Frank Anthony Ferrone

The understanding of how molecular motors work in living systems has been immeasurably advanced by the use of focused light beams to manipulate nano-scale objects in a tool known as optical tweezers. Steven M. Block has been a leader in developing these tools, developing methods of observing molecular motions at the nanometer scale, and measuring piconewton forces in the process. His work on understanding kinesin as well as RNA polymerase revealed fascinating behavior that generated profound insight into how these motors behave, and how other molecular motors might also carry out their tasks.

Sickle hemoglobin differs from normal adult hemoglobin by a single point mutation in two of its four subunits, and this renders it prone to self-assembly. This first-order phase transition generates stiff, 14-stranded fibers that are generated in dense arrays via a double-nucleation mechanism. The strands are only roughly equivalent, and they interact adhesively as well as obstructively to generate a solid in a process commonly referred to as gelation. This review will recount the essential features of this gelation process, as well as the successes of a mean-field approach to describing the interactions and kinetics of this first molecular disease.

In sickle cell anemia, deoxygenation causes erythrocytes to distort, while reoxgenation permits them to recover a normal biconcave disk shape. Irreversibly sickled cells (ISCs) cells remain distorted when reoxygenated and have been thought to have among the highest intracellular hemoglobin concentration of the sickle red cell population and therefore the greatest vulnerability to vaso-occlusion. Using a new optical method, which we describe, we have made precise measurements of the intracellular hemoglobin concentration, and intracellular O saturation, of ISCs, as well as oxygenated sickle cells with a normal biconcave disc shape, and cells with shapes distorted by the sickle fibers they contain. This method also provides good estimates of cell volumes, and hemoglobin per red cell. The concentration distribution of the ISCs is found to be similar to normal, discoid cells. Average ISC volumes exceed their discoid counterparts, with a much broader distribution, arguing against dehydration as their origin. The concentration distribution of the polymer-laden sickled cells is significantly higher in mean value, and their volume distributions indicate some dehydration. Previous assumptions about ISCs may have thus been colored by the presence of sickle cells that did contain polymer, and that true ISC's may be much more benign than once thought, which underscores the importance of accurate measurement on individual cells. This method could be used to follow changes in individual cell properties under various specific perturbations, and where characterization by flow cytometry is infeasible.

Adults with sickle cell disease bear a mutation in the beta-globin gene, leading to the expression of sickle hemoglobin (HbS; alpha 2 beta S2). Adults also possess the gene for gamma-globin, which is a component of fetal hemoglobin (HbF, alpha 2 gamma 2); however, gamma-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 gamma chain or introduce a gene encoding a modified beta chain having gamma-like character. Here, we show that a single-site modification on the alpha chain, alpha Pro114Arg, 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.

The drug voxelotor (commercially known as Oxbryta) has been approved by the 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 (HbS) 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 HbS 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.

Voxelotor is designed to impeded the polymerization of sickle haemoglobin. Although FDA-approved, its place in the treatment of sickle cell anaemia remains controversial. A report in this issue describes preliminary studies with a new drug GBT021601. Working in virtually the same fashion as voxelotor, the drug therefore immediately recapitulates on-going concern and controversy over the original agent.
Oxygen delivery in humans is elegantly efficient. The sigmoidal oxygen saturation curve insures that as tissue pO2 drops, more oxygen can be delivered. Haemoglobin's exchange of a single oxygen molecule with solution is common and expected. The departure of a second O2 from Hb signals that local oxygen is becoming sparse, and as a result the Hb switches over to a state of lower oxygen affinity, anticipating future demands. The remaining two oxygen molecules can then dissociate with relative ease.
Interaction between haemoglobin's subunits is responsible for this behaviour. Hb has two αβ dimers that can pack in different fashion depending on whether the molecule is in its high affinity (R) or low affinity (T) state. The T state is stabilized by eight salt bridges, six of which are between subunits and two of which involve the terminus of the α chain.1 The R-state packing is a ‘marriage of convenience’, with no special stabilization. Unsurprisingly, more than one R-state packing is known, and multiple packings are present in solution.2-4 In the absence of ligands, the T-state is some 64 000 times more abundant than the R thanks to these T-state bonds.5
Sickle cell anaemia is not primarily a disease of anaemic compromise, but one of circulatory catastrophe. A seemingly small mutation on the surface of the β chains allows HbS to form large and extensive arrays of polymers. These render the cell incompressible6 and make its escape from the capillaries problematic (a) if the polymers have formed during transit, and (b) if the exit to the venuoles is compromised by the presence of adherent cells that the usually pliable red cells could slip past but rigid sickled cells cannot.7 This clog of a single capillary is not an ischemic event of itself, since tissues are fed by multiple capillaries, and simulations we have conducted show that, relative to the rate of sickling, it is rare to recruit enough obstructed capillaries to draw down local O2 far enough for tissue damage, even though this is the eventual outcome. (Reese, Jiang and Ferrone, unpublished). O2 binding to the polymers can lead to polymer dissolution and thus reopen blocked capillaries. But polymers can only form when Hb is in the T-state.5, 8 Thus, the allosteric change subsequent to deoxygenation is required for polymerization.
This motivates the therapeutic approach of restraining T-state formation so as to block polymer formation, which is the premise of voxelotor, and its successor GBT021601.9 By binding to the terminus of the α chain, the drug obstructs one of the salt bridges that stabilize the T structure, and by stretching across to the other α chain, also occludes its symmetry-related partner. (It is a bit of a misnomer to say that the R-state has become more stable.) But this strategy represents a trade-off, for in maintaining the R-state, the ingenious mechanism of enhanced haemoglobin O2 delivery is compromised (Figure 1).

Sickle cell disease is the consequence of a single point mutation on the surface of the β 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.

We have characterized the imbibed horizontal flow of sickle blood into 100-mu m-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 S beta(+) 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.