. At moderately higher than physiological pH value, this propensity is inhibited: The rate of aggregate nucleation becomes exceedingly small and solubility after polymerization increases. These order-of-magnitude effects on polymer nucleation rates and concurrent relatively modest changes of solubility after polymerization are here shown to be related to both pH-induced changes of location and shape of the liquid-liquid demixing (LLD) region. This allows establishment of a self-consistent contact between the thermodynamics of the solution as such (i.e., the LLD region), the kinetics of fiber nucleation, the theory of percolation, and the thermodynamics of gelation. The observed pH-induced changes are largely attributable to strong perturbations of hydrophobic hydration configurations and related free energy by electric charges. Similar mechanisms of effective control of aggregate nucleation rates by means of agents such as cosolutes, pH, salts, and additives, shifting the LLD and associated regions of anomalous fluctuations, promise to be relevant to the whole field of protein aggregation pathologies.     

Gene therapy for sickle cell disease: An update

Sickle cell disease (SCD) is one of the most common life-threatening monogenic diseases affecting millions of people worldwide. Allogenic hematopietic stem cell transplantation is the only known cure for the disease with high success rates, but the limited availability of matched sibling donors and the high risk of transplantation-related side effects force the scientific community to envision additional therapies. Ex vivo gene therapy through globin gene addition has been investigated extensively and is currently being tested in clinical trials that have begun reporting encouraging data. Recent improvements in our understanding of the molecular pathways controlling mammalian erythropoiesis and globin switching offer new and exciting therapeutic options. Rapid and substantial advances in genome engineering tools, particularly CRISPR/Cas9, have raised the possibility of genetic correction in induced pluripotent stem cells as well as patient-derived hematopoietic stem and progenitor cells. However, these techniques are still in their infancy, and safety/efficacy issues remain that must be addressed before translating these promising techniques into clinical practice.     

Mechanisms of homogeneous nucleation of polymers of sickle cell anemia hemoglobin in deoxy state

The primary pathogenic event of sickle cell anemia is the polymerization of the mutant hemoglobin (Hb) S within the red blood cells, occurring when HbS is in deoxy state in the venous circulation. Polymerization is known to start with nucleation of individual polymer fibers, followed by growth and branching via secondary nucleation, yet the mechanisms of nucleation of the primary fibers have never been subjected to dedicated tests. We implement a technique for direct determination of rates and induction times of primary nucleation of HbS fibers, based on detection of emerging HbS polymers using optical differential interference contrast microscopy after laser photolysis of CO-HbS. We show that: (i). nucleation throughout these determinations occurs homogeneously and not on foreign substrates; (ii). individual nucleation events are independent of each other; (iii). the nucleation rates are of the order of 10(6)-10(8)cm(-3)s(-1); (iv). nucleation induction times agree with an a priori prediction based on Zeldovich's theory; (v). in the probed parameter space, the nucleus contains 11 or 12 molecules. The nucleation rate values are comparable to those leading to erythrocyte sickling in vivo and suggest that the mechanisms deduced from in vitro experiments might provide physiologically relevant insights. While the statistics and dynamics of nucleation suggest mechanisms akin to those for small-molecule and protein crystals, the nucleation rate values are nine to ten orders of magnitude higher than those known for protein crystals. These high values cannot be rationalized within the current understanding of the nucleation processes.     

Responses of normal and sickle cell hemoglobin to S-nitroscysteine: implications for therapeutic applications of NO in treatment of sickle cell disease

Factors which govern transnitrosation reactions between hemoglobin (Hb) and low molecular weight thiols may define the extent to which S-nitrosated Hb (SNO-Hb) plays a role in NO in the control of blood pressure and other NO-dependent reactions. We show that exposure to S-nitrosylated cysteine (CysNO) produces equivalent levels of SNO-Hb for Hb A(0) and sickle cell Hb (Hb S), although these proteins differ significantly in the electron affinity of their heme groups as measured by their anaerobic redox potentials. Dolphin Hb, a cooperative Hb with a redox potential like that of Hb S, produces less SNO-Hb, indicating that steric considerations outweigh effects of altered electron affinity at the active-site heme groups in control of SNO-Hb formation. Examination of oxygen binding at 5-20 mM heme concentrations revealed increases due to S-nitrosation in the apparent oxygen affinity of both Hb A(0) and Hb S, similar to increases seen at lower heme concentrations. As observed at lower heme levels, deoxygenation is not sufficient to trigger release of NO from SNO-Hb. A sharp increase in apparent oxygen affinity occurs for unmodified Hb S at concentrations above 12.5 mM, its minimum gelling concentration. This affinity increase still occurs in 30 and 60% S-nitrosated samples, but at higher heme concentration. This oxygen binding behavior is accompanied by decreased gel formation of the deoxygenated protein. S-nitrosation is thus shown to have an effect similar to that reported for other SH-group modifications of Hb S, in which R-state stabilization opposes Hb S aggregation.     

The role of fibrocytes in sickle cell lung disease

Background

Interstitial lung disease is a frequent complication in sickle cell disease and is characterized by vascular remodeling and interstitial fibrosis. Bone marrow-derived fibrocytes have been shown to contribute to the pathogenesis of other interstitial lung diseases. The goal of this study was to define the contribution of fibrocytes to the pathogenesis of sickle cell lung disease.

Methodology/Principal Findings

Fibrocytes were quantified and characterized in subjects with sickle cell disease or healthy controls, and in a model of sickle cell disease, the NY1DD mouse. The role of the chemokine ligand CXCL12 in trafficking of fibrocytes and phenotype of lung disease was examined in the animal model. We found elevated concentration of activated fibrocytes in the peripheral blood of subjects with sickle cell disease, which increased further during vaso-occlusive crises. There was a similar elevations in the numbers and activation phenotype of fibrocytes in the bone marrow, blood, and lungs of the NY1DD mouse, both at baseline and under conditions of hypoxia/re-oxygenation. In both subjects with sickle cell disease and the mouse model, fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on most fibrocytes, and CCR2 and CCR7 expressed on a smaller subset of cells. Depletion of the CXCR4 ligand, CXCL12, in the mouse model resulted in a marked reduction of fibrocyte trafficking into the lungs, reduced lung collagen content and improved lung compliance and histology.

Conclusions

These data support the notion that activated fibrocytes play a significant role in the pathogenesis of sickle cell lung disease.     

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.     

Plasma annexin A5 and microparticle phosphatidylserine levels are elevated in sickle cell disease and increase further during painful crisis

Expression of phosphatidylserine (PS) on the membrane surface of red blood cells and circulating microparticles (MP) plays an important role in etiology of the hypercoagulable state of sickle cell disease (SCD), as well as in the reduced red cell life span and adhesive interactions between red cells and endothelium. Annexin A5, an intracellular protein abundantly present in endothelial cells and platelets, exhibits high affinity for PS and has been shown to inhibit several of these PS-mediated pathophysiological processes. We determined plasma annexin A5 levels and MP-associated procoagulant activity, a measure of MP-PS exposure, in 17 sickle cell patients (12 HbSS and 5 HbSC) in steady state and at presentation with a painful crisis. Twenty-five HbAA blood donors served as controls.Both annexin A5 and MP-PS were highest in HbSS patients (5.7 ng/mL, IQR 3.7-7.6 and 37.9 nM, IQR 31.9-69.8) as compared to HbSC patients (1.8 ng/mL, IQR 1.7-7.6 and 20.9 nM, IQR 10.9-29.6) and healthy controls (2.5 ng/mL, IQR 1.4-4.4 and 13.1 nM, IQR 9.5-18.5) (p = 0.01 and p < 0.001, respectively). At presentation with a painful crisis, annexin A5 and MP-PS had increased in 16 of 17 patients (p = 0.001 and p < 0.001, respectively). Most interestingly, in 7 HbSS patients the proportional increase in MP-PS exposure was higher than the proportional increase in plasma annexin A5 concentration, leading to lower annexin A5/MP-PS ratio of HbSS patients during crisis than HbAA controls (0.0027 (0.0017-0.0049) vs 0.0048 (0.0027-0.0085), p = 0.05). In conclusion, patients with SCD have elevated plasma levels of annexin A5- and PS-exposing MP. During crisis both levels increase, but in most HbSS patients MP-PS exposure increases more than annexin A5. Future studies must address a potential role of annexin A5 in modulating PS-related pathophysiological processes in SCD.