Polymerization Of Sickle Cell Hemoglobin Research Articles

In Vitro Biological Activities of Drepanoalpha® Ethanolic Extract, A Justicia Secunda and Moringa Oleifera-Based Phytomedicine Proposed for The Symptomatic Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is an autosomal recessive blood disorder characterized by red blood cells that assume an abnormal, rigid sickle shape under low-oxygen conditions. These sickle-shaped erythrocytes tend to lyse, aggregate, and obstruct small blood vessels, leading to major complications. The present study aims to investigate properties that may underlie the activity of Drepanoalphaâ, an antisickling herbal formulation developed in the Democratic Republic of Congo (DRC) for the prevention and symptomatic treatment of sickle cell disease crises. The Drepanoalpha® Ethanolic Extract (DEE) is a dry extract (drug-extract ratio, DER, 100/11) prepared from ethanol (96 %, v/v) percolation of a 1:1 mixture of 2 food plants, Justicia secunda Vahl and Moringa oleifera Lam. Sickling was classically measured by light microscopy on diluted washed erythrocytes obtained from homozygote patients; erythrocytes were treated with 2 % Na2S2O5 in the presence of DEE (suspension in 9 ‰ NaCl), 9 ‰ NaCl (negative control) or disodium cromoglycate (DSCG, positive control). For all tested conditions, the sickle hemoglobin polymerization, the Fe2+/Fe3+ ratio, and the median corpuscular fragility were measured by spectrophotometry. The DEE reversed sickling by 89.1 %, comparable to DSCG (87.7 %; 60.3 µg/mL), inhibiting sickle cell hemoglobin polymerization of 77.8 % and 74.4 %, respectively. The Fe2+/Fe3+ ratio was improved by 18.0 % for DEE and 15.9 % for DSCG. The median corpuscular fragility values were 0.602, 0.714, and 0.732 for NaCl 9 ‰, DSCG, and DEE, respectively. The measured in vitro parameters validate an effective antisickling effect of DEE and confirm the value of this improved traditional herbal formulation for the management of SCD.

Antiviral Response and HIV-1 Inhibition in Sickle Cell Disease

BACKGROUND: A number of host restriction factors controls HIV-1 infection. While many of these factors have been investigated in vitro, their role in controlling HIV-1 infection in vivo remains to be elucidated. Here, we investigate a naturally occurring HIV-1 restriction in Sickle cell disease (SCD), a hereditary disorder characterized by sickle cell hemoglobin polymerization and red blood cells hemolysis. We previously showed that SCD PBMCs express higher levels of ferroportin (FPN), and that reduced intracellular iron leads to the upregulation of antiviral restriction factors. Previously, HIV-1 infection was shown to inversely correlate with the expression levels of transferrin receptor (TFR), a marker of reduced intracellular iron. Iron chelation also potently inhibits HIV-1 replication as shown by us and others. Thus, iron metabolism is intrinsically connected to the HIV-1 infection and disease progression. Our study in vitro suggests that SCD environment leads to the antiviral state of T cells and macrophages, and suppression of HIV-1 infection. Here we analyzed HIV infection of SCD mice using mouse adapted EcoHIV and compared antiviral factors in SCD mouse spleenocytes and human SCD PBMCs. METHODS: The study was approved by Howard University IRB and IACUC committees. Townes SCD mice carrying human normal α (hα) and sickle hemoglobin beta (βS) genes and control Townes mice carrying human normal hα and βA genes were used in the study. EcoHIV virus obtained from David Volsky (Albert Einstein University) was constructed in pNL4-3 vector with the substitution of gp120 with the envelope-coding gp80 region from ectopic MLV. Whole blood samples were obtained from 9 SCD patients and 9 healthy individuals. RNA Seq analysis was conducted on Illumina® NextSeq 500 and the data were mapped and analyzed using Dragen software. Normalized gene counts were used for gene set enrichment analysis (GSEA) and Gene ontology (GO) analysis. RNA expression was measured by quantitative RT-PCR (Roche). Protein expression in mouse splenocytes was determined by ELISA. RESULTS: Infection of control mice with EcoHIV virus, which contained MLV envelope and was able to infect mouse cells, led to robust viral replication in spleen at 7 days post infection, as detected by the expression of HIV-1 Nef mRNA (Fig. 1) and Gag mRNA. We also detected HIV-1 p24 in splenic tissue. Comparing to the control, SCD mice showed significantly reduced EcoHIV replication both at the levels of RNA expression (Figs 1) and p24 production. GSEA analysis against the 72 genes that included all known HIV-1 restriction factors revealed strong enrichment of these genes in the human activated SCD PBMCs compared to the activated control PBMCs (Fig.2). These genes included APOBEC3A, HMOX1, CH25H, IFIT3, CDKN1A, APOBEC3B, IFITM3, SAMHD1 and TSPO. GSEA analysis also showed upregulation of several pathogen-associated molecular patterns sensing genes including NOD1, NLRC4, TRL7 and TLR9 in human PBMCS. In SCD mice splenocytes, we detected overexpression of FPN, and transferrin receptor (Tfr) genes indicating upregulation of iron metabolism similar to previously observed in human SCD PBMCs. In mouse splenocytes we detected upregulation of CDKN1A, HMOX1, IFITM3, CH25H and TSPO mRNAs. Protein expression analysis showed upregulation of FPN, TFR, HO-1, APOBEC, CH25H, IFIT3 and TSPO in SCD mouse splenocytes. CONCLUSION: Together, these data reveal, for the first time, that HIV-1 infection is suppressed in vivo in SCD mouse infected with the mouse-adapted EcoHIV. SCD mouse splenocytes expressed higher levels of FPN and TFR, and exhibited higher expression of antiviral factor, which were also observed in human SCD PBMCs. The observed upregulation of similar sets of antiviral factors in SCD PBCMs and SCD mice splenocytes suggests that these factors upregulation might be driven by the SCD-related processes such as hemolysis. Taken together, our findings point to a novel mechanism of upregulation of antiviral factors mediated by SCD that included induction of antiviral, heme- and iron- regulatory pathways. ACKNOWLEDGEMENTS: We thank Dr. David Volsky for the gift of EcoHIV and the George Washington University SPH Genomics Core for library preparation and sequencing. This work was supported by NIH grants 1R01HL125005, 5U54MD007597, 1P30AI117970-06 and 1SC1HL150685. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Drug discovery by a basic research scientist.

I was fortunate to do my military service during the Vietnam era as a medical officer at the National Institutes of Health (NIH) in Bethesda, Maryland. My first research at NIH was concerned with making a variety of optical measurements on nucleic acid bases and proteins, including single crystal spectra in linearly polarized light and near infrared circular dichroism, interpreting the spectra using molecular orbital and crystal field theories. What I do now is drug discovery, a field at the opposite end of the scientific spectrum. This article gives a brief account of my transition from spectroscopy to sickle cell hemoglobin polymerization to protein folding to drug discovery for treating sickle cell disease. My lab recently developed a high throughput assay to screen the 12,657 compounds of the California Institute of Biomedical Research ReFrame drug repurposing library. This is a precious library because the compounds have either been FDA approved or have been tested in clinical trials. Since the 1970s numerous agents have been reported in the literature to inhibit HbS polymerization and/or sickling with only one successful drug, hydroxyurea, and another of dubious value, voxelotor, even though it has been approved by the FDA. Our screen has discovered 106 anti-sickling agents in the ReFrame compound library. We estimate that as many as 21 of these compounds could become oral drugs for treating sickle cell disease because they inhibit at concentrations typical of the free concentrations of oral drugs in human serum.

Omega 3 and Azadirachta Indica J. Seed Oil Inhibit Hemoglobin Polymerization and Modulate Erythrocyte Membrane Atpases in Sickle Cell Disease (SCD)

Back ground: Functional foods/Nutraceuticals efficacy depends on both its composition and the physicochemical properties of the components that used in their formulation. Vegetable oil can play an important role. Several studies are shown that omega 3 (0.2%) and Azadirachta indica J. seed oil (0.4%) have antisickling, antioxidant and anti-haemolytic properties. Objective: The purpose of this present study was conducted to determine effects of omega 3 and Azadirachta indica J. seed oil on hemoglobin polymerization and erythrocyte membrane ATPases in Sickle Cell Disease (SCD). Method: The ability for the omega 3 and Azadirachta indica J. seed oil to inhibit sickle cell hemoglobin polymerization and improve the Fe2+/Fe3+ ratio of HbS blood were assed using spectroscopic method. The activities of three ATPases (Na+/K+; Ca2+; and Mg2+-ATPases ) were investigated by the kinetic method following the release of inorganic phosphate (Pi) by ATP taking into account the determination of the total membrane proteins of erythrocytes by Bradford’s method. Results: The lowest potential was obtained with Azadirachta indica J. seed oil (AO: 0.4% v/v) corresponding significantly (p <0.05) to the highest potential of inhibition of polymerization (83.11 ± 8%) compared to that obtained with omega 3 (ω3 0.2% v/v) estimated at 68.15±7%. The greatest Fe2+Fe3+ ratio was obtained (6.65±1.68) with AO (0.4% v/v) corresponding significantly (p <0.05) to the greatest rate of increase of oxyhemoglobin (79.05±3%) compared to that obtained with ω3 (0.2% v/v) estimated at 70.58±5%. As for the activities of Na+/K+, Ca2+ and Mg2+-ATPases expressed in μmole Pi/mg protein/hour x 10-3, there was an increase in the activities of Na+/K+ and Ca2+-ATPases (203.82±41 and 190.48±32) with AO (0.4% v/v), but without any significant difference (p>0.05) compared to those obtained with ω3 (0.2% v/v) (188.66±9 and 235.61±52). However, for Mg2+-ATPase, there was rather a significant decrease (p <0.05) in these activities ranging from 181.9±32 for ω3 (0.2% v/v) to 124.00±21 for AO (0.4% v/v). Conclusion: The Azadirachta indica J. seed oil at a concentration of 0.4% v/v offered the best potential inhibition of hemoglobin S polymerization, oxyhemoglobin increase rate and the best ATPastic activities. Keywords: Omega 3 - Azadirachta indica J. seed oil - sickle cell disease - polymerization - Fe2+/Fe3+ ratio - ATPase activities.

Amino Acids Extract from Black Bean Seeds (Phaseolus Vulgaris L.)Inhibit Hemoglobin Polymerizationand Modulate Erythrocyte Membrane Atpases In Sickle Cell Disease (SCD)

Back ground: The basic extracts involved in the formulation of functional foods/nutraceutics must first be the subject of preclinical and clinical trials. The several studies were shown that amino acids extract from black bean seeds (Phaseolus vulgaris L.) have antisickling, antioxidant and anti-haemolitic properties. Objective: The objective of this study is to investigate the effect of amino acids extract from black bean seeds (Phaseolus vulgaris L.) on hemoglobin polymerization and erythrocytes membrane ATPases activities in Sickle Cell Disease (SCD). Method: The ability for the amino acids extract to inhibit sickle cell hemoglobin polymerization and improve the Fe2+/Fe3+ ratio of HbSS blood were assed using spectroscopic method. The activities of three ATPases (Na+/K+; Ca2+; and Mg2+-ATPases) were investigated by the kinetic method following the release of inorganic phosphate (Pi) by ATP taking into account the determination of the total membrane proteins of erythrocytes by Bradford’s method. Result: The lowest potential of inhibition was obtained (39.98±2%) with the amino acids extract at 12 mg/mL corresponding significantly (p<0.05) to the highest potential of inhibition of polymerization (71.16±2%). The greatest Fe2+Fe3+ ratio was obtained (3.94±2%) with the amino acids extract at 12 mg/mL corresponding significantly (p<0.05) to the best rates increase of oxy-hemoglobin (48.73±2%). The red blood cells membranes ATPase activity were estimated and expressed as μmol of Pi/mg of protein/hour x 10-3 there was an increase in the activities of Na+/ K+ and Ca2+-ATPases (255.98±62 and 282.98±36 respectively) at the concentration of 12 mg/mL of extract compared to the negative control (167.36±60 and 236.91±62 respectively). However, for Mg2+-ATPase, there is rather a significant reduction (p<0.05) in this activity (286.71±51 against 146.53±35) obtained in the presence of the amino acid extract at 12 mg/mL). Conclusion: The amino acids extract of black bean seeds (Phaseolus vulgaris L.) at a concentration of 12 mg/mL offered the best inhibition potentials of hemoglobin S polymerization, increase rates of oxyhemoglobin and the best ATPasic activities. Keywords: Amino acid extracts; black bean seeds; sickle cell disease; polymerization; Fe2+/Fe3 + ratio; ATPase activities

In vitro Investigation of the Anti-sickling and Erythrocyte Membrane Stabilizing Potentials of Elaesis guineensis Jacq Flower

Anti-sickling activity of Elaeis guineensis jacq flowers was investigated to determine the capability of the plant to inhibit the polymerization of sickle cell hemoglobin, maintain membrane osmotic fragility and recover the Fe2+/Fe3+ ratio. Spectrophotometric technique was employed to determine the rate of sodium metabisulphite provoked HbSS erythrocytes polymerization. The profile for HbSS erythrocytes polymerization of test and control specimens demonstrated growing level of polymerization at three investigational concentrations (20mg/ml, 40mg/ml and 60mg/ml) while (80mg/ml, 100mg/ml, 120mg/ml) concentrations of the extracts showed pronounced anti-sickling activity through inhibition of HbSS gelation. The estimation of mean corpuscular fragility (MCF), which is the concentration of saline causing 50% hemolysis of the erythrocytes, revealed that the plant extract decreased the MCF values of the HbSS erythrocytes at all concentrations in comparison to the control. The Fe2+/Fe3+ investigation revealed an increase in the test groups when compared to the control. The aqueous extracts of Elaeis guineensis jacq flowers demonstrated towering potency in altering the polymerization of sickle cell hemoglobin at increased concentration, enhancement in Fe2+/Fe3+ ratio and maintaining erythrocyte membrane integrity.

Hemoglobin S Oxidation Promotes Plasma-Derived Microparticle Membrane Alterations and Toxicity

Polymerization of sickle cell hemoglobin S (HbS) is recognized as a key event in the pathophysiology of sickle cell disease (SCD). Repeated HbS polymerization promotes an altered red blood cell (RBC) membrane, hemolysis, and microparticle (MP) formation, which have been shown to play significant roles in the interaction of RBCs with vascular endothelium and progression of vaso-occlusive events. Circulating RBC-derived MPs are elevated in SCD patients and they release a significant portion of their contents including oxidized HbS and heme to the cells of the vasculature. We have recently reported that free HbS oxidizes faster, remains locked in a highly oxidizing form (ferryl) longer, and loses heme faster than normal HbA (Kassa et al., J Biol Chem 290: 27939, 2015). The contributions of HbS higher oxidation states (ferric and ferryl heme) to MP formation, membrane alterations, and heme loss are poorly defined in SCD. RBC-derived MPs (ranging in size between 100-300 nm in diameter) generated by sheer stress or isolated by ultracentrifugation from the plasma (circulating) of SCD patients (N=6), ethnically matched control subjects (N=5), humanized transgenic sickle mice (Townes-SS, N=4), and control wild-type mice (Townes-AA, N=4) were identified by flow cytometry using CD235a glycophorin antibody and annexin V for externalized phosphatidylserine (PS). Time courses of Hb oxidation, obtained during 30 hour incubations of mouse or human MPs were biphasic. The initial levels of oxidized (ferric) Hb (30 to 45%) were slightly reduced within the first ~10 hours, likely due to the presence of RBC residual reductive enzymes within MPs. This was followed by a second phase in which Hb oxidation (ferric Hb) increased linearly and uncontrollably to 65 to 75% of total Hb. SCD MP's contained highly reactive ferryl Hb intermediates, carbonylated membrane proteins, and phosphorylated band 3 proteins. Quantitative proteomic analysis indicated a higher level of protein oxidation in MPs derived from SCD mice and patients. Five-fold higher levels of irreversibly oxidized βCys93 oxidation were found in untreated versus hydroxyurea-treated SCD patients. Intriguingly, HbS β subunits from SCD MPs were ubiquitinated and MPs isolated from untreated SCD patients had 25-fold higher ubiquitination levels than hydroxyurea-treated SCD patients that were comparable to normal controls. MP ubiquitination levels were correlated with HbS and an overall increase in MP oxidative stress, and inversely correlated with HbF. Compared to respective control MPs, incubation of either mice or human SCD MPs with human endothelial cells (HUVEC) activated apoptotic pathways and impacted cellular bioenergetic parameters by lowering mitochondrial oxygen consumption rates to a greater degree in a manner that was correlated with the redox state of Hb iron within MPs. Human endothelial cells incubated with SCD MPs showed greater intracellular reactive oxygen species production and heme oxygenase-1 induction. In summary, Hb transformation to higher oxidation forms is markedly increased in MPs generated from SCD mice and patients, which when incubated with endothelial cells, lead to mitochondrial dysfunction and apoptotic cell death. These mechanistic analyses of RBC-derived SCD microparticles suggest potential anti-oxidative reducing modalities that may interrupt MP heme-mediated pathophysiology in patients with SCD. DisclosuresBelcher:Cydan/Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.

Lack of Dependence of the Sizes of the Mesoscopic Protein Clusters on Electrostatics

Protein-rich clusters of steady submicron size and narrow size distribution exist in protein solutions in apparent violation of the classical laws of phase equilibrium. Even though they contain a minor fraction of the total protein, evidence suggests that they may serve as essential precursors for the nucleation of ordered solids such as crystals, sickle-cell hemoglobin polymers, and amyloid fibrils. The cluster formation mechanism remains elusive. We use the highly basic protein lysozyme at nearly neutral and lower pH as a model and explore the response of the cluster population to the electrostatic forces, which govern numerous biophysical phenomena, including crystallization and fibrillization. We tune the strength of intermolecular electrostatic forces by varying the solution ionic strength I and pH and find that despite the weaker repulsion at higher I and pH, the cluster size remains constant. Cluster responses to the presence of urea and ethanol demonstrate that cluster formation is controlled by hydrophobic interactions between the peptide backbones, exposed to the solvent after partial protein unfolding that may lead to transient protein oligomers. These findings reveal that the mechanism of the mesoscopic clusters is fundamentally different from those underlying the two main classes of ordered protein solid phases, crystals and amyloid fibrils, and partial unfolding of the protein chain may play a significant role.

Thermodynamic mechanism of free heme action on sickle cell hemoglobin polymerization

For insights into the mechanisms of heme action on the rate of sickle cell hemoglobin polymerization, we determine the erythrocytic concentration of free heme using a novel method based on enzymatic catalysis and luminescence. We find in sickle cell patients 44 ± 10 µM, in sickle trait individuals, 33 ± 4 µM, and in healthy adults, 21 ± 2 µM. We test the applicability of two mechanisms of heme action: a kinetic one, whereby heme aggregates serve as heterogeneous nucleation centers, and a thermodynamic pathway, in which free heme enhances the attraction between sickle hemoglobin (HbS) molecules in solution. We show that the latter mechanism exclusively operates. The enhanced attraction leads to increase of the total volume of a population of dense liquid clusters by about two orders of magnitude. As the dense liquid clusters serve as locations and precursors to the formation of the HbS polymer nuclei, their increased volume directly leads to faster polymer nucleation. © 2015 American Institute of Chemical Engineers AIChE J, 61: 2861–2870, 2015

Guilt by Association: The Physical Chemistry and Biology of Protein Aggregation

The aggregation of proteins and polypeptides is a major problem in the laboratory and in biotechnology, and is a serious issue in biology and human health.1−4 Protein aggregation is a topic that is ripe for investigation by physical chemical and biophysical methods, as well as by molecular dynamics (MD) simulations, and by more coarse grained modeling and analytical theory. Much recent attention in the physical chemistry community has focused on the process of amyloid fibril formation, motivated in part by its role in human disease. More than 30 different diseases involve amyloid formation, including devastating neurodegenerative disorders, but the mechanism of amyloid formation is not well understood, and there are very few viable therapeutic approaches. Amyloids are partially ordered aggregates of proteins that contain significant β-sheet structure arrayed in a cross-β structure in which the individual peptide chains are oriented perpendicular to the long axis of the fibril (Figure ​(Figure1). The1). The formation of amyloid is a fascinating and challenging problem in molecular self-assembly. However, it is worthwhile reminding ourselves that the importance of amyloids is directly related to their role in human disease, and thus experiments and simulations conducted under simplified conditions need to connect to biology to remain relevant. Fortunately, recent developments in methodology and experimental design, as well as growing collaborations between biophysicists and biologists hold the promise of providing a more rigorous, biologically relevant description of amyloid formation. Several of these advances are highlighted in the Perspectives included in this issue. Protein aggregation is not limited to amyloid formation, and new methods and approaches are also needed to study nonamyloidogenic aggregation. For example, considerable resources are invested in optimizing protein solubility and preventing aggregation of potential therapeutic proteins. Aggregation and poor solubility have limited the development of many monoclonal antibodies and other protein-based drugs and can lead to problems with immunogenicity, as well as loss of active protein.4 Protein aggregation can take other guises in biology beyond just amyloid. A classical example, and still arguably the best understood case of pathological protein aggregation, is the polymerization of sickle cell hemoglobin to form polymeric fibers. Elegant spectroscopic measurements together with detailed modeling have defined the mechanism of sickle cell polymerization, illustrating the power of physical chemical approaches.5 There are numerous other examples of deleterious aggregation that does not involve amyloid, including interesting mechanisms by which certain viruses subvert host defenses.

The Effects of Extracts of Christmas Bush (Alchornea cordefolia) on Sickle Cell Hemoglobin

The study aims at determining the antisicking effectiveness of the extracts of the leaves of a herbal medicinal plant Alchornea cordefolia. The plant is widely distributed in the South-eastern region of Nigeria. The study is designed to involve phytochemical exploration, the nutrient and mineral compositions, the free amino acid, ascorbic acid and the amino acid constituents. Apart from these, the rates of sickle cell hemoglobin polymerization were assayed with different fractions of extracts and compared with the control to ascertain their antisickling effectiveness. Sickle cell blood samples were donated by a total of forty patients (25 males and 15 females) of ages (17-32 years) whose sickle cell status were confirmed by electrophoresis of sickle cell blood by staff of the hematology unit of the centre . The donors were co-opted into the study by the personnel of the sickle cell unit of the Federal Medical Centre, Owerri, Nigeria. The determination of the Fe2+/Fe3+ ratio was to assess the oxygen affinity of sickle cells or drepanocytes. The antisickling properties of leaf extracts of Christmas bush (Alchornea cordefolia) were investigated. Results from phytochemical analysis of crude aqueous extract (CAE) revealed the presence of flavonoids (4.2±0.1%), alkaloids (5.7±0.12%), saponins (4.60±0.10%), tannins (6.50±0.1%), phenol (3.0±0.10%) and oxalate (5.47±0.1%). Proximate composition showed the following results: moisture (11.05±0.0%), ash (6.8±0.1%), crude fat (6.03±0.0%), protein (6.10±0.0%), fiber (24.5±0.2%) and carbohydrates (44.50±0.2%). Assay of mineral composition, revealed a preponderance of such, which include: Potassium (150.30 mg/100g), Sodium (228.20 mg/100g), Calcium (1.60 mg/100g), Magnesium (2.40 mg/100g) and Phosphorus (1.00 mg/100g) of dry weight of sample. The determination of the antisickling effects of the extracts of Alchornea cordifolia was assessed based on the inhibition of sickle cell hemoglobin polymerization and the improvement of Fe2+/Fe3+ ratio. Fifty grams (50 g) of the powdered sample was used for the batch extraction procedures with chloroform, methanol, butanol and distilled water to obtain the fat soluble fraction (FAS), the butanol soluble(BUS) and water soluble fractions (WAS) respectively. The FAS, BUS and WAS fractions exhibited profound antisickling effectiveness by inhibiting the HbSS polymerization to varying degrees from (47.50% for the BUS to 98.12% for the WAS fractions respectively in 20 min. The WAS and FAS fractions improved the Fe2+/Fe3+ ratio remarkably, except the BUS fraction . Thin layer chromatographic (TLC) analysis revealed the following amino acids - Phenylalanine, Alanine, Glutamate, Histidine, etc. The total free amino acid concentration of the fractions were as follows: the FAS (526.8 mg/100g); the WAS (79.33mg/100 g and the BUS (15.65 mg/100 g). The total vitamin C concentration was found to be 1929.18 mg/100 g of sample. Alchornea cordefolia leaf extracts, with the preponderance of micro and macro-nutrients, vitamins, amino acids and others, may be very beneficial for the management of sickle cell disease.

Oxygen-Dependent Depolymerization of Sickle Cell Hemoglobin Polymers in the Lungs: Kinetic Mechanisms and their Significance for Pathogenesis and its Prevention

Our kinetic studies have characterized the mechanisms of deoxyhemoglobin S polymer depolymerization when exposed to CO (used as a model for oxygen because rates can be controlled photolytically). Fibers dissolve slowly, losing monomers from their ends at low partial pressure, and very rapidly at higher partial pressures that induce fiber fracture and therefore many new ends. Slow dissolution that is not complete in the time red cells traverse the pulmonary microvasculature will generate residual arterial polymers (RAPs), enhancing pathogenesis by seeding nucleation of new polymers, accelerating repolymerization and increasing its extent.We now demonstrate, to the best of our knowledge for the first time by direct observation, that anaerobically drawn arterial blood of sickle patients shows birefringence in many red cells and therefore RAPs exist, which we confirm by EM observation of aligned polymers. RAPs exist not only under hypoxemic conditions, when they can be explained by limited solubility due to the presence of deoxyHbS, but also when hypoxemia is absent. RAPs without hypoxemia imply that slow depolymerization kinetics are responsible. One minute of voluntary hyperventilation and (separately) brief nasal oxygen greatly decrease RAPs. RAPs increase during sleep. We attribute these results to accelerated depolymerization at increasing levels of oxygen that cooperatively induce polymer fracture (fracture, using CO, exhibits a 4.7 power dependence on pCO). These results and the interdependent progress of oxygen saturation, partial pressure, fracture rate and remaining polymer that we model bear on pathogenesis and particularly on vaso-occlusive crises, which result from red cell rigidification and from cellular adhesion due to polymer-dependent cellular damage. Under these mechanisms, the lungs may play an important role in initiating pathology; and remediation of dysfunction by breathing assists is potentially prophylactic.

Control of the nucleation of sickle cell hemoglobin polymers by free hematin

The polymerization of sickle cell hemoglobin (HbS) in the erythrocytes of sickle cell anemia patients is the primary event in the pathophysiology of this debilitating and deadly disease. Correspondingly, the majority of the current clinical treatments rely on delaying HbS polymerization. In search of pathways towards novel, more efficient treatment strategies, we explore the mechanism of nucleation of the HbS polymers. Previous work has shown that this nucleation follows a two-step mechanism, whereby the polymers nucleate inside dense liquid clusters suspended in the solution and occupying about 10−5 of the solution volume. We show that free hematin, which is spontaneously released by HbS due to its intrinsic instability to autoxidation, accelerates by ∼100× the rates of both nucleation and growth of the polymers and that its removal leads to complete arrest of HbS polymerization. Exploring the mechanism underlying these hematin effects, we show that hematin enhances the attraction between the hemoglobin molecules in the solution and this yields ∼100× higher volume of the dense liquid clusters in which nucleation occurs. These findings suggest that the nucleation of sickle cell hemoglobin polymers and the ensuing pathology can be suppressed by controlling the release and concentration of hematin in the red blood cells.

Minimalistic Approach to Protein Assembly Modelling/Application to the Sickle Cell Hemoglobin Polymerization

Aberrant assembly of proteins into oligomers and fibrils is associated with many diseases such as Alzheimer's disease, Parkinson's disease, Huntington disease, amyotrophic lateral sclerosis, type II diabetes, systemic amyloidosis, sickle cell anemia, etc. Due to limitations of fully atomistic computational approaches, coarse-grained models provided important insights into protein assembly in general as well as in particular as in the case of amyloid beta-protein, for which discrete molecular dynamics (DMD) using a four-bead protein model with implicit solvent successfully elucidated oligomer formation. Here we addressed the question of how to construct a minimal model of protein assembly using the DMD computational method. We found that a tetrahedron model of a monomer with both effective hydrophobic and hydrophilic interactions accounts for a description of an entire pathway of assembly from monomeric, through transient oligomeric to elongated ordered assemblies.A more specific and challenging problem is the model for the sickle cell hemoglobin polymerization. A genetic mutation (E6V) on the two beta chains of the hemoglobin tetramer allows effective hydrophobic interactions at these sites with other hydrophobic sites. As a result, the sickle cell hemoglobin polymerizes into long fibers that eventually change the shape of the red blood cell. There are experimentally identified lateral and axial contacts on the mutated hemoglobin that contribute to assembly into a 14-strand fiber structure. Currently there are no computational models to predict the assembly of sickle cell hemoglobin into helical fibers. We have developed a 9-bead DMD model for the sickle cell hemoglobin, based on the crystal-structure sites known to be involved in the assembly. The relative location of the axial and lateral contacts determines the structure of the final fiber as well as intermediate structures observed on the assembly pathway.

Sodium metabisulfite-induced polymerization of sickle cell hemoglobin incubated in the extracts of three medicinal plants (Anacardium occidentale, Psidium guajava, and Terminalia catappa)

Background:The exploitation and utilization of vast varieties of herbal extracts may serve as alternative measures to deter aggregation of deoxygenated sickle cell hemoglobin (deoxyHbS) molecules.Objective:The present in vitro study ascertained the capacity of three medicinal plants, namely, Anacardium occidentale, Psidium guajava, and Terminalia catappa, to alter polymerization of HbS.Materials and Methods:Spectrophotometric method was used to monitor the level of polymerization of hemolysate HbS molecules treated with sodium metabisulfite (Na2 S2 O5) at a regular interval of 30 s for a period of 180 s in the presence of separate aqueous extracts of A. occidentale, P. guajava, and T. catappa. At time intervals of 30 s, the level of polymerization was expressed as percentage of absorbance relative to the control sample at the 180th s.Results:Although extracts of the three medicinal plants caused significant (P < 0.05) reduction in polymerization of deoxyHbS molecules, the corresponding capacity in this regard diminished with increase in incubation time. Aqueous extract of P. guajava exhibited the highest capacity to reduced polymerization of deoxyHbS molecules. Whereas at t > 60 s, extract concentration of 400 mg% of A. occidentale activated polymerization of deoxyHbS molecules by 6.23±1.34, 14.53±1.67, 21.15±1.89, and 24.42±1.09%, 800 mg% of T. catappa at t > 30 s gave values of 2.50±1.93, 5.09±1.96, 10.00±0.99, 15.38±1.33, and 17.31±0.97%.Conclusion:The capacity of the three medicinal plants to interfere with polymerization of deoxyHbS molecules depended on the duration of incubation and concentration of the extracts.

Free Heme and the Polymerization of Sickle Cell Hemoglobin

In search of novel control parameters for the polymerization of sickle cell hemoglobin (HbS), the primary pathogenic event of sickle cell anemia, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that the concentration of free heme in HbS solutions typically used in the laboratory is 0.02–0.04 mole heme/mole HbS. We show that dialysis of small molecules out of HbS solutions arrests HbS polymerization. The addition of 100–260 μM of free heme to dialyzed HbS solutions leads to rates of nucleation and polymer fiber growth faster by two orders of magnitude than before dialysis. Toward an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at a concentration of 66 μM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that spikes of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a significant factor in the complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell therapy.

The Heme and Sickle Cell Hemoglobin Polymerization

In search of novel control parameters for the polymerization of sickle cell hemoglobin (HbS), the primary pathogenic event of sickle cell anemia, we explore the role of free heme, which may be excessively released in sickle erythrocytes. We show that concentration of free heme in HbS solutions typically used in studies of polymerization kinetics is ∼0.04 - 0.05 mole heme/mole HbS. We show that dialysis of small-molecules out of HbS solutions completely prevents HbS polymerization. We show that that after dialysis, no apo-globin forms and HbS is largely intact. The addition of 100 - 300 μM of free heme to dialyzed HbS solutions leads to nucleation rates and delay times and polymer fiber growth rates faster by two orders of magnitude than prior to dialysis. Towards an understanding of the mechanism of nucleation enhancement by heme, we show that free heme at concentration 66 μM increases by two orders of magnitude the volume of the metastable clusters of dense HbS liquid, the locations where HbS polymer nuclei form. These results suggest that variation of the free heme concentration in the erythrocytes of sickle cell anemia patients may be a major factor for the puzzling complexity of the clinical manifestations of sickle cell anemia. The prevention of free heme accumulation in the erythrocyte cytosol may be a novel avenue to sickle cell disease therapy.

Determination of the Transition-State Entropy for Aggregation Suggests How the Growth of Sickle Cell Hemoglobin Polymers can be Slowed

Sickle cell anemia is associated with the mutant hemoglobin HbS, which forms polymers in red blood cells of patients. The growth rate of the polymers is several micrometers per second, ensuring that a polymer fiber reaches the walls of an erythrocyte (which has a 7-μm diameter) within a few seconds after its nucleation. To understand the factors that determine this unusually fast rate, we analyze data on the growth rate of the polymer fibers. We show that the fiber growth follows a first-order Kramers-type kinetics model. The entropy of the transition state for incorporation into a fiber is 95 J mol−1 K−1, very close to the known entropy of polymerization. This agrees with a recent theoretical estimate for the hydrophobic interaction and suggests that the gain of entropy in the transition state is due to the release of the last layer of water molecules structured around contact sites on the surface of the HbS molecules. As a result of this entropy gain, the free-energy barrier for incorporation of HbS molecules into a fiber is negligible and fiber growth is unprecedentedly fast. This finding suggests that fiber growth can be slowed by components of the red cell cytosol, native or intentionally introduced, which restructure the hydration layer around the HbS molecules and thus lower the transition state entropy for incorporation of an incoming molecule into the growing fiber.

Two-Step Mechanism of Homogeneous Nucleation of Sickle Cell Hemoglobin Polymers

Sickle cell anemia is a debilitating genetic disease that affects hundreds of thousands of babies born each year worldwide. Its primary pathogenic event is the polymerization of a mutant, sickle cell, hemoglobin (HbS); and this is one of a line of diseases (Alzheimer's, Huntington's, prion, etc.) in which nucleation initiates pathophysiology. We show that the homogeneous nucleation of HbS polymers follows a two-step mechanism with metastable dense liquid clusters serving as precursor to the ordered nuclei of the HbS polymer. The evidence comes from data on the rates of fiber nucleation and growth and nucleation delay times, the interaction of fibers with polarized light, and mesoscopic metastable HbS clusters in solution. The presence of a precursor in the HbS nucleation mechanism potentially allows low-concentration solution components to strongly affect the nucleation kinetics. The variations of these concentrations in patients might account for the high variability of the disease in genetically identical patients. In addition, these components can potentially be utilized for control of HbS polymerization and treatment of the disease.

The Kinetics of Nucleation and Growth of Sickle Cell Hemoglobin Fibers

Polymerization of sickle cell hemoglobin (HbS) in deoxy state is one of the basic events in the pathophysiology of sickle cell anemia. For insight into the polymerization process, we monitor the kinetics of nucleation and growth of the HbS polymer fibers. We define a technique for the determination of the rates J and delay times θ of nucleation and the fiber growth rates R of deoxy-HbS fibers, based on photolysis of CO-HbS by laser illumination. We solve numerically time-dependent equations of heat conductance and CO transport, coupled with respective photo-chemical processes, during kinetics experiments under continuous illumination. After calibration with experimentally determined values, we define a regime of illumination ensuring uniform temperature and deoxy-HbS concentration, and fast (within < 1 s) egress to steady conditions. With these procedures, data on the nucleation and growth kinetics have relative errors of < 5% and are reproducible within 10% in independent experiments. The nucleation rates and delay times have steep, exponential dependencies on temperature. In contrast, the average fiber growth rates only weakly depend on temperature. The individual growth rates vary by up to 40% under identical conditions. These variations are attributed to instability of the coupled kinetics and diffusion towards the growing end of a fiber. The activation energy for incorporation of HbS molecules into a polymer is E A = 50 kJ mol − 1 , a low value indicating the significance of the hydrophobic contacts in the HbS polymer. More importantly, the contrast between the strong θ( T) and weak R( T) dependencies suggests that the homogenous nucleation of HbS polymers occurs within clusters of a precursor phase. This conclusion may have significant consequences for the understanding of the pathophysiology of sickle cell anemia and should be tested in further work.