Extracellular Hemoglobin Research Articles

Role of Extracellular Hemoglobin in Thrombosis and Vascular Occlusion in Patients with Sickle Cell Anemia

Sickle cell anemia (SCA) is a common hemolytic disorder caused by a gene mutation in the β-globin subunit of hemoglobin (Hb) and affects millions of people. The intravascular hemolysis releases excessive amount of extracellular hemoglobin (ECHb) into plasma that causes many cellular dysfunctions in patients with SCA. ECHb scavenges NO which promotes crisis events such as vasoconstriction, thrombosis and hypercoagulation. ECHb and its degradation product, heme, are known to cause oxidative damage to the vessel wall and stimulate the expression of adhesive protein ligands on vascular endothelium. Our study shows that ECHb binds potently to VWF—largest multimeric glycoprotein in circulation—through the A2-domain, and significantly inhibits its cleavage by the metalloprotease ADAMTS13. Furthermore, a subpopulation of VWF multimers bound to ECHb exists in significant amount, accounting for about 14% of total plasma VWF, in SCD patients. The Hb-bound VWF multimers are resistant to ADAMTS13, and are hyperactive in aggregating platelets. Thus, the data suggest that Hb-bound VWF multimers are ultralarge and hyperactive because they are resistant to the protease. The Hb-bound VWF multimers are elevated parallely with the level of ECHb in patients' plasma, and is associated with the pathogenesis of thrombosis and vascular occlusion in SCA.

Molecular masses and sedimentation coefficients of extracellular hemoglobin of Glossoscolex paulistus: Alkaline oligomeric dissociation

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) has a molecular mass ( M) of 3600 ± 100 kDa and a standard sedimentation coefficient ( s 20 , w 0 ) of 58 S, estimated by analytical ultracentrifugation (AUC). In the present work, further AUC studies were developed for HbGp, at pH 10.0, which favors oligomeric dissociation into lower M species. The HbGp oligomer is formed by globin chains a , b , c and d plus the linker chains. The pure monomeric fraction, subunit d , and HbGp at pH 10.0, in the presence of β-mercaptoethanol, were also studied. Our results indicate that for samples of pure subunit d , besides the monomeric species with s 20 , w 0 of 2.0 S, formation of dimer of subunit d is observed with s 20 , w 0 of around 2.9 S. For the whole HbGp at pH 10.0 contributions from monomers, trimers and linkers are observed. No contribution from 58 S species was observed for the sample of oxy-HbGp at pH 10.0, showing its complete dissociation. For cyanomet-HbGp form a contribution of 17% is observed for the un-dissociated oligomer, consistent with data from other techniques that show the cyanomet-form is more stable as compared to oxy-HbGp. Masses of HbGp subunits, especially trimer abc and monomeric chains a , b , c and d , were also estimated from sedimentation equilibrium data, and are in agreement with the results from MALDI-TOF-MS.

Crystallization and preliminary structural analysis of the giant haemoglobin from Glossoscolex paulistus at 3.2 Å.

Glossoscolex paulistus is a free-living earthworm encountered in south-east Brazil. Its oxygen transport requirements are undertaken by a giant extracellular haemoglobin, or erythrocruorin (HbGp), which has an approximate molecular mass of 3.6 MDa and, by analogy with its homologue from Lumbricus terrestris (HbLt), is believed to be composed of a total of 180 polypeptide chains. In the present work the full 3.6 MDa particle in its cyanomet state was purified and crystallized using sodium citrate or PEG8000 as precipitant. The crystals contain one-quarter of the full particle in the asymmetric unit of the I222 cell and have parameters of a = 270.8 Å, b = 320.3 Å and c = 332.4 Å. Diffraction data were collected to 3.15 Å using synchrotron radiation on beamline X29A at the Brookhaven National Laboratory and represent the highest resolution data described to date for similar erythrocruorins. The structure was solved by molecular replacement using a search model corresponding to one-twelfth of its homologue from HbLt. This revealed that HbGp belongs to the type I class of erythrocruorins and provided an interpretable initial electron density map in which many features including the haem groups and disulfide bonds could be identified.

The Role of Endothelial Cell Injury in Thrombotic Microangiopathy

Thrombotic microangiopathy (TMA) refers to a clinical and pathologic syndrome in which endothelial injury results in the manifestations of thrombocytopenia, microangiopathic hemolytic anemia, and kidney injury. A host of causes may induce endothelial injury and TMA, including enteric bacterial toxins, deficiency or dysfunction of complement regulatory proteins, deficiency or inhibition of von Willebrand factor-cleaving proteases, and factors that inhibit endothelial cell proliferation and turnover. This has led specialists to concentrate on these specific inciting factors in terms of designing treatment and management. However, a key and less recognized factor is the underlying level of endothelial health. Many persons with hereditary causes may remain disease free for years or may never develop disease. Others with acute inciting events, such as Escherichia coli O157 enteritis, never manifest TMA. Experimental studies document the importance of specific factors, such as endothelial nitric oxide levels, in helping protect animals from TMA. This suggests that one might approach the management of TMA not simply with specific treatments aimed at the underlying hereditary cause or inciting event, but rather at general measures that may improve overall endothelial health. We propose studies to determine whether interventions that improve endothelial health, such as the administration of angiotensin-converting enzyme inhibitors, statins, vitamin C, allopurinol, or nitric oxide-producing drugs, may be able to prevent TMA, even in persons with underlying hereditary conditions that otherwise would predispose them to these diseases.

MR Imaging Depicts Oxidative Stress Induced by Methemoglobin

To correlate the effect of red blood cell hemoglobin on signal generation during magnetic resonance (MR) imaging and local oxidation of low-density lipoprotein (LDL). Informed consent was obtained from all volunteers participating in this study, which was approved by the research ethics board. T1 relaxometry of blood samples from six volunteers was performed. Lipid peroxidation was assayed by using thiobarbituric acid reactive species (TBARS) and fluorescence quenching of cis-parinaric acid. Two-tailed Student t tests were used to detect differences between means. A Pearson correlation coefficient was calculated to determine the linearity of the data. Lipid oxidation was significantly enhanced after addition of blood, according to results of the TBARS assay; greater oxidation occurred with ferric than with ferrous blood. The cis-parinaric acid assay demonstrated increased oxidative stress caused by extracellular as compared with intracellular ferric hemoglobin. MR imaging measures showed a T1 relaxivity that was 10 times higher for ferric than for ferrous forms of hemoglobin. Extracellular ferric hemoglobin is significantly more pro-oxidant and has higher T1 relaxivity than its ferrous counterparts. These results support the hypothesis that ferric methemoglobin-generated T1 high signal intensity reflects a pro-oxidant environment that, in the setting of vessel wall disease, might be proatherogenic.

Thermal stability of extracellular hemoglobin of Glossoscolex paulistus: Determination of activation parameters by optical spectroscopic and differential scanning calorimetric studies

Glossoscolex paulistus hemoglobin (HbGp) was studied by dynamic light scattering (DLS), optical absorption spectroscopy (UV–VIS) and differential scanning calorimetry (DSC). At pH 7.0, cyanomet-HbGp is very stable, no oligomeric dissociation is observed, while denaturation occurs at 56 °C, 4 °C higher as compared to oxy-HbGp. The oligomeric dissociation of HbGp occurs simultaneously with some protein aggregation. Kinetic studies for oxy-HbGp using UV–VIS and DLS allowed to obtain activation energy ( E a ) values of 278–262 kJ/mol (DLS) and 333 kJ/mol (UV–VIS). Complimentary DSC studies indicate that the denaturation is irreversible, giving endotherms strongly dependent upon the heating scan rates, suggesting a kinetically controlled process. Dependence on protein concentration suggests that the two components in the endotherms are due to oligomeric dissociation effect upon denaturation. Activation energies are in the range 200–560 kJ/mol. The mid-point transition temperatures were in the range 50–65 °C. Cyanomet-HbGp shows higher mid-point temperatures as well as activation energies, consistent with its higher stability. DSC data are reported for the first time for an extracellular hemoglobin.

Vasospasm after subarachnoid hemorrhage in haptoglobin 2-2 mice can be prevented with a glutathione peroxidase mimetic

Vasospasm after subarachnoid hemorrhage (SAH) is attributable to inflammation and oxidative stress associated with extracellular hemoglobin (Hb). Haptoglobin (Hp) binds free Hb and the Hp–Hb complex is cleared by macrophages, and the Hp-2 isoform of Hp is associated with more oxidative stress and more severe vasospasm. We hypothesized that treatment with an anti-oxidant, the glutathione peroxidase mimetic SYI-2074, would reduce vasospasm after SAH in Hp-2 mice. We found that SAH induced significant vasospasm in Hp-2 mice (lumen patency reduced to 65.9%), but no vasospasm was seen in mice that received SYI-2074 after SAH (lumen patency of 98.7%). We conclude that vasospasm after SAH in Hp-2 mice can be prevented with SYI-2074, suggesting that oxidative stress contributes significantly to vasospasm.

Isoelectric Point Determination for Glossoscolex paulistus Extracellular Hemoglobin: Oligomeric Stability in Acidic pH and Relevance to Protein−Surfactant Interactions

The extracellular hemoglobin from Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 MDa. It has a high oligomeric stability at pH 7.0 and low autoxidation rates, as compared to vertebrate hemoglobins. In this work, fluorescence and light scattering experiments were performed with the three oxidation forms of HbGp exposed to acidic pH. Our focus is on the HbGp stability at acidic pH and also on the determination of the isoelectric point (pI) of the protein. Our results show that the protein in the cyanomet form is more stable than in the other two forms, in the whole pH range. Our zeta-potential data are consistent with light scattering results. Average values of pI obtained by different techniques were 5.6 +/- 0.5, 5.4 +/- 0.2 and 5.2 +/- 0.5 for the oxy, met, and cyanomet forms. Dynamic light scattering (DLS) experiments have shown that, at pH 6.0, the aggregation (oligomeric) state of oxy-, met- and cyanomet-HbGp remains the same as that at pH 7.0. The interaction between the oxy-HbGp and ionic surfactants at pH 5.0 and 6.0 was also monitored in the present study. At pH 5.0, below the protein pI, the effects of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium chloride (CTAC) are inverted when compared to pH 7.0. For CTAC, in acid pH 5.0, no precipitation is observed, while for SDS an intense light scattering appears due to a precipitation process. HbGp interacts strongly with the cationic surfactant at pH 7.0 and with the anionic one at pH 5.0. This effect is due to the predominance, in the protein surface, of residues presenting opposite charges to the surfactant headgroups. This information can be relevant for the development of extracellular hemoglobin-based artificial blood substitutes.

Ligand changes in ferric species of the giant extracellular hemoglobin ofGlossoscolex paulistusas function of pH: correlations between redox, spectroscopic and oligomeric properties and general implications with different hemoproteins

The present review is focused on the relationship between oligomeric and heme properties of HbGp, emphasizing the characteristics that can be generalized to other hemoproteins. This study represents the state-of-the-art with respect to the approaches for investigating giant extracellular hemoglobins as well as the correlation between oligomeric assembly alterations and their consequent changes in the first coordination sphere. A wide introduction focused on the properties of this hemoglobin is developed. Indeed, this hemoprotein is considered an interesting prototype of blood substitute and biosensor due to its peculiar properties, such as resistance to autoxidation and oligomeric stability. Previous studies by our group employing UV-vis, EPR and CD spectroscopies have been revised in a complete approach, in agreement with recent and relevant data from the literature. In fact, a consistent and inter-related spectroscopic study is described propitiating a wide assignment of "fingerprint" peaks found in the techniques evaluated in this paper. This review furnishes physicochemical information regarding the identification of ferric heme species of hemoproteins and metallic complexes through their spectroscopic bands. This effort at the attribution of UV-vis, EPR and CD peaks is not restricted to HbGp, and includes a comparative analysis of several hemoproteins involving relevant implications regarding several types of iron-porphyrin systems.

Extracellular haemoglobin, oxidative stress and quality of red blood cells relative to perioperative blood salvage

The quality of blood-pack units in perioperative blood salvage was studied with respect to the presence of extracellular haemoglobin (Hb) and in terms of oxidative stress and the mechanical properties of red blood cells (RBC). The study was performed on blood-pack units and patients' blood after retransfusion. Results were compared to those obtained in patients prior to autotransfusion. Free Hb, non-protein thiols (as antioxidant marker) and markers of oxidative stress (conjugated dienes, thiobarbituric reactive substances and protein carbonyl content) were measured. The mechanical properties of RBC were evaluated by their osmotic fragility, membrane fluidity by measuring the fluorescence anisotropy of an extrinsic probe and permeability by measurement of extracellular K(+) and lactate dehydrogenase. Despite washing, extracellular Hb concentrations remained high (up to 0.7 g/L in blood-pack units) and was associated with a decrease of haptoglobin in patients, despite a concomitant inflammatory syndrome. In blood-pack units, we observed a decrease in antioxidant markers along with an increase in oxidative stress markers in association with an alteration of RBC membrane properties. Haemolysis must be limited during perioperative blood salvage in order to prevent exposure to oxidative stress and improve the efficiency of autotransfusion.

Differential Induction of Renal Heme Oxygenase and Ferritin in Ascorbate and Nonascorbate Producing Species Transfused with Modified Cell-Free Hemoglobin

Abstract Heme catabolism and iron sequestration systems play an important role in regulating the response to extracellular hemoglobin (Hb). We previously reported that extracellular Hb oxidizes more readily in the circulation of guinea pigs, a nonascorbate (AA)-producing species with similar plasma and tissue antioxidant status to humans, compared to rats, an AA-producing species. To determine whether these two species exhibit differences in heme catabolism and iron sequestration at the level of the kidney, we examined heme oxygenase (HO), H- and L-ferritin expression, nonheme iron deposition, and renal AA content following transfusion with polymerized bovine hemoglobin (HbG). Both species showed similar rates of hemoglobinuria but urinary HbG was significantly more oxidized in guinea pigs. HbG enhanced HO activity in both species but appeared greater and more sustained in guinea pigs. Conversely, rats showed a greater and more rapid induction of H- and L-ferritin as well as greater iron accumulation and AA content. Furthermore, ferrous and ferric iron deposits were detected in rats while only ferric iron was observed in guinea pigs. These findings suggest significant differences in the renal handling of HbG which may be important for understanding how endogenous antioxidant defenses may modulate the renal response to extracellular Hb.

Origin and Evolution of the Unique Tetra-Domain Hemoglobin from the Hydrothermal Vent Scale Worm Branchipolynoe

Hemoglobin is the most common respiratory pigment in annelids. It can be intra or extracellular, and this latter type can form large multimeric complexes. The hydrothermal vent scale worms Branchipolynoe symmytilida and Branchipolynoe seepensis express an extracellular tetra-domain hemoglobin (Hb) that is unique in annelids. We sequenced the gene for the single-domain and tetra-domain globins in these two species. The single-domain gene codes for a mature protein of 137 amino acids, and the tetra-domain gene codes for a mature protein of 552 amino acids. The single-domain gene has a typical three exon/two intron structure, with introns located at their typical positions (B12.2 and G7.0). This structure is repeated four times in the tetra-domain gene, with no bridge introns or linker sequences between domains. The phylogenetic position of Branchipolynoe globins among known annelid globins revealed that, although extracellular, they cluster within the annelid intracellular globins clade, suggesting that the extracellular state of these Hbs is the result of convergent evolution. The tetra-domain structure likely resulted from two tandem duplications, domain 1 giving rise to domain 2 and after this the two-domain gene duplicated to produce domains 3 and 4. The high O(2) affinity of Branchipolynoe extracellular globins may be explained by the two key residues (B10Y and E7Q) in the heme pocket in each of the domains of the single and tetra-domain globins, which have been shown to be essential in the oxygen-avid Hb from the nematode Ascaris suum. This peculiar globin evolutionary path seems to be very different from other annelid extracellular globins and is most likely the product of evolutionary tinkering associated with the strong selective pressure to adapt to chronic hypoxia that characterizes hydrothermal vents.

Sequestration of extracellular hemoglobin within a haptoglobin complex decreases its hypertensive and oxidative effects in dogs and guinea pigs

Release of hemoglobin (Hb) into the circulation is a central pathophysiologic event that contributes to morbidity and mortality in chronic hemolytic anemias and severe malaria. These toxicities arise from Hb-mediated vasoactivity, possibly due to NO scavenging and localized tissue oxidative processes. Currently, there is no established treatment that targets circulating extracellular Hb. Here, we assessed the role of haptoglobin (Hp), the primary scavenger of Hb in the circulation, in limiting the toxicity of cell-free Hb infusion. Using a canine model, we found that glucocorticoid stimulation of endogenous Hp synthesis prevented Hb-induced hemodynamic responses. Furthermore, guinea pigs administered exogenous Hp displayed decreased Hb-induced hypertension and oxidative toxicity to extravascular environments, such as the proximal tubules of the kidney. The ability of Hp to both attenuate hypertensive responses during Hb exposure and prevent peroxidative toxicity in extravascular compartments was dependent on Hb-Hp complex formation, which likely acts through sequestration of Hb rather than modulation of its NO- and O2-binding characteristics. Our data therefore suggest that therapies involving supplementation of endogenous Hb scavengers may be able to treat complications of acute and chronic hemolysis, as well as counter the adverse effects associated with Hb-based oxygen therapeutics.

Issuing of blood components dispensed in syringes and bar code–based pretransfusion check at the bedside for pediatric patients

The pretransfusion check at the bedside is the most critical step for the prevention of mistransfusion in pediatric patients, as well as in adults. The objective of this study was to assess whether a bar code-based patient-blood unit identification system could be applied to the pretransfusion check at the bedside for the issuing of blood dispensed in syringes. The issuing of blood components dispensed in syringes and the bar code-based pretransfusion check at the bedside were initiated for pediatric patients in May 2003. The number of blood components transfused to pediatric patients and rate of compliance with electronic bedside verification for blood dispensed in syringes were determined. Several variables in blood samples that were freshly collected, irradiated, split into 20-mL aliquots, and stored in syringes at 4 degrees C were measured. Between May 2003 and April 2007, a total of 3957 blood components (10% of all transfusions) were administered to pediatric patients without a single mistransfusion, of which 871 (22%) were issued by dispensing in syringes. The compliance rate with electronic bedside verification for blood dispensed in syringes was 99%. The supernatant potassium concentrations and extracellular free hemoglobin in blood samples stored in syringes at 4 degrees C for 24 hours were 14 +/- 1.3 mmol/L and less than 0.3 g/L, respectively, and these were considered to be acceptable for transfusion to pediatric patients. The bar code-based identification system that we used was fully applicable to the pretransfusion check at the bedside for blood dispensed in syringes.

Enhanced expression of haemoglobin scavenger receptor in accumulated macrophages of culprit lesions in acute coronary syndromes

Effective clearance of extracellular haemoglobin (Hb) is thought to limit systemic oxidative heme toxicity, which is presumed to contribute to the pathogenesis of plaque instability. We immunohistochemically examined the relationship between intraplaque haemorrhage, 4-HNE (4-hydroxy-2-nonenal), an index of lipid peroxidation, and the Hb scavenger receptor (CD163), using coronary atherectomy specimens from 74 patients with stable angina pectoris (SAP, n = 39) or unstable angina pectoris (UAP, n = 35). Atherectomy samples were stained with antibodies against glycophorin A (a protein specific to erythrocyte membranes), CD31, 4-HNE, and CD163. Quantitative analysis demonstrated that glycophorin A-positive areas, 4-HNE-positive macrophage score, and CD163-positive macrophage score in UAP patients were significantly higher (glycophorin A, P < 0.0001; 4-HNE-positive macrophage score, P < 0.0001; CD163-positive macrophage score, P < 0.0005) than in SAP patients. The percentage of the glycophorin A-positive area showed a significant positive correlation with the number of CD31-positive microvessels and the 4-HNE-positive macrophage score (microvessels, R = 0.59, P < 0.0001; 4-HNE, R = 0.59, P < 0.0001). Moreover, the CD163-positive macrophage score was positively correlated with glycophorin A-positive area and the 4-HNE-positive macrophage score (glycophorin A, R = 0.58, P < 0.0001; 4-HNE, R = 0.53, P < 0.0001). These findings suggest a positive association among intraplaque haemorrhage, enhanced expression of Hb scavenger receptor, and lipid peroxidation in human unstable plaques.

Erythrocyte Hemolysis and Hemoglobin Oxidation Promote Ferric Chloride-induced Vascular Injury

The release of redox-active iron and heme into the blood-stream is toxic to the vasculature, contributing to the development of vascular diseases. How iron induces endothelial injury remains ill defined. To investigate this, we developed a novel ex vivo perfusion chamber that enables direct analysis of the effects of FeCl(3) on the vasculature. We demonstrate that FeCl(3) treatment of isolated mouse aorta, perfused with whole blood, was associated with endothelial denudation, collagen exposure, and occlusive thrombus formation. Strikingly exposing vessels to FeCl(3) alone, in the absence of perfused blood, was associated with only minor vascular injury. Whole blood fractionation studies revealed that FeCl(3)-induced vascular injury was red blood cell (erythrocyte)-dependent, requiring erythrocyte hemolysis and hemoglobin oxidation for endothelial denudation. Overall these studies define a unique mechanism of Fe(3+)-induced vascular injury that has implications for the understanding of FeCl(3)-dependent models of thrombosis and vascular dysfunction associated with severe intravascular hemolysis.

Attenuated erythrocytic ATP release is caused by extracellular hemoglobin.

Local regulation of microvascular flow is a complex process evolved to match oxygen supply to the metabolic demands of the tissue. The RBC has been proposed as an O2 sensor through a direct link between the desaturation of intracellular hemoglobin (Hb) and ATP release, leading to vasodilation1. This theory suggests a link between alterations of O2 transport parameters in the plasma space and vasoactivity. Based on theoretical calculations of O2 extraction from RBCs in the presence of Hb‐based oxygen carriers (HBOCs), we hypothesized that that the addition of cell‐free Hb to the extracellular space provides a supplementary O2 source that reduces RBC desaturation and ATP release. In this study, the total O2 concentration of RBC/HBOC suspensions was lowered by additions of deoxygenated saline, and then assayed for ATP production via the luciferin‐luciferase bioluminescence assay. When an acellular Hb cross‐linked between α subunits (αα Hb, p50 = 33 mmHg) was added to the red cell suspension, ATP production was significantly less in the absence of HBOC. These results provide a possible mechanism for HBOC‐induced vasoactivity through delayed release of red cell ATP.1 Ellsworth, ML (2000) Acta Physiol Scand 168: 551‐559.

On the Fate of Extracellular Hemoglobin and Heme in Brain

Intracerebral hemorrhage (ICH) is a major cause of disability in adults worldwide. The pathophysiology of this syndrome is complex, involving both inflammatory and redox components triggered by the extravasation of blood into the cerebral parenchyma. Hemoglobin, heme, and iron released therein seem be important in the brain damage observed in ICH. However, there is a lack of information concerning hemoglobin traffic and metabolism in brain cells. Here, we investigated the fate of hemoglobin and heme in cultured neurons and astrocytes, as well as in the cortex of adult rats. Hemoglobin was made traceable by conjugation to Alexa 488, whereas a fluorescent heme analogue (tin-protoporphyrin IX) was prepared to allow heme tracking. Using fluorescence microscopy we observed that neurons were more efficient in uptake hemoglobin and heme than astrocytes. Exposure of cortical neurons to hemoglobin or heme resulted in an oxidative stress condition. Viability assays showed that neurons were more susceptible to both hemoglobin and heme toxicity than astrocytes. Together, these results show that neurons, rather than astrocytes, preferentially take up hemoglobin-derived products, indicating that these cells are actively involved in the ICH-associated brain damage.

Haptoglobin preserves the CD163 hemoglobin scavenger pathway by shielding hemoglobin from peroxidative modification

Detoxification and clearance of extracellular hemoglobin (Hb) have been attributed to its removal by the CD163 scavenger receptor pathway. However, even low-level hydrogen peroxide (H(2)O(2)) exposure irreversibly modifies Hb and severely impairs Hb endocytosis by CD163. We show here that when Hb is bound to the high-affinity Hb scavenger protein haptoglobin (Hp), the complex protects Hb from structural modification by preventing alpha-globin cross-links and oxidations of amino acids in critical regions of the beta-globin chain (eg, Trp15, Cys93, and Cys112). As a result of this structural stabilization, H(2)O(2)-exposed Hb-Hp binds to CD163 with the same affinity as nonoxidized complex. Endocytosis and lysosomal translocation of oxidized Hb-Hp by CD163-expressing cells were found to be as efficient as with nonoxidized complex. Hp complex formation did not alter Hb's ability to consume added H(2)O(2) by redox cycling, suggesting that within the complex the oxidative radical burden is shifted to Hp. We provide structural and functional evidence that Hp protects Hb when oxidatively challenged with H(2)O(2) preserving CD163-mediated Hb clearance under oxidative stress conditions. In addition, our data provide in vivo evidence that unbound Hb is oxidatively modified within extravascular compartments consistent with our in vitro findings.

Characterization of morphological response of red cells in a sucrose solution

The dynamics of red cell shape changes following transfer into sucrose media having a low chloride content was studied. Based on a large number of measurements, six types of morphological response (MR), differing both in the degree of shape changes and the time course of the process, were identified. The most prominent type of response is a triphasic sequence of shape changes consisting of a fast transformation into a sphere (phase 1), followed by restoration of the discoid shape (phase 2) and final transformation into spherostomatocytes (phase 3), with individual parameters which could vary significantly. It was found that individual morphological response exhibited day to day variations, depending on the initial state of the red blood cells and the donor, but to a larger extent depended on the composition of the sucrose solution, such as concentration and type of buffers, the presence of EDTA, calcium, as well as very small amounts of extracellular hemoglobin. MR shows strong pH and ionic strength dependence. Low pH inhibited phase 1 and high pH changed dramatically the time course of the response. Increasing ionic strength inhibited all phases of MR, and at concentrations above 10–20 mM NaCl it was fully suppressed. Tris and phosphate were also inhibitory whereas HEPES, MOPS and Tricine were less effective. MR occurred also in hypertonic or hypotonic sucrose solutions, with exception of extreme hypotonicity due to volume restrictions. It is concluded that strong membrane depolarization per se is not a causal factor leading to MR, and its different phases could be regulated independently. For some types of morphological response the fast shape transformation from sphere to disc and back to sphere occurs within a 10 s time interval and could be accelerated several fold in the presence of a small amount of hemoglobin. It is suggested that MR represents a type of general cell reaction that occurs upon exposure to low ionic strength.