An α-chain modification rivals the effect of fetal hemoglobin in retarding the rate of sickle cell fiber formation.

Adults with sickle cell disease bear a mutation in the ß-globin gene, leading to the expression of sickle hemoglobin (HbS; α2ßS2). Adults also possess the gene for γ-globin, which is a component of fetal hemoglobin (HbF, α2γ2); however, γ-chain expression normally ceases after birth. As HbF does not form the fibers that cause the disease, pharmacological and gene-modifying interventions have attempted to either reactivate expression of the γ chain or introduce a gene encoding a modified ß chain having γ-like character. Here, we show that a single-site modification on the α chain, αPro114Arg, retards fiber formation as effectively as HbF. Because this addition to the repertoire of anti-sickling approaches acts independently of other modifications, it could be coupled with other therapies to significantly enhance their effectiveness.

Voxelotor does not inhibit sickle hemoglobin fiber formation upon complete deoxygenation.

The drug voxelotor (commercially known as Oxbryta) has been approved by the US Food and Drug Administration for the treatment of sickle cell disease. It is known to reduce disease-causing sickling by inhibiting the transformation of the non-polymerizing, high-oxygen-affinity R quaternary structure of sickle hemoglobin into its polymerizing, low-affinity T quaternary structure. It has not been established whether the binding of the drug has anti-sickling effects beyond restricting the change of quaternary structure. By using a laser photolysis method that employs microscope optics, we have determined that fully deoxygenated sickle hemoglobin will assume the T structure. We show that the nucleation rates essential to generate the sickle fibers are not significantly affected by voxelotor. The method employed here should be useful for determining the mechanism of sickling inhibition for proposed drugs.

The Sickle-Cell Fiber Revisited.

Sickle cell disease is the consequence of a single point mutation on the surface of the ß chains of the hemoglobin molecule leading to the formation of rigid polymers that disrupt circulation. It has long been established that the polymers are comprised of seven pairs of double strands that are twisted replicas of the double strands found in crystals. Here, we review several newer developments that elaborate on that simple model and provide deeper insights into the process.

The flow of sickle blood in glass capillaries: Fundamentals and potential applications.

We have characterized the imbibed horizontal flow of sickle blood into 100-µm-diameter glass capillaries. We find that blood containing sickled cells typically traverses the capillaries between three and four times as slowly as oxygenated cells from the same patient for all genotypes tested, including SS, AS, SC and Sß+ thalassemia blood. Blood from SS patients treated with hydroxyurea has a viscosity intermediate between the SS and AA values. Blood containing cells that are not rigidified, such as normal red cells or oxygenated sickle cells, follows a simple Lucas-Washburn flow throughout the length of the 3-cm capillary. By fitting the flexible-cell data to the Lucas-Washburn model, a viscosity can be derived that is in good agreement with previous measurements over a range of volume fractions and is obtained using an apparatus that is far more complex. Deoxygenation sickles and thus rigidifies the cells, and their flow begins as Lucas-Washburn, albeit with higher viscosity than flexible cells. However, the flow further slows as a dense mass of cells forms behind the meniscus and increases in length as flow progresses. By assuming that the dense mass of cells exerts a frictional force proportional to its length, we derive an equation that is formally equivalent to vertical imbibition, even though the flow is horizontal, and this equation reproduces the observed behavior well. We present a simple theory using activity coefficients that accounts for this viscosity and its variation without adjustable parameters. In the course of control experiments, we have found that deoxygenation increases the flexibility of normal human red cells, an observation only recently published for mouse cells and previously unreported for human erythrocytes. Together, these studies form the foundation for an inexpensive and rapid point-of-care device to diagnose sickle cell disease or to determine blood viscosity in resource-challenged settings.

Water, Ions, and Hemoglobin: Effects on Allostery and Polymerization.

Proteins that function in aqueous solution can be perturbed by the solvent. Here we present experimental studies on two such interactions in the hemoglobin molecule. (1) Hemoglobin's oxygen binding is altered by introduction of crowding species or osmoticants, such as sucrose, through the linked binding of ions such as Cl or CO2, but not otherwise. This rules out a significant role of buried surface in the allosteric energetics. (2) Sickle hemoglobin (HbS) polymerizes more readily in high concentrations of phosphate buffer. Such polymerization is analyzed quantitatively here for the first time in terms of the double nucleation mechanism. The changes in solubility are found to account for the increase in monomer addition rates and nucleation rates without requiring additional parameter adjustments. In the analysis, we also show how the analytical formulation of HbS nucleation may be adapted to include water that occupies the interstices between the assembled molecules. While such a "correction" has been applied to the equilibrium process, it has not previously been applied to the nucleation process.

Solid nuclei and liquid droplets: A parallel treatment for 3 phase systems.

For solid phase self assembly into crystals or large diameter polymers, the presence of a liquid-liquid demixing transition has been known to have an accelerating effect on the nucleation process. We present a novel approach to the description of accelerated nucleation in which the formation of solid phase aggregates and liquid-like aggregates compete as parallel pathways to formation of dense phases. The central idea is that the small aggregates that would ultimately form the liquid phase are sufficiently labile to sample the configurations that would form the solid, so that the growing cluster begins as a liquid, and switches into growth as a solid when the aggregates have equal free energies. This can accelerate the reaction even when the liquid-demixed state is thermodynamically unfavorable. The rate-limiting barrier is therefore the energy at which there is a transition between liquid and solid, and the effective nucleus size is then concentration independent, even though for both nucleated demixing and nucleated crystallization, the nucleus size does depend on concentration. These ideas can be expressed in a chemical potential formalism that has been successfully used in nucleation of sickle hemoglobin, but not to our knowledge previously employed in describing LLD processes. The method is illustrated by considering existing data on Lysozyme.

Targeting HbS Polymerization.

The mutation of ß6 from glu to val in hemoglobin is responsible for the polymer formation that leads to vaso-occlusion, and a range of severe consequences in sickle cell disease. The treatment of the disease can be addressed in many ways, but the prevention of polymer formation is one of the most fundamental approaches one can take. Such prevention includes affecting the polymer structure, or dilution of the fraction of polymerizable hemoglobin. The latter approach includes (1) induction of HbF, which does not itself, nor in hybrid form, join sickle polymers, or (2) restricting the allosteric change in hemoglobin that occurs in oxygen delivery, and which is required for polymer formation. These approaches will be critically reviewed, as well as the most recent developments that show the benefits of simply swelling the volume of the red cell.

Note: Professional grade microfluidics fabricated simply.

Microfluidics has found increasingly wide usage in the research and teaching laboratory, but setting up a facility for its production has typically required either significant capital expense or sacrifice of quality. We present an approach to produce devices, without a clean room, using LEDs and spin-coaters, and plasma bonded using a commercial microwave oven. Submicron features can be readily reproduced with good fidelity.

Sickle cell disease: Its molecular mechanism and the one drug that treats it.

Sickle cell disease is probably the first known assembly disease, and its mechanism has been extensively studied. It arises because of the expression of a mutant hemoglobin that can polymerize, and which does so by a double nucleation mechanism that is now seen to operate in other diseases. The polymers so formed lead to circulatory obstruction in the microcirculation. The accuracy of the description that has been developed is sufficient to describe precisely the impact of molecules that cannot join polymers but that still crowd the solution, including fetal hemoglobin. The one approved drug, hydroxyurea, is thought to achieve its benefit by enhancing the production of fetal hemoglobin, but the effects of the drug on polymerization exceed what the added fetal hemoglobin can accomplish. While some possible answers to this mystery are suggested, no mechanism has been conclusively established for the remarkably efficacy of the one drug available to treat this disease.

Calibrating Sickle Cell Disease.

Sickle cell disease is fundamentally a kinetic disorder, in which cells containing the mutated hemoglobin (hemoglobin S; HbS) will cause occlusion if they sickle in the microvasculature, but have minimal (or no) consequences if they sickle in the venous return. Physiologically, sickling always occurs when some ligands are present; nonetheless, the kinetics in the presence of ligands are virtually unstudied. Sickling arises from nucleation-controlled polymer formation, triggered when the HbS loses ligands (e.g., oxygen). Thus, understanding how nucleation responds to the presence of oxygen is the key to understanding how sickling proceeds in a physiological context. We have measured the rate of nucleus formation in HbS partially liganded with NO or CO, which we find have equivalent effects in reducing the nucleation rates. We find that hemoglobin must be in the T (tense) quaternary structure for nucleation, but the presence of ligands inhibits nucleus formation even when the correct quaternary structure is present. From these results, we can predict the fraction of cells that will sickle at any given partial ligand saturations. The ability to make such predictions may prove especially useful in designing future therapies, particularly those where the oxygen affinity is perturbed.

Universality of supersaturation in protein-fiber formation.

The thermodynamics and kinetics of the aggregation of sickle-cell hemoglobin into fibers have been studied in great detail under a wide range of solution conditions. The stability of the fiber is measured by the solubility; the kinetics is characterized by a delay before the appearance of fibers. A review of data in the literature shows that there is no correlation of the delay time with fiber stability and only a weak correlation with the initial protein concentration. There is, however, a striking collapse of all the data onto a single universal curve when the delay time is plotted versus the supersaturation, which is the ratio of the initial protein concentration to the solubility, expressed as activities. Collapse onto the same universal curve is also obtained when using delay times calculated from the double-nucleation theoretical model.

The delay time in sickle cell disease after 40 years: A paradigm assessed.

Sickle hemoglobin polymerization commences with a striking latency period, called a "delay time" followed by abrupt polymer formation. The delay time is exceedingly concentration dependent. This discovery (40 years ago) led to the "kinetic hypothesis," that is, that the pathophysiology was related to the relationship between the delay time and the capillary transit. The delay time is well described by a double-nucleation mechanism of polymer formation. In macroscopic volumes, the delay time is highly reproducible, but in small volumes such as erythrocytes, under certain conditions, the intrinsic delay time can be augmented by a stochastic delay owing to random waiting times for the first nucleus to form. This lengthens the average delay and adds further protection from vaso-occlusion. When oxygen removal is not sudden, the growth of polymers after the delay time is limited by the rate of oxygen removal, further lengthening the time before occlusion may occur. This is important if some polymers have remained in the cell after pulmonary transit as their presence otherwise would obliterate any delay. The difficulty of deforming a cell once polymerized rationalizes the "two-step" model of vaso-occlusion in which a postcapillary adhesion event is followed by a sickling logjam. The delay time that is required is therefore generalized to be the delay time for an erythrocyte to move beyond regions in the venuoles where adherent cells have reduced the available lumen. The measurements of delay times correlate well with the severity of sickling syndromes. They also correlate with the improvements owing to the administration of hydroxyurea.

Assembly of Aß proceeds via monomeric nuclei.

Aggregation of amyloid-ß (Aß) peptides is fundamental to Alzheimer's disease. It has now been shown that nucleated proliferation of Aß fibrils utilizes a secondary mechanism with existing fibrils catalyzing the formation of new ones. Here it is shown that the data for Aß40 and Aß42 require that the nuclei be monomeric; that is, an initial, unfavorable conformational change is rate limiting for the processes that appear to be nucleation. Following the conformational change, the assembly process is "downhill" despite clear lag times and significant concentration dependence. The similarity to polyglutamine nucleation suggests that monomeric nuclei may be widespread in amyloid formation.

Dissecting the energies that stabilize sickle hemoglobin polymers.

Sickle hemoglobin forms long, multistranded polymers that account for the pathophysiology of the disease. The molecules in these polymers make significant contacts along the polymer axis (i.e., axial contacts) as well as making diagonally directed contacts (i.e., lateral contacts). The axial contacts do not engage the mutant ß6 Val and its nonmutant receptor region on an adjacent molecule, in contrast to the lateral contacts which do involve the mutation site. We have studied the association process by elastic light scattering measurements as a function of temperature, concentration, and primary and quaternary structure, employing an instrument of our own construction. Even well below the solubility for polymer formation, we find a difference between the association behavior of deoxy sickle hemoglobin molecules (HbS), which can polymerize at higher concentration, in comparison to COHbS, COHbA, or deoxygenated Hemoglobin A (HbA), none of which have the capacity to form polymers. The nonpolymerizable species are all quite similar to one another, and show much less association than deoxy HbS. We conclude that axial contacts are significantly weaker than the lateral ones. All the associations are entropically favored, and enthalpically disfavored, typical of hydrophobic interactions. For nonpolymerizable Hemoglobin, ΔH(o) was 35 ± 4 kcal/mol, and ΔS was 102.7 ± 0.5 cal/(mol-K). For deoxyHbS, ΔH(o) was 19 ± 2 kcal/mol, and ΔS was 56.9 ± 0.5 cal/(mol-K). The results are quantitatively consistent with the thermodynamics of polymer assembly, suggesting that the dimer contacts and polymer contacts are very similar, and they explain a previously documented significant anisotropy between bending and torsional moduli. Unexpectedly, the results also imply that a substantial fraction of the hemoglobin has associated into dimeric species at physiological conditions.

Ratchets, red cells, and metastability.

Sickle cell disease is a genetic disorder in which a negatively charged glutamic acid is replaced by a hydrophobic valine on the surface of the hemoglobin molecule, leading to polymerization of the deoxygenated form, and resulting in microvascular obstruction. Because of the high volume occupancy under which polymerization occurs physiologically, this process has been an exemplar in the study of excluded volume effects on assembly. More recently, we have identified yet another type of crowding effect involving the obstruction of the ends at which the polymers grow as a consequence of the dense arrays in which these polymers form. This makes such solutions metastable, and leads to Brownian ratchet behavior in which pressure is exerted outward when the gel occupies a finite volume, as in an emulsion or red cell. Such behavior is capable of holding sickled cells in place in the microcirculation against weak pressure differentials (hundreds of Pa), but not against the typical pressures found in vivo.

The physical foundation of vasoocclusion in sickle cell disease.

The pathology of sickle cell disease arises from the occlusion of small blood vessels because of polymerization of the sickle hemoglobin within the red cells. We present measurements using a microfluidic method we have developed to determine the pressure required to eject individual red cells from a capillary-sized channel after the cell has sickled. We find that the maximum pressure is only ∼100 Pa, much smaller than typically found in the microcirculation. This explains why experiments using animal models have not observed occlusion beginning in capillaries. The magnitude of the pressure and its dependence on intracellular concentration are both well described as consequences of sickle hemoglobin polymerization acting as a Brownian ratchet. Given the recently determined stiffness of sickle hemoglobin gels, the observed obstruction seen in sickle cell disease as mediated by adherent cells can now be rationalized, and surprisingly suggests a window of maximum vulnerability during circulation of sickle cells.