HbE/β-Thalassemia and Oxidative Stress: The Key to Pathophysiological Mechanisms and Novel Therapeutics

Abstract

Significance: Oxidative stress and generation of free radicals are fundamental in initiating pathophysiological mechanisms leading to an inflammatory cascade resulting in high rates of morbidity and death from many inherited point mutation-derived hemoglobinopathies. Hemoglobin (Hb)E is the most common point mutation worldwide. The β^E-globin gene is found in greatest frequency in Southeast Asia, including Thailand, Malaysia, Indonesia, Vietnam, Cambodia, and Laos. With the wave of worldwide migration, it is entering the gene pool of diverse populations with greater consequences than expected.

Critical issues: While HbE by itself presents as a mild anemia and a single gene for β-thalassemia is not serious, it remains unexplained why HbE/β-thalassemia (HbE/β-thal) is a grave disease with high morbidity and mortality. Patients often exhibit defective physical development, severe chronic anemia, and often die of cardiovascular disease and severe infections. Recent Advances: This article presents an overview of HbE/β-thal disease with an emphasis on new findings pointing to pathophysiological mechanisms derived from and initiated by the dysfunctional property of HbE as a reduced nitrite reductase concomitant with excess α-chains exacerbating unstable HbE, leading to a combination of nitric oxide imbalance, oxidative stress, and proinflammatory events.

Future directions: Additionally, we present new therapeutic strategies that are based on the emerging molecular-level understanding of the pathophysiology of this and other hemoglobinopathies. These strategies are designed to short-circuit the inflammatory cascade leading to devastating chronic morbidity and fatal consequences. Antioxid. Redox Signal. 26, 794-813.

Keywords: hemoglobin E; hypoxia; inflammation; nitric oxide; oxidative stress; β-thalassemia.

FIG. 1.

Model of the Human Normal RBC and the proposed oxidative stressed HbE/β⁰-thal RBC. (a) The redox system of the normal RBC contains intracellular oxidative reactions during the RBC 120-day lifetime. (b) Proposed pathophysiological mechanism of the HbE/β⁰-thal RBC. A small amount of excess α chains arising from heterozygous thalassemia produce heme, hemichromes, labile iron, ROS, and HDP that bind to the lipid bilayer and Band 3 (108). Membrane dysfunction, including ion transport [e.g., Band 3 (the anion transporter)], alters normal NO cell physiology, as proposed (95). These compounds may then heighten HbE instability by further elevating heme, hemichromes, labile Fe, and free globin chain levels and consequently over-riding the RBC redox system by diminishing GSH and altering the expression of RBC protective redox enzymes, all of which result in (1) cross-linking of membrane proteins by hemin, cross-linking/clustering of Band 3 by hemichromes, altered anion transport, and other membrane ion transport proteins; (2) membrane lipid peroxidation; and (3) greater PS exposure. Further consequences are RBC shape changes, decreased RBC deformability, and ultimately hemolysis liberating these highly reactive species that minimize NO bioavailability and endothelial proinflammatory effects (168a), leading to endothelial dysfunction, subsequent cardiac damage, iron overload, and other organ damage along with an increase in RBC microparticles and increased platelet activation. Quite significantly, the presence of HbE in the HbE/β⁰-thal RBC diminishes NO by a minimized NR reaction compounding NO and NOx reduction. Haptoglobin and hemopexin are indicated in this figure as general plasma proteins that (respectively) bind free Hb and heme. Under chronic and large-scale hemolysis, these proteins become limited in their ability to quench these free species that intrinsically are highly reactive and cause oxidative damage when circulating freely in plasma (for recent reviews, see 4,48) Notably, haptoglobin levels are significantly reduced in HbE/β-thal patients (5). To the best of our knowledge, hemopexin levels have not been reported for HbE/β-thal. 6PG, 6-phosphoglucono-d-lactone, CAT, catalase; CYB5R1, NADH/NADPH cytochrome b₅ reductase; G6P, glucose-6-phosphate; GPX, glutathione peroxidase; GR, glutathione reductase; Hb, hemoglobin; ROS, reactive oxygen species; SOD, superoxide dismutase; TrxR, thioredoxin reductase. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 2.

HbE acts as a β⁺-thalassemia because of reduced synthesis of the β^E-globin chain due to the mutation, GAG to AAG at codon 26, of the β-globin gene, which activates a cryptic splice site and gives rise to reduced amounts of β^E-mRNA. In exon 1 of the β-globin gene, the G → A mutation at codon 26 (GAG → AAG) results in the Glu to Lys substitution in β^E-globin. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 3.

HbE/β^E-thal is a life-threatening disease. Infants are asymptomatic because HbF levels are high at birth. Adults with higher levels of HbF exhibit more moderate clinical manifestations. Death often results from cardiac failure and infections in childhood.

FIG. 4.

HbE instability makes for double oxidative jeopardy in an HbE/β-thal RBC. HbE results from a single-nucleotide substitution that gives rise to both a hemoglobin mutant with altered properties and reduced production of an unstable β-globin mRNA with aberrant splicing that explains the thalassemic-like nature of the gene. When combined with a gene for β-thal, greater RBC oxidative jeopardy ensues, leading to significant hemolysis and a cascade of cardiovascular pathophysiology and ultimate organ damage (39, 106, 136, 163, 164).

FIG. 5.

Superposition of COHbE and COHbA (1.8Å resolution). The structural superposition of human R-state COHbA and human COHbE near the βLys26 (E26) substitution is shown. The α-chain is highlighted in light green, while the β-chain is shown in gray. The red spheres denote water molecules. Side chains of the HbA structure are shown in pink. Significantly noted for COHbE is the loss of stabilizing H-bonds from βLys26 (E26) to βArg30 (R30), residues critical for Hb assembly, stability, and oxidation state. Details are presented in Roche et al. (152). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 6.

Blood smear from adult HbE/β-thal patient, showing marked anisopoikilocytosis with profound hypochromic red cells, target cells, and red cell fragmentation. (Courtesy of S. Fucharoen). (Table 2 for hematological parameters.) To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 7.

Increased platelet activation in HbE/β-thalassemia. Hemolysis and microparticle formation are increased in thalassemia and it is proposed here that the unstable nature of HbE and its inadequate NR production of NO result in an unusually large depletion of NO that further enhances factors contributing to RBC-platelet activation. The model depicts that PS on the RBC surface can catalyze production of thrombin, which is a platelet agonist. Hemolysis releases Hb-bound ADP that can activate platelets and arginase enzyme that degrades L-arginine; thus depleting substrate for NO synthesis. Increased cell-free Hb scavenges NO and enhances ROS formation and oxidative stress that can facilitate platelet activation and damage endothelial cells. ROS decrease DDAH activity, an enzyme important in removing ADMA (an endogenous NOS inhibitor as a product of protein catabolism), leading to accumulation of ADMA. Taken together, endothelial dysfunction with decreased NO, increased ROS, microparticles, and PS-exposed RBCs contribute to increased platelet activation in thalassemia. ADP, adenosine diphosphate; ADMA, asymmetric dimethylarginine; DDAH, dimethylarginine dimethylaminohydrolase; Hb, hemoglobin; NOS, nitric oxide synthase; NR, nitrite reductase; PS, phosphatidylserine; ROS, reactive oxygen species. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

FIG. 8.

Initial rate of NR reaction for HbE and HbA (pH 7.0 (0.2 mM heme, ∼1:1 nitrite, 0.05 M bis Tris, with 1 mM dithionite). The down-pointing arrow on the right indicates the time-dependent decrease in absorbance at 430 nm. The data show the first functional difference for HbE: HbE in vitro exhibits minimal NR activity compared with HbA [from Roche et al. (152)]. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars