Because the β^6Glu→Val substitution entails a loss of negative charge and gain in hydrophobicity, HbS exhibits three abnormal molecular behaviors of direct relevance to pathophysiology. (See box on Relationship of HbS Molecular Behaviors to Disease Features .)

Hemoglobin S Charge and Tetramer

Assembly The formation of Hb tetramers requires the proximate assembly of stable dimers from unlike monomers (e.g., α + β → α β ), an event governed by electrostatic attraction. The normal α and β chains are positively and negatively charged, respectively. In heterozygous states for β -globin mutants, β -chain competition for dimer assembly is a determinant of the relative proportions of the Hb variants. 1 Mutant β chains with less negative charge form α β dimers more slowly; the relative rates for dimer association are α β A > α β S > α β C, with α β A dimers formed about twice as rapidly as α β S dimers. This explains why those with HbAS typically have only 40% HbS and why the proportion of HbS exceeds this in HbSC disease. It also explains the effect of con current α -thalassemia on the proportion of HbS in sickle trait—avail ability of α chains becomes limiting, the percentage of HbS typically drops from 40% to 35% (one α deletion), 30% (two α deletions), or less than 25% (three α deletions).

Hemoglobin S Stability and Oxidant Formation

 HbS is modestly unstable, observed in vitro as instability to various applied stresses. Two stresses that are most clearly physiologic involve Hb oxidation.  HbS has an abnormal redox potential compared with HbA that may underlie its modestly (~40%) increased auto oxidation rate. Yet, HbS exhibits markedly (~340%) augmented instability and oxidation upon interaction with aminophospholipids of the membrane’s inner leaflet. This instability leads to the accumulation of various Hb and iron forms at the cytosol–membrane interface.  The resulting localization of abnormal oxidative biochemistry promotes a number of prominent defects of the sickle RBC membrane. Other contributors to oxidant stress within sickle RBC are addressed later.

Hemoglobin S and Polymerization

 OxyHbS, oxyHbA, and deoxyHbA have very high solubilities, but deoxyHbS aggregates into densely packed polymers, a process that is fully reversible upon reoxygenation. This abnormal property, the fundamental pathogenesis of the sickling disorders, causes the eponymous RBC shape to change because of polymer-mediated distortion (see Fig.1).

Fig1. SICKLE RED BLOOD CELL (RBC) MORPHOLOGIES. (A) A sickle cell anemia blood smear made directly from venous blood not exposed to oxygen. Smoothly elongated, raisin-shaped, and normal-appearing sickle RBC are present. (B) The same blood fully oxygenated reveals morphology that is largely normal, except for a single irreversibly sickled cell (ISC). (C) Cells from panel B that have now been fully deoxygenated, causing most RBCs to become sickled. The physical–chemical basis for these shapes is presented in Fig. 4. (Reproduced with permission from Obata K, Mattiello J, Asakura K, et al. Exposure of blood from patients with sickle cell disease to air changes the morphological, oxygen-binding, and sickling properties of sickled erythrocytes. Am J Hematol. 2006;81:26.)

Polymer Structure

 Deoxygenation transforms soluble HbS into a highly viscous and semi solid gel that behaves thermodynamically similar to a crystal in equilibrium with a solution of individual tetrameric Hb molecules. Even complete deoxygenation does not convert all deoxyHbS to polymers. The insoluble phase is a collection of domains of aligned polymers, the basic unit of which is a double-strand in which two strings of deoxyHb tetramers make multiple contacts with each other (Fig. 2).

Fig2. DEOXYGENATED HEMOGLOBIN S (HBS) POLYMER. (A) Electron micrograph of a fiber of polymerized HbS. (B) Electron density surface map, modeled from authentic HbS fibers, shows pairings that create double strands plus a helical twist. (C) Model of the HbS fiber, with Hb tetramers rendered as solid spheres. (D) Protein backbone shows tetramer staggering in the HbS crystal. Heme in red, and β 6 valines in blue. (E) Schematic representation of a double-strand (without the twist), emphasizing that only one of the two β 6 valines residing (in red) in each HbS tetramer participates in critical lateral contacts. (F) Sickled red blood cells, showing various morphologies (top to bottom): granular, holly leaf-shaped, classically sickled, and irreversibly sickled. (G) Electron microscopy of sickled RBC cytoplasm reveals highly ordered polymer domains, as seen from the side (bottom) and on the end (middle), or highly disorganized domains from rapid polymerization (top). (A and C, Reproduced with permission from Dykes G, Crepeau RH, Edelstein SJ. Three-dimensional reconstruction of the fibres of sickle cell hemoglobin. Nature. 1978;272:506. B, Reproduced with permission from Carragher B, Bluemke DA, Becker M, et al. Structural analysis of polymers of sickle cell hemoglobin. J Mol Biol. 1988;199:315. D, Reproduced with permission from Harrington DJ, Adachi K, Royer WE, Jr. The high-resolution crystal structure of deoxyhemoglobin S. J Mol Biol. 1997;272:398. F and G, Courtesy Dr. James G. White and reproduced with permission from White JG. Ultrastructural features of erythrocyte and hemoglobin sickling. Arch Intern Med. 1974;133:545.)

Each HbS tetramer has two β S chains, the β 1 and β 2 . DeoxyHbS undergoes a slight structural shift so that the A helix β^6Val “donor” site of the β 2 chain in one tetramer can contact an EF helix “acceptor” site (formed mainly by β^85Phe, β^88Leu, and β^70Ala) in the β 1 chain of a tetramer in the neighboring single string. This critical, lateral association can be made only when HbS is in its deoxy confor mation. In HbA this EF helix hydrophobic pocket is not a favorable acceptor site for the charged β 6^Glu of the β^A. In HbS, the β^6Val in the β1 subunit is located so it cannot participate in such contacts. However, the β2 chain of the second single string can form chemically similar β^6Val-dependent contacts with the β1 chain of the first single string. There are multiple additional axial and lateral contacts, but these are largely the same for deoxyHbA and deoxyHbS and are not themselves sufficient to stabilize a polymeric structure.

In the physiologic form of the polymer, the component strings of HbS molecules in a double-strand are half-staggered and have a slight twist, creating a fiber that is approximately 21 nM in diameter and is composed of one central and six peripheral double strands. The crystal formed in vitro lacks the twist, but its molecular structure is known in great detail.

HbS Solubility

The RBC’s hydration state dominates the physical-chemical behavior of HbS. The solubility of deoxyHbS (approximately 16 g/dL, measured under laboratory conditions) is much lower than the RBC mean cell Hb concentration (MCHC , ~34 g/dL). So, even partial cellular deoxygenation can raise deoxyHbS concentration above its solubility limit, allowing polymerization to occur. The biophysical effect of macromolecular crowding (boosting a protein’s activity far above that predicted from concentration alone) confers nonideal behavior upon cytoplasmic constituents, augmenting the likelihood for polymerization at any given degree of deoxygenation.

Equilibrium and Polymerization

In vitro studies carried out under (nonphysiologic) equilibrium conditions of stable oxygen tension and long-time scale corroborate crystallographic identification of critical amino acids involved in atomic contacts by revealing the influence of other Hbs on HbS solubility (Fig. 3). When different Hbs are mixed together, the tetramers dissoci ate into dimers that intermix and randomly reassemble in a binomial distribution to reform tetramers. This clarifies the impact of naturally occurring, intracellular Hb mixtures. In a mixture of HbS and HbA, overall solubility is improved because the hybrid α β S/ α β A tetramer integrates into polymer only one half as well as the α β S/ α β S tetramer (see Fig. 3A). The addition of HbF to HbS has a greater sparing effect because neither the α γ / α γ nor the hybrid α β S/ α γ tetramer can be incorporated into polymers. In this regard, HbC has the same effect as HbA, and HbA 2 has the same effect as HbF. This sparing effect of HbA is such that much lower Hb oxygen saturation is required for a polymer to form in HbAS than in HbSS RBCs (see Fig.3B).

Fig3. DEOXYHEMOGLOBIN S SOLUBILITY, DEFINED BY EQUILIBRIUM STUDIES. (A) Under laboratory conditions, solubility of 100% deoxyHbS is ~16 g/dL. An admixture of other hemoglobins with HbS raises overall solubility in absence of oxygen. The x-axis indicates the pro portion of admixed non-sickle Hb. (B) The hemoglobin oxygen saturation required to initiate intracellular polymer formation (i.e., polymer fraction) is much lower for HbAS RBC than for HbSS RBC. (A, Reproduced with permission from Poillon WN, Kim BC, Rodgers GP, et al. Sparing effect of hemoglobin F and hemoglobin A2 on the polymerization of hemoglobin S at physiologic ligand saturations. Proc Natl Acad Sci U S A. 1993;90:5039. B, Reproduced with permission from Schechter AN, Noguchi CT. Sickle hemoglobin polymer: structure-function correlates. In: Embury SH, Hebbel RP, Mohandas N, Steinberg MH, eds. Sickle Cell Disease: Basic Principles and Clinical Practice. New York: Raven Press; 1994.)

Kinetics and Polymerization

Laboratory measurements of polymerization kinetics, enabled by inducing (nonphysiologic) near-instantaneous and complete conversion of HbS from R (oxy) to T (deoxy) state, reveal a delay until polymer forms explosively.  This inherent delay time is inversely related to an extraordinarily high power of the initial Hb concentration. For example, the delay is 10 ms at HbS 40 g/dL, but it is 100,000 seconds at HbS 20 g/dL (Fig. 4A). HbS solutions and sickle RBCs behave similarly in this regard. Delay times must vary enormously from cell to cell because they are dominated by the marked heterogeneity in MCHC (i.e., shorter delay for more dehydrated cells) and are influenced by the presence of any non-S Hb (i.e., longer delay for the presence of HbA, C, or F) (see Fig. 4E). Admixture of 20% to 30% HbA with HbS (simulating HbS- β +-thalassemia) increases the delay time 10- to 100-fold, and admixture of 20% to 30% HbF with HbS increases it by 10³- to 10⁴-fold.

Fig4. KINETICS OF HEMOGLOBIN S POLYMERIZATION AFTER NEAR-INSTANTANEOUS AND COMPLETE DEOXYGENATION. (A) Extreme dependence of delay time on hemoglobin concentration. (B − D) Kinetic progress curves for HbS polymer formation. (B) Long delay times are highly variable, tend to involve a single elongated polymer domain and result in sickle-shaped RBC. (C and D) Short delay times are far more reproducible, tend to involve multiple polymer domains and result in holly leaf or granular shaped RBC. (E) Delay times for individual RBCs are influenced by substituent hemoglobins. (F) A double nucleation process underlies polymer formation, with unfavored homogeneous nucleation (top) followed by explosive heterologous nucleation (bottom). (G) Delay times that are short relative to the physiological rate of deoxygenation rate (i.e., ~1 second) are pathophysiologically irrelevant. (A–E, Reproduced with permission from Eaton WA, Hofrichter J. Hemoglobin S gelation and sickle cell disease. Blood. 1987;70:1245. F, Reproduced with permission from Ferrone FA, Hofrichter J, Eaton WA. Kinetics of sickle hemoglobin polymerization II. A double nucleation mechanism. J Mol Biol. 1985;183:611. G, Reproduced with permission from Ferrone FA. Oxygen transits and transports. In Embury S, Hebbel RP, Mohandas N, Steinberg MH, eds. Sickle Cell Disease: Basic Principles and Clinical Practice. New York: Raven Press; 1994.)

The mechanism of such polymer formation is understood to proceed by a two-step, double-nucleation process (see Fig. 4F). Accordingly, the initial homogeneous nucleation takes place in bulk solution, during which small numbers of tetramers associate, with accumulation not favored until a critical nucleus size, develops (esti mated to be 30 to 50 tetramers). Only then can new tetramers be added lengthwise to form a large polymer. After this occurs, heterogeneous nucleation causes explosive, autocatalytic polymer formation as new fibers form and extend on the surface of the preexisting polymer. It is the time until this explosive formation occurs that laboratory experiments detect as the inherent “delay time.”

The striking irreproducibility of long delay times reflects the underlying stochastic formation of a single (or very few) homogeneous nucleation event(s) in cells that form polymer slowly— and thus assume a sickled shape reflecting membrane deformation by highly elongated polymer (see Fig. 4B). In contrast, short delay times are highly reproducible and reflect the simultaneous formation of multiple nucleation sites in cells that polymerize rapidly—and thus assume a less dramatic, irregular shape reflecting membrane deformation by multiple short polymer domains (see Fig. 4C and D).

Polymerization Under Physiologic Conditions

 In physiologic conditions, however, sickle RBCs are neither at equilibrium with constant oxygen tension nor undergoing instantaneous complete deoxygenation. Rather, irrespective of the inherent delay time addressed above, the rate of deoxy-HbS polymer growth in vivo is limited by the rate at which RBC deoxygenation develops during the microvascular passage. Since this transit time is on the order of ~1 second, it effectively renders irrelevant any inherent delay times of more than ~1 second (see Fig. 4G).  Thus kinetic considerations argue that most RBCs in patients with HbSS are unlikely to sickle during their passage through the microcirculation unless something, such as RBC–endothelial adhesion, slows their transit.

Predictability is complicated by the marked heterogeneity among sickle RBCs in both MCHC and HbF content, as well as the natural biologic variability in capillary transit times. A good qualitative correspondence between polymerization in solution and within RBCs argues that the fundamental polymerization mechanism is not altered by RBC membranes. Yet, emerging evidence suggests that the notably abnormal sickle RBC membrane can accelerate nucleation, in essence eliminating the inherent delay time. A similar effect would be exerted by any preexisting polymer not completely melted during prior pulmonary transit (expected for fewer than 1% of RBCs). However, neither of these effects would alter the physiologic constraint that bulk polymer growth rate can only parallel RBC deoxygenation rate.

In vitro, sickle RBC can become classically sickled or assume holly leaf or granular forms, depending on deoxygenation rate (slow to rapid, respectively), which determines the number of nucleation domains created (see Fig. 4B–D). In the microcirculation, granular forms are probably most likely to occur; in contrast, frankly sickled forms are probably more likely to develop in veins. The RBC shape per se is not a determinant of RBC deformability, but rigidification caused by polymer does and, therefore, affects microvascular passage.

HbF and Its Protective Effect

In HbSS, HbF in RBC lysates averages 5% to 8% (range 1% to 25%). However, this HbF is not distributed evenly amongst RBCs.  Rather, its heterocellular expression is evident in the presence of F cells (RBC particularly enriched in HbF) that comprise anywhere between 2% and 80% of all RBCs. For most patients, only the small proportion of their F cells that contain at least ~10 pg HbF (roughly one-third of RBC Hb content) are predicted to be protected from polymerization under physiologic conditions.  On average, F cells remain better hydrated and exhibit better survival.

Alternative Ligands: CO and NO

Patients with HbSS can have nontrivial elevations of CO-Hb levels (reportedly as high as 7.6% in children) because of hemolysis. Hb that is partially liganded with CO is shifted to the R state conformation but has lost a portion of its oxygen-carrying capacity. RBC and Hb appear to participate in NO transport to the microcirculation, although both magnitude of the effect and mechanisms involved are debated. NO is asserted to improve RBC deformability and impair HbS polymerization. The reaction of NO with oxyHb causes Hb oxidation to metHb and reciprocal consumption of NO.

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مواضيع ذات صلة

Severe Iodine Deficiency and natural Goitrogens

Iatrogenic Hypothyroidism

Pathobiology of Sickle Cell Disease: The Basis of Phenotypic Diversity

The Unique Systems Biology of Sickle Cell Anemia

Abnormalities of Sickle Red Blood Cells

Clinical Assessment and Systemic Manifestations of Hypothyroidism: Diagnostic Accuracy

Clinical Aspects of Hypothyroidism due to Different Aetiologies

Clinical Aspects of Hypothyroidism at Different Ages

Thalassemic Structural Variants

Chronic Care of the Adult Patient With Thalassemia : Novel Therapeutic Approaches

Chronic Care of the Adult Patient With Thalassemia : Hematopoietic Stem Cell Transplantation

Survival in Patients With Thalassemia Major