Even oxygenated sickle RBCs exhibit a variety of cellular and mem brane abnormalities that contribute directly to pathophysiology. Some are the direct consequence of polymer formation, some result from oxidative biochemistry, and some even reflect RBC response to its environment. An overarching theme in sickle disease pathobiology is that individual sickle RBC exhibit remarkable heterogeneity in various cellular characteristics. Their striking variabilities in hydration status (MCHC) and HbF content are particularly important. (See box on Major Sickle RBC Membrane Defects .)

Membrane Iron and Oxidant Generation

 An abnormal oxidative biochemistry takes place at the cytosol– membrane interface of the sickle RBC. 2 The avidity of HbS for bilayer lipid, and perhaps its modestly enhanced auto-oxidation in solution, resulting in the augmented formation of superoxide and met-Hb. This, in turn, can readily lose its heme to the lipid bilayer, where it is easily destroyed by lipid hydroperoxides to liberate “free” iron. Forms of iron on the sickle RBC membrane are catalytically active, generating highly reactive oxidants. Also, membrane “free” iron can form a redox couple with soluble oxyHb to promote further Hb oxidation, denaturation, and deposition. The sickle RBC membrane thereby acquires abnormal amounts of various iron forms: Hb, denatured hemichrome, free heme, and nonheme iron.

The membrane association of catalytic iron establishes in sickle RBC a unique oxidant risk (not present in normal RBC) because it effectively targets oxidative damage to membrane components. Further, the juxtaposition of iron with bilayer lipid allows re-initiation of peroxidative chain reactions, effectively bypassing protection by vitamin E. Deficient levels of antioxidants (e.g., vitamin E, glutathione, ascorbic acid) in sickle RBC, caused by oxidative consumption and dietary insufficiencies, contribute. The result is abnormal oxidation of membrane protein thiols and peroxidation of membrane lipids. Among the many sickle membrane defects, evidence for an oxidative origin or contribution is strongest for Band 3 clustering, abnormal stiffness, formation of irreversibly sickled cells (ISCs), aberrant cation homeostasis, microvesiculation tendency, abnormal mechanosensitivity, and erythrophagocytosis.

Other sources of oxidant generation by sickle RBC include the reaction of H₂O₂ with metHbS to form membrane-damaging fer ryl-HbS. Enhanced activity of sickle RBC nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase exhibits responsiveness to certain plasma substances, for example, endothelin-1 (ET-1). And some oxidant generation derives from retained mitochondria. within sickle RBC.

Cation Homeostasis and Dehydrated Cells

 For normal RBCs, MCHC averages ~32 g/dL and varies from 27 to 38 g/dL, with fewer than 1% of cells having MCHC greater than 38 g/dL. In contrast, the MCHC of sickle RBCs averages ~34 g/dL and ranges from 23 to 50 g/dL, with up to 40% of cells having MCHC greater than 38 g/dL. This extreme density heterogeneity results from reticulocytosis (low-density, low-MCHC cells) and dehydrating mechanisms (higher density, high-MCHC cells) (Fig. 1).

Fig1. MARKED HETEROGENEITY IN SICKLE RED BLOOD CELL HYDRATION. Left: Density gradient separations of normal (NL) and sickle (SS) RBCs illustrate that many sickle RBC are abnormally dense (at bottom), and an increased number are low-density RBCs (at top). The brackets indicate the (approximate) subpopulations for which data are presented. Note that most sickle RBCs have abnormally elevated MCHC. Right: Morphology of these sickle RBC density-separated subpopulations, under oxygenated versus deoxygenated conditions. (Density gradients reproduced with permission from Ballas SK and Mohandas N: Sickle red cell rheology and sickle blood rheology. Microcirculation. 2004;11:209. The displayed data and RBC images are extracted with permission from Kaul DK, Fabry ME, Windisch P, Baez S, Nagel RL. Erythrocytes in sickle cell anemia are heterogeneous in their rheological and hemodynamic characteristics. J Clin Invest. 1983;72:22.)

The most dramatic ion-handling abnormality of the sickle RBC is sickling-induced permeabilization of the RBC membrane to cations (Na +, K +, Ca 2+).  Since this depends on cell deformation, it prob ably partly reflects, in part, the sickle RBC’s exaggerated leak susceptibility to deformation (mechanosensitivity).  Sickling induces calcium influx and slight acidification, occurring stochastically and only in some cells at any one time. This results in net potassium and water loss mediated mostly by activation of a Ca 2+ activated (Gardos) K + channel and potassium chloride (KCl) cotransport. The latter can be activated by lowered pH, ET-1, thiol oxidation, and a mem brane interaction effect of Hbs that are relatively positively charged (HbC > HbS > HbA). It is influenced by macromolecular crowding of cytosolic proteins caused by the high MCHC. Via its greater membrane association, HbC promotes greater cation loss via KCl cotransport than HbS does, probably the reason HbSC is more severe than HbAS. Even at a steady state, sickle RBCs can contain increased Ca 2+ because it can be sequestered in cytoplasmic inside-out mem brane vesicles, a footprint of prior cytosolic Ca 2+ transients.

These aberrancies lead to decrements in RBC hydration and deformability.  Consequently, hyperdense RBCs—mostly ISCs—are not necessarily older cells with longer histories of sickling and unsickling. Rather, they can develop via a rapid reticulocyte-to-ISC transformation, with those RBC having lower HbF levels being particularly susceptible. It is unclear whether this rapid induction of cation loss or the gradualism of classic interpretations is the dominant mechanism underlying sickle RBC dehydration. Dehydrated RBCs have diminished deformability and an increased propensity for polymerization, the mutually promotive effects of dehydration and sickling comprising a vicious cycle. RBC dehydration is particularly likely to be exaggerated by the renal medullary environment and possibly by nocturnal arterial desaturation accompanying disordered sleep.

Deformability, Fragility, and Vesiculation

Even oxygenated sickle RBCs are poorly deformable. 7 The dominant cause of this is the abnormally high cytoplasmic viscosity of dehydrated cells. Additional factors include abnormal stiffness of the RBC membrane caused, in part, by protein thiol oxidation and, in part, by a poorly understood direct rigidifying effect of HbS upon the membrane. Upon RBC deoxygenation in vitro, there is a temporal correspondence between the appearance of polymer-induced shape change and deterioration of deformability, as measured by micropipette and laser diffractometer. On the other hand, filtration studies found decreased deformability before the morphologic change, and viscometry reveals a large increase in bulk viscosity caused by deoxygenated dense discocytes that show little shape change.

Sickle RBCs are somewhat mechanically fragile, which may be a consequence of dehydration and a weakening of critical skeletal associations caused by oxidative protein damage. The tendency of sickled RBCs to lose membrane microvesicles reflects the separation of the bilayer from the underlying skeleton due to spicules of polymerized Hb, with enhanced susceptibility caused by protein thiol oxidation.

Membrane Proteins and Lipids

Sickle RBC membrane protein function is adversely affected by excess thiol oxidation and possibly other oxidative protein modifications. 2 Ankyrin interactions with spectrin and Band 3 are abnormal, glycophorin and Band 3 exhibit decreased mobility, and thiol-oxidized β -actin displays abnormal associations in the spectrin–actin-4.1 com plex. Band 3 is abnormally clumped from the binding of denatured HbS, which enables the attraction of naturally occurring anti-Band 3 immunoglobulin (Fig. 2). In turn, this promotes engulfment of sickle RBC by macrophages.

Fig2. BAND 3 AND IMMUNOGLOBULIN COCLUSTERING. Denatured Hb on the RBC membrane causes clumping of Band 3 and opsonization by naturally occurring anti-Band 3 antibodies. Clusters (white spots) of band 3 colocalized with immunoglobulin on the membranes of sickle RBC (left). The drawing shows the colocalization scheme (right). Not shown here is that these changes attract complement and promote erythrophagocytosis by macrophages. (Reproduced with permission from Schluter K, Drenckhahn D: Co-clustering of denatured hemoglobin with band 3: its role in binding of autoantibodies against band 3 to abnormal and aged erythrocytes. Proc Natl Acad Sci U S A. 1986;83:6137.)

Normal enforcement of bilayer phospholipid asymmetry is impaired in sickle RBCs. Sickling promotes PS externalization, especially in ISCs, but also in some reticulocytes. A scramblase that moves phosphatidylserine (PS) outward is activated by calcium transits, and a translocase that restores PS inwardly can be inhibited by thiol oxidation.

Other changes include the presence of peroxidation byproducts such as malondialdehyde (MDA) that can cross-link proteins and promote erythrophagocytosis. Notably, the increased presence of bilayer lipid hydroperoxides appears to account for the sickle RBC membrane’s abnormal mechanosensitivity, evident in its enhanced cation leak response to a deforming stress. Perhaps this abnormal deformation susceptibility augments other aberrancies, for example, microvesiculation tendency.

Irreversibly Sickled Cells

 The elongated RBCs seen on a typically-obtained blood smear are mostly partially deoxygenated RBCs and ISCs. The latter’s permanent shape abnormality is caused not by persistent polymer but rather by membrane retention of an elongated shape, explained by thiol oxidation of β -actin such that the spectrin–actin-4.1 complex exhibits abnormally slow dissociation. Otherwise, ISCs are similar to other equally dense RBC in having high MCHC, poor deformability, externalized PS, and low HbF content (Fig. 1). ISC counts on average are higher in male patients, perhaps reflecting their average lower levels of HbF. The fundamental requirements for ISC formation seem to be RBC dehydration, prolonged deoxygenation, and the assumption of a fixed membrane shape. We suspect that the latter is fostered by a prior deforming and “conditioning” residence in the microcirculation.

The clinical importance of ISCs lies in their ability to prompt diagnosis of a sickling disorder when seen on blood smear, their short life span contributing to overall hemolytic rate, and their participation in the RBC logjam involved in occlusion. Although still adhesive to endothelium, ISCs are less so than other sickle subpopulations, but they exhibit greater adherence to macrophages.

Endothelial Adhesivity

Sickle RBCs are abnormally adhesive to vascular endothelial cells (Fig. 3). In vitro studies have implicated about two dozen candidate mechanisms, most involving adhesion molecules on endothelium and sickle RBCs, with or without intermediate bridging by adhesogenic plasma proteins.  Some mechanisms require RBC signaling responses to plasma factors, for example, epinephrine, for activation. Some described mechanisms are most evident for reticulocytes and are probably high affinity, identified using flowing conditions. Yet, microcirculatory blood flow can be intermittent and involves constraining vascular diameters that enable greater contact surface area between RBC and endothelium. So it seems plausible that low-affinity adhesive mechanisms could gain relevance in capillaries.  To date, only sickle RBC/endothelial adhesion mediated by α v β 3 and P-selectin and ICAM-4 (LW) has been verified under flow in vivo in the sickle mouse. An unanswered question is whether or not RBC adhesion in vivo occurs via a single dominant mechanism—or via different mechanisms that can vary amongst patients, locations, and clinical circumstances.

Fig3. SICKLE RBC ADHESION TO ENDOTHELIUM. RBCs adhere to the vascular wall endothelium under flowing conditions in the microcirculation of a rat infused with human sickle RBC. Immobile RBCs are on walls of the postcapillary venule that still is flowing (long arrow). The smaller feeder microvessels (small arrows) have no flow because of the log jam of RBC. (Reproduced with permission from Kaul DK, Fabry ME, Nagel RL. Microvascular sites and characteristics of sickle cell adhesion to vascular endothelium in shear flow conditions: pathophysiological implications. Proc Natl Acad Sci U S A. 1989;86:3356.)

Macrophage Interaction Sickle RBCs adhere to and are readily phagocytosed by macrophages because of RBC membrane modifications by malondialdehyde, PS externalization, and opsonization by immunoglobulin. The latter process is triggered by abnormal clustering of membrane protein Band 3 (see Fig. 2) and possibly by modification induced by malondialdehyde. The densest cells have the most surface immunoglobulin and higher PS externalization, and they exhibit the greatest interaction with macrophages and the potential for erythrophagocytosis.

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قسم المعارف: كتاب (الكواكب السماوية) يقدم مادة علمية تخدم الباحثين في مجالات الأدب والعقيدة والبلاغة

قسم الصيانة يجري أعمال الإدامة الدورية لجدران حرم مرقد أبي الفضل العباس (عليه السلام) وصحنه الشريف

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السجائر الإلكترونية والإقلاع عن التدخين.. نتائج متباينة تثير الجدل العلمي

مصر تحقق إنجازاً دولياً جديداً في منظمة الصحة العالمية

دراسة: السباحة فعّالة أكثر من الجري في الحفاظ على صحة القلب

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أحلامك ليست عشوائية.. إليك ما يفعله دماغك أثناء نومك

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سلمي أو دموي.. هكذا تنتقل السلطة في مجتمع الجرذان

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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

Abnormal Molecular Behaviors of Sickle Hemoglobin

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