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Brugnara, Carlo

Background: Deoxygenation of sickle erythrocytes activates a cation permeability of unknown molecular identity (Psickle), leading to elevated intracellular [Ca2+] ([Ca2+]i) and subsequent activation of KCa 3.1. The resulting erythrocyte volume decrease elevates intracellular hemoglobin S (HbSS) concentration, accelerates deoxygenation-induced HbSS polymerization, and increases the likelihood of cell sickling. Deoxygenation-induced currents sharing some properties of Psickle have been recorded from sickle erythrocytes in whole cell configuration. Methodology/Principal Findings: We now show by cell-attached and nystatin-permeabilized patch clamp recording from sickle erythrocytes of mouse and human that deoxygenation reversibly activates a Ca2+- and cation-permeable conductance sensitive to inhibition by Grammastola spatulata mechanotoxin-4 (GsMTx-4; 1 µM), dipyridamole (100 µM), DIDS (100 µM), and carbon monoxide (25 ppm pretreatment). Deoxygenation also elevates sickle erythrocyte [Ca2+]i, in a manner similarly inhibited by GsMTx-4 and by carbon monoxide. Normal human and mouse erythrocytes do not exhibit these responses to deoxygenation. Deoxygenation-induced elevation of [Ca2+]i in mouse sickle erythrocytes did not require KCa3.1 activity. Conclusions/Significance: The electrophysiological and fluorimetric data provide compelling evidence in sickle erythrocytes of mouse and human for a deoxygenation-induced, reversible, Ca2+-permeable cation conductance blocked by inhibition of HbSS polymerization and by an inhibitor of strctch-activated cation channels. This cation permeability pathway is likely an important source of intracellular Ca2+ for pathologic activation of KCa3.1 in sickle erythrocytes. Blockade of this pathway represents a novel therapeutic approach for treatment of sickle disease.

SUMMARY Defects in the availability of heme substrates or the catalytic activity of the terminal enzyme in heme biosynthesis, ferrochelatase (Fech), impair heme synthesis, and thus cause human congenital anemias1,2. The inter-dependent functions of regulators of mitochondrial homeostasis and enzymes responsible for heme synthesis are largely unknown. To uncover this unmet need, we utilized zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anemia, pinotage (pnt tq209). We now report a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize heme. The loss of Atpif1 impairs hemoglobin synthesis in zebrafish, mouse, and human hematopoietic models as a consequence of diminished Fech activity, and elevated mitochondrial pH. To understand the relationship among mitochondrial pH, redox potential, [2Fe-2S] clusters, and Fech activity, we used (1) genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, and (2) pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to Atpif1-regulated mitochondrial pH and redox potential perturbations. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize heme, resulting in anemia. The novel mechanism of Atpif1 as a regulator of heme synthesis advances the understanding of mitochondrial heme homeostasis and red blood cell development. A deficiency of Atpif1 may contribute to important human diseases, such as congenital sideroblastic anemias and mitochondriopathies.

Although effective low-cost interventions exist, child mortality attributable to sickle cell disease (SCD) remains high in low-resource areas due, in large part, to the lack of accessible diagnostic methods. The presence of dense (ρ > 1.120 g/cm3) cells is characteristic of SCD. The fluid, self-assembling step-gradients in density created by aqueous multiphase systems (AMPSs) identifies SCD by detecting dense cells. AMPSs separate different forms of red blood cells by density in a microhematocrit centrifuge and provide a visual means to distinguish individuals with SCD from those with normal hemoglobin or with nondisease, sickle-cell trait in under 12 min. Visual evaluation of a simple two-phase system identified the two main subclasses of SCD [homozygous (Hb SS) and heterozygous (Hb SC)] with a sensitivity of 90% (73–98%) and a specificity of 97% (86–100%). A three-phase system identified these two types of SCD with a sensitivity of 91% (78–98%) and a specificity of 88% (74–98%). This system could also distinguish between Hb SS and Hb SC. To the authors’ knowledge, this test demonstrates the first separation of cells by density with AMPSs, and the usefulness of AMPSs in point-of-care diagnostic hematology.

Iron deficiency anemia (IDA) is a nutritional disorder that impacts over one billion people worldwide, it causes permanent cognitive impairment in children, fatigue in adults, and suboptimal outcomes in pregnancy. IDA can be diagnosed by detection of red blood cells (RBCs) that are characteristically small (microcytic) and deficient in hemoglobin (hypochromic), typically by examining the results of a complete blood count performed by a hematology analyzer. These instruments are expensive, not portable, and require trained personnel; they are therefore, unavailable in many low-resource settings. This paper describes a low-cost and rapid method to diagnose IDA using aqueous multiphase systems (AMPS)—thermodynamically stable mixtures of biocompatible polymers and salt that spontaneously form discrete layers having sharp steps in density. AMPS are preloaded into a microhematocrit tube and used with a drop of blood from a fingerstick. After only two minutes in a low-cost centrifuge, the tests (n = 152) were read by eye with a sensitivity of 84% (72-93%) and a specificity of 78% (68-86%), corresponding to an area under the curve (AUC) of 0.89. The AMPS test outperforms diagnosis by hemoglobin alone (AUC = 0.73) and is comparable to methods used in clinics like reticulocyte hemoglobin concentration (AUC = 0.91). Standard machine learning tools were used to analyze images of the resulting tests captured by a standard desktop scanner to 1) slightly improve diagnosis of IDA—sensitivity of 90% (83-96%) and a specificity of 77% (64-87%), and 2) predict several important red blood cell parameters, such as mean corpuscular hemoglobin concentration. These results suggest that the use of AMPS combined with machine learning provides an approach to developing point-of-care hematology.