Extracellular Hemoglobin Research Articles

Mechanisms of haptoglobin protection against hemoglobin peroxidation triggered endothelial damage

Extracellular hemoglobin (Hb) has been recognized as a disease trigger in hemolytic conditions such as sickle cell disease, malaria, and blood transfusion. In vivo, many of the adverse effects of free Hb can be attenuated by the Hb scavenger acute-phase protein haptoglobin (Hp). The primary physiologic disturbances that can be caused by free Hb are found within the cardiovascular system and Hb-triggered oxidative toxicity toward the endothelium has been promoted as a potential mechanism. The molecular mechanisms of this toxicity as well as of the protective activities of Hp are not yet clear. Within this study, we systematically investigated the structural, biochemical, and cell biologic nature of Hb toxicity in an endothelial cell system under peroxidative stress. We identified two principal mechanisms of oxidative Hb toxicity that are mediated by globin degradation products and by modified lipoprotein species, respectively. The two damage pathways trigger diverse and discriminative inflammatory and cytotoxic responses. Hp provides structural stabilization of Hb and shields Hb's oxidative reactions with lipoproteins, providing dramatic protection against both pathways of toxicity. By these mechanisms, Hp shifts Hb's destructive pseudo-peroxidative reaction to a potential anti-oxidative function during peroxidative stress.

A combination of dynamic light scattering and polarized resonance Raman scattering applied in the study of Arenicola Marina extracellular hemoglobin

Arenicola Marina extracellular hemoglobin (Hbl Hb) is considered to be a promising candidate as a blood substitute. To entangle some of the properties of extracellular giant hexagonal bilayer hemoglobin (Hbl Hb) of Arenicola Marina, we combined polarized resonance Raman scattering (532 nm excitation) with dynamic light scattering (DLS) (632.8 nm). An analysis of the depolarization ratio of selected a(2g) skeletal modes of the heme in native Hbl Hb and porcine Hb, shows that the distortion of the heme group away from its ideal fourfold symmetry is much smaller for heme groups bound in the Hbl Hb than for heme groups bound in porcine Hb. Using DLS, the average hydrodynamic diameter (<d(h)>) of Hbl Hb was measured at pH = 5, 7, 8, 9, and 10. At pH = 5 to 7, the Hbl Hb was found in its native form with <d(h)> equal to 24.2 nm, while at pH = 8 and 9, a dissociation process starts to take place resulting in <d(h)> = 9 nm. At pH = 10, only large aggregates of fragmented Hbl Hb with <d(h)> larger than 1000 nm was detected, however, a comparison of the DLS results with the polarized resonance Raman scattering (RRS) revealed that the coupling between the fragments did not involve direct interaction between the heme groups, but also that the local heme environment seems to be comparable in the aggregates and in the native Hbl Hb. By comparing the unpolarized RRS results obtained for erythrocytes (RBC) with those for Hbl Hb, led us to the important conclusion that Hbl Hb is much easier photolyzed than porcine RBC.

Hemoglobin induces inflammation after preterm intraventricular hemorrhage by methemoglobin formation

BackgroundCerebral intraventricular hemorrhage (IVH) is a major cause of severe neurodevelopmental impairment in preterm infants. To date, no therapy is available that prevents infants from developing serious neurological disability following IVH. Thus, to develop treatment strategies for IVH, it is essential to characterize the initial sequence of molecular events that leads to brain damage. In this study, we investigated extracellular hemoglobin (Hb) as a causal initiator of inflammation in preterm IVH.MethodsUsing a preterm rabbit pup model, we investigated the molecular mechanisms and events following IVH. We also characterized the concentrations of cell-free Hb metabolites and pro-inflammatory mediators in the cerebrospinal fluid (CSF) of preterm human infants and rabbit pups. Finally, Hb metabolites were evaluated as causal initiators of inflammation in primary rabbit astrocyte cell cultures.ResultsFollowing IVH in preterm rabbit pups, the intraventricular CSF concentration of cell-free methemoglobin (metHb) increased from 24 to 72 hours and was strongly correlated with the concentration of TNFα at 72 hours (r2 = 0.896, P <0.001). Also, the mRNA expression of TNFα, IL-1β, and Toll-like receptor-4 and TNFα protein levels were significantly increased in periventricular tissue at 72 hours, which was accompanied by extensive astrocyte activation (that is, glial fibrillary acidic protein (GFAP)staining). Furthermore, exposure of primary rabbit astrocyte cell cultures to metHb caused a dose-dependent increase in TNFα mRNA and protein levels, which was not observed following exposure to oxyhemoglobin (oxyHb) or hemin. Finally, a positive correlation (r2 = 0.237, P <0.03) between metHb and TNFα concentrations was observed in the CSF of preterm human infants following IVH.ConclusionsFollowing preterm IVH, increased metHb formation in the intraventricular space induces expression of pro-inflammatory cytokines. Thus, the formation of metHb might be a crucial initial event in the development of brain damage following preterm IVH. Accordingly, removal, scavenging, or neutralization of Hb could present a therapeutic opportunity and plausible approach to decreasing the damage in the immature brain following preterm IVH.

Autoxidation and Oxygen Binding Properties of Recombinant Hemoglobins with Substitutions at the αVal-62 or βVal-67 Position of the Distal Heme Pocket

The E11 valine in the distal heme pocket of either the α- or β-subunit of human adult hemoglobin (Hb A) was replaced by leucine, isoleucine, or phenylalanine. Recombinant proteins were expressed in Escherichia coli and purified for structural and functional studies. (1)H NMR spectra were obtained for the CO and deoxy forms of Hb A and the mutants. The mutations did not disturb the α1β2 interface in either form, whereas the H-bond between αHis-103 and βGln-131 in the α1β1 interfaces of the deoxy α-subunit mutants was weakened. Localized structural changes in the mutated heme pocket were detected for the CO form of recombinant Hb (rHb) (αV62F), rHb (βV67I), and rHb (βV67F) compared with Hb A. In the deoxy form the proximal histidyl residue in the β-subunit of rHb (βV67F) has been altered. Furthermore, the interactions between the porphyrin ring and heme pocket residues have been perturbed in rHb (αV62I), rHb (αV62F), and rHb (βV67F). Functionally, the oxygen binding affinity (P50), cooperativity (n50), and the alkaline Bohr Effect of the three α-subunit mutants and rHb (βV67L) are similar to those of Hb A. rHb (βV67I) and rHb (βV67F) exhibit low and high oxygen affinity, respectively. rHb (βV67F) has P50 values lower that those reported for rHb (αL29F), a B10 mutant studied previously in our laboratory (Wiltrout, M. E., Giovannelli, J. L., Simplaceanu, V., Lukin, J. A., Ho, N. T., and Ho, C. (2005) Biochemistry 44, 7207-7217). These E11 mutations do not slow down the autoxidation and azide-induced oxidation rates of the recombinant proteins. Results from this study provide new insights into the roles of E11 mutants in the structure-function relationship in hemoglobin.

Hydroxyurea reduces leukocyte interactions with the vessel wall in a haemolytic mouse model via a possible NO/cGMP-mediated effect

Background Haemolysis occurs in a number of haematologic and non-haematologic diseases, including sickle cell disease, malaria and sepsis. Elevated extracellular haemoglobin (Hb) can trigger specific events that are associated with adverse clinical outcomes. For example, in sickle cell disease, cell-free haemoglobin has been proposed to reduce nitric oxide (NO) bioavailability and induce vascular oxidative stress and, possibly, inflammation [1]. Hydroxyurea, a drug used commonly as a therapy for sickle cell disease, may exert some of its effects by acting as a NO donor, in vivo [2]. This study aimed to compare the effects of haemolytic and inflammatory stimuli on blood vessel leukocyte recruitment in vivo. The effects of the acute administration of hydroxyurea (HU) or an NO donor on these alterations were also investigated. Methods Inflammatory or hemolytic processes were induced in C57BL/6 mice (2-4 months old) and cremaster muscles of mice were prepared for intravital microscopy (IVM), according to protocols described in figure 1. Plasma-free Hb was measured with Drabkin’s solution. Results C57BL/6 mice that received water i.v. (H2O mice) presented marked vascular haemolysis after 15 min; levels of plasma Hb were more than doubled in H2O mice, compared to mice that receiving PBS i.v. (2.57±0.43g/dL, 1.08 ±0.08g/dL Hb, respectively, p<0.01, n=3-5), and resembled those of SCD mice (data not shown). In contrast, C57BL/6 mice (n=3) that received an inflammatory stimulus (TNFa) showed no alteration in plasma free Hb levels (1.29 ±0.43 g/dL, 1.23±0.09 g/dL; PBS and TNF-a, respectively). IVM demonstrated that both the inflammatory and haemolytic stimuli induced leukocyte adhesion to vessel walls (3.57±0.84; 11.07±0.92; 7.25±1.20 [100µm -1 ]f or PBS, TNF-a and H2O, respectively, p<0.05, N=15-30 venules), as well as leukocyte extravasation (1.07±0.17; 4.02±0.39; 2.97±0.49 [per 100 µm x 50 µm] for PBS, TNF-a and H2O, respectively, p<0.05, N=15-30 venules). Surprisingly, when HU was given simultaneously with TNF-a or following H2O, this drug was able to prevent leukocyte recruitment in both models, reducing both leukocyte adhesion (7.40±0.47; 2.79±0.34 [100 µm -1 ] for TNF-a+PBS and TNF-a+HU, respectively, p<0.0001, n=30-60 venules; and 7.84±0.73; 2.41±0.37 for H2O+PBS and H2O+HU, p<0.0001, n=20-30 venules) and leukocyte extravasation (3.88±0.26; 1.22±0.18 for TNF-a+PBS and TNF-a+HU, p<0.0001, n=30-60 venules and 2.98±0.32; 1.94±0.21 for H2O+PBS and H2O+HU, p<0.0001, n=20-30 venules) and increased leukocyte rolling (18.11±1.58; 25.48±5.01 for TNF-a+PBS and TNF-a+HU, respectively, p<0.05, n=30-60 venules and 11.56±1.47; 32.47±3.54 for H2O+PBS and H2O+HU, p<0.0001, n=20-30 venules). Additionally, DEANO was also able to reverse the inflammatory process installed by TNF-a, reducing leukocyte adhesion (10.24 ±1.49; 11.45±1.40 for TNF-a before and after vehicle control, n=21-22 venules; 7.63±0.63; 3.76±0.41 for TNF-a, before and after DEANO, p<0.01; n=29-35 venules) and extravasation (1.95±0.29; 3.23±0.44 before and after vehicle control, p<0.05, n=21-22 venules; 2.49±0.25; 2.24±0.24 before and after DEANO, n=29-35 venules).

The impact of anti-inflammatory cytokines provoked by CD163 positive macrophages on ventricular functional recovery after myocardial infarction

Present study aimed to investigate the impact of anti-inflammatory cytokines provoked by the hemoglobin scavenger receptor, CD163, on left ventricular (LV) functional recovery after successful reperfusion in patients with acute myocardial infarction (AMI). Intraplaque hemorrhage accelerates plaque destabilization. Extracellular hemoglobin is cleared by CD163, a macrophage scavenger receptor. This process provokes secretion of anti-inflammatory atheroprotective cytokine, interleukin (IL)-10. In 40 patients with the first AMI, coronary atherothrombotic debris was retrieved during percutaneous coronary intervention (PCI), stained with antibodies to CD163 and IL-10. LV function was determined by echocardiography before PCI and 6months after PCI. %CD163 was defined as ratio of CD163 (+)-cells to whole cells. %IL-10 was expressed as the ratio of positively stained areas per total tissue. Patients were divided into two groups depending on the amount of CD163 (+)-cells: CD163>10% (CD163high, n=20) and CD163≤10% (CD163low, n=20). CD163high group had significantly higher %IL-10. Final thrombolysis in myocardial infarction (TIMI) flow grade was significantly lower in CD163high group. In subgroups with the final TIMI-3 flow (CD163high-Reflow, n=15 and CD163low-Reflow, n=20), the time to reperfusion, infarct size, LV dimensions and fractional shortening (%FS) before PCI were similar. Significant correlation was observed between %IL10 and changes in LV dimensions (diastole, r=-0.49, P=0.01; systole, r=-0.65, P<0.01) or %FS (r=0.51, P<0.01) at 6months after PCI. Plaque with CD163(+)-macrophages could impair distal flow after primary PCI. However, CD163(+)-macrophages enhance the anti-inflammatory cytokine expression that aids in ventricular functional recovery if distal flow can be achieved by successful reperfusion.

PH effect upon HbGp oligomeric stability: characterization of the dissociated species by AUC and DLS studies

Glossoscolex paulistus (HbGp) extracellular hemoglobin is a giant oligomeric protein. It is constituted by 144 heme containing subunits and non-heme structures (linkers), with a total molecular mass of 3.6MDa. AUC and DLS studies were developed for three HbGp forms, oxy-, met- and cyanomet-, at several pH values, in order to characterize the species in solution upon oligomeric dissociation. Isolated SEC fractions, trimer and dodecamer, are less stable as compared to the whole oxy-HbGp. The monomer d displays a large thermal stability up to 59°C. Hydrodynamic properties of the isolated subunits are very similar to those described for them in the whole protein, in the presence of urea or at pH 10.0. The degree of HbGp oligomeric dissociation, in alkaline pH, depends significantly on the iron oxidation state. Also on the ligand coordinated to the heme iron. Thus, at pH 8.0, the oxy-HbGp is partially dissociated, while the met-form is fully dissociated. The cyanomet-HbGp remains undissociated. Our present results show that the effect of pH on the HbGp oligomeric stability is similar to that associated to the urea-induced unfolding. Simultaneous use of AUC and DLS allowed the characterization of the species in the SEC fractions of isolated HbGp subunits.

Perioperative release of pro-regenerative biochemical signals from human renal allografts subjected to ischemia–reperfusion injury

Complement-derived molecules modulate the intensity of renal ischemia-reperfusion injury and may lead to the generation of biochemical signals [such as stromal-derived factor-1 (SDF-1) or sphingosine-1-phosphate (S1P)], which stimulate tissue/organ regeneration after injury. We tested the association between perioperative C5b-9/membrane attack complex (MAC) levels and intensified erythrocyte lysis, and asked whether significant changes in the levels of pro-regenerative substances occur during the early phase of renal allograft reperfusion. Seventy-five recipients were enrolled and divided into the early, slow, and delayed graft function (DGF) groups. Perioperative blood samples were collected from the renal vein during consecutive minutes of reperfusion. Extracellular hemoglobin (eHb), albumin (plasma S1P transporter), 8-iPF2α-III isoprostane, SDF-1 and S1P concentrations were measured. Throughout the reperfusion period, erythrocyte lysis intensified and was most pronounced in the DGF group. However, perioperative eHb levels did not correlate significantly with C5b-9/MAC values, but rather with the intensity of oxidative stress. No significant changes were observed in S1P, its plasma transporter (albumin) or SDF-1 levels, which were relatively low in all groups throughout the reperfusion period. Our study therefore demonstrates that no known biochemical signal for bone marrow-derived stem cell mobilization is released from human renal allografts to the periphery during the early phase of reperfusion.

Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins

AbstractHemolysis occurs in many hematologic and nonhematologic diseases. Extracellular hemoglobin (Hb) has been found to trigger specific pathophysiologies that are associated with adverse clinical outcomes in patients with hemolysis, such as acute and chronic vascular disease, inflammation, thrombosis, and renal impairment. Among the molecular characteristics of extracellular Hb, translocation of the molecule into the extravascular space, oxidative and nitric oxide reactions, hemin release, and molecular signaling effects of hemin appear to be the most critical. Limited clinical experience with a plasma-derived haptoglobin (Hp) product in Japan and more recent preclinical animal studies suggest that the natural Hb and the hemin-scavenger proteins Hp and hemopexin have a strong potential to neutralize the adverse physiologic effects of Hb and hemin. This includes conditions that are as diverse as RBC transfusion, sickle cell disease, sepsis, and extracorporeal circulation. This perspective reviews the principal mechanisms of Hb and hemin toxicity in different disease states, updates how the natural scavengers efficiently control these toxic moieties, and explores critical issues in the development of human plasma–derived Hp and hemopexin as therapeutics for patients with excessive intravascular hemolysis.

A shortcut mechanism of innate immune defense by a solitary PRR

Event Abstract Back to Event A shortcut mechanism of innate immune defense by a solitary PRR Jeak Ling Ding1* 1 National University of Singapore, Biological Sciences, Singapore The accepted paradigm about an innate immune response is a multi-stepped process - from recognition of the invading microbe by pathogen-recognition receptors (PRRs) to signal transduction, to the production of antimicrobial effectors by the immune cells. Contrary to this belief, we found that a PRR can act singly via a direct shortcut process, bypassing multiple cascades of reactions. We showed that the extracellular hemoglobin (Hb) acts as a solitary frontline defense PRR, directly recognizing pathogens and eliciting powerful antimicrobial potencies. This innate immune response phenomenon is evolutionarily entrenched for 500 million years, from the limulus to humans. While the invading microbe cleaves the cell-free hemoglobin to extract an iron-rich meal for its survival and further invasion, the host exploits the intruding microbe’s proteases and PAMPs to produce toxic reactive oxygen species (ROS) that effectively kills the pathogen. In this process, the hemoglobin structure-function is rapidly reprogrammed to expose multiple dual antimicrobial activities and its redox reactivity is subsequently suppressed by plasma antioxidants, thus protecting the host from ROS-induced cytotoxicity. Furthermore, we demonstrated that the monocytes efficiently import the redox active Hb via a (a) novel endocrine loop of CD163 receptor recycling and (b) paracrine communication with vascular endothelial cells. Acknowledgements We thank the MoE and A*STAR BMRC for financial support. References Jiang N, Tan NS, Ho B and Ding JL. Respiratory protein-generated reactive oxygen species as an antimicrobial strategy. Nature Immunology 8 (2007): 1114-1122 Du RJ, Ho B and Ding JL. Rapid reprogramming of haemoglobin structure-function exposes multiple dual-antimicrobial potencies. EMBO J 29 (2010): 632-642 Bahl N, Du R, Winarsih I, Ho B, Tucker-Kellogg L, Ho B and Ding JL. Delineation of LPS-binding sites of hemoglobin - from in silico predictions to biophysical characterization. J. Biol. Chem 286 (2011): 37793-37803. Du R, Winarsih I, Ho B and Ding JL. Lipid-free apolipoprotein A1 exerts an antioxidative role against cell-free hemoglobin. Am. J. Clin. Exp. Immunol. 1 (2012): 33-48. Subramanian K, Du RJ, Tan NS, Ho B and Ding JL. CD163 and IgG co-defend against cytotoxic hemoglobin via autocrine and paracrine mechanisms. J. Immunol. (in press) Keywords: Innate immune defense, Pathogen pattern-recognition receptors (PRRs), Hemoglobin, Reactive Oxygen Species, Host-Pathogen Interactions Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Ding J (2013). A shortcut mechanism of innate immune defense by a solitary PRR. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00433 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 02 Apr 2013; Published Online: 22 Aug 2013. * Correspondence: Prof. Jeak Ling Ding, National University of Singapore, Biological Sciences, Singapore, 117543, Singapore, dbsdjl@nus.edu.sg Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Jeak Ling Ding Google Jeak Ling Ding Google Scholar Jeak Ling Ding PubMed Jeak Ling Ding Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Chloroquine Interference with Hemoglobin Endocytic Trafficking Suppresses Adaptive Heme and Iron Homeostasis in Macrophages: The Paradox of an Antimalarial Agent

The CD163 scavenger receptor pathway for Hb:Hp complexes is an essential mechanism of protection against the toxicity of extracellular hemoglobin (Hb), which can accumulate in the vasculature and within tissues during hemolysis. Chloroquine is a lysosomotropic agent, which has been extensively used as an antimalarial drug in the past, before parasite resistance started to limit its efficacy in most parts of the world. More recent use of chloroquine is related to its immunomodulatory activity in patients with autoimmune diseases, which may also involve hemolytic disease components. In this study we examined the effects of chloroquine on the human Hb clearance pathway. For this purpose we developed a new mass-spectrometry-based method to specifically quantify intracellular Hb peptides within the endosomal-lysosomal compartment by single reaction monitoring (SRM). We found that chloroquine exposure impairs trafficking of Hb:Hp complexes through the endosomal-lysosomal compartment after internalization by CD163. Relative quantification of intracellular Hb peptides by SRM confirmed that chloroquine blocked cellular Hb:Hp catabolism. This effect suppressed the cellular heme-oxygenase-1 (HO-1) response and shifted macrophage iron homeostasis towards inappropriately high expression of the transferrin receptor with concurrent inhibition of ferroportin expression. A functional deficiency of Hb detoxification and heme-iron recycling may therefore be an adverse consequence of chloroquine treatment during hemolysis.

A Perspective on the Role of Extracellular Hemoglobin on the Innate Immune System

Host cell-derived danger-associated molecular patterns (DAMPs), such as the hemoglobin (Hb) can interact with the innate immune system either directly or through binding to pathogen-associated molecular patterns (PAMPs). Hemolysis occurs under various pathological conditions, leading to hemoglobinemia. In the extracellular environment, the Hb becomes a redox-reactive DAMP molecule. In severe hemolysis, the massive level of extracellular pro-oxidative Hb generates reactive oxygen species (ROS), which perturbs the innate immune homeostasis. The Hb also binds to PAMPs and triggers Toll-like receptor-mediated signal transduction. In this perspective, we review the roles of cell-free Hb in the innate immune system, focusing on the plausible interactions among Hb, pathogens, host cell components, and innate immune cells, all of which remain to be explored with experiential detail.

Understanding the Erythrocyte Storage Lesion

Understanding the Erythrocyte Storage Lesion

Prognostic Value of Total Bilirubin in Patients With ST-Segment Elevation Acute Myocardial Infarction Undergoing Primary Coronary Intervention

Previous studies have shown that the serum total bilirubin (TB) concentration was inversely related with stable coronary artery disease, diabetes mellitus, hypertension, and metabolic syndromes. The relation between TB levels and in-hospital and long-term outcomes in patients with ST-segment elevation myocardial infarction (STEMI) who undergo primary percutaneous coronary intervention (PCI) is not known. Data from 1,624 consecutive patients with STEMI who underwent primary PCI were evaluated. TB was measured after primary PCI, and the study population was divided into tertiles. The high TB group (n=450) was defined as a value in the upper third tertile (>0.9 mg/dl) and the low TB group (n= 1,174) as any value in the lower 2 tertiles (≤0.9 mg/dl). The in-hospital mortality rate was significantly greater in the high TB group than in the low TB group (4% vs 1.5%, p= 0.003). In the multivariate analyses, a significant association was noted between high TB levels and the adjusted risk of in-hospital cardiovascular mortality (odds ratio 3.24, 95% confidence interval 1.27 to 8.27, p= 0.014). In the receiver operating characteristic curve analysis, TB >0.90 mg/dl was identified as an effective cutpoint in patients with STEMI for in-hospital cardiovascular mortality (area under the curve 0.66, 95% confidence interval 0.55 to 0.76, p= 0.001). The mean follow-up period was 26.2 months. No differences wereseen in the long-term mortality rates between the 2 groups. In conclusion, high TBisindependently associated with in-hospital adverse outcomes in patients with STEMIwhoundergo primary PCI. However, no association was found with long-term mortality.

Urea-induced unfolding of Glossoscolex paulistus hemoglobin, in oxy- and cyanomet-forms: A dissociation model

The urea effect on the giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) stability was studied by analytical ultracentrifugation (AUC) and small angle X-ray scattering (SAXS). AUC data show that the sedimentation coefficient distributions curves c (S), at 1.0mol/L of urea, display a single peak at 57 S, associated to the undissociated protein. The increase in urea concentration, up to 4.0mol/L, induces the appearance of smaller species, due to oligomeric dissociation. The sedimentation coefficients and molecular masses are 9.2S and 204kDa for the dodecamer (abcd)3, 5.5S and 69kDa for the tetramer (abcd), 4.1S and 52kDa for the trimer (abc) and 2.0 S and 17kDa for the monomer d, respectively. SAXS data show initially a decrease in the I(0) values due to the oligomeric dissociation, and then, above 4.0mol/L of denaturant, for oxy-HbGp, and above 6.0mol/L for cyanomet-HbGp, an increase in the maximum dimension and gyration radius is observed, due to the unfolding process. According to AUC and SAXS data the HbGp unfolding is described by two phases: the first one, at low urea concentration, below 4.0mol/L, characterizes the oligomeric dissociation, while the second one, at higher urea concentration, is associated to the unfolding of dissociated species. Our results are complementary to a recent report based on spectroscopic observations.

Molecular Link between Intravascular Hemolysis and Vascular Occlusion in Sickle Cell Disease

Intravascular hemolysis is a major component of anemia in sickle cell disease (SCD). Plasma extracellular hemoglobin (ECHb) liberated by intravascular hemolysis has deleterious effects on the vasculature. ECHb scavenges nitric oxide (NO) and promotes the pathogenesis of several clinical events including pulmonary hypertension, priapism and non-hemorrhagic strokes. ECHb reduces the bioavailability of NO which down-regulates platelet activation, leading to platelet aggregation and vascular clot formation. Recently we have identified an additional mechanism whereby increased hemolysis can lead to a prothrombotic state in SCD by increasing the activity of von Willebrand factor (VWF), a multimeric plasma glycoprotein secreted by the endothelium. Our studies show that ECHb binds to the A2-domain on VWF at the proteolytic site of the metalloprotease, ADAMTS13, and blocks VWF cleavage in vitro. Elevated ECHb is associated with high levels of ultralarge and hyperactive VWF multimers in SCD patients' plasma. A sub-population of VWF multimers, bound to ECHb is hyperactive, and exists in greater quantity in SCD patients' plasma compared to normal controls. These results suggest a possible role for plasma ECHb in the accumulation of hyperactive VWF multimers in vivo that may mediate thrombotic and vasoocclusive complications in SCD patients.

Oxygen delivery during extreme anemia with ultra-pure earthworm hemoglobin

AimLumbricus terrestris (earthworm) erythrocruorin (LtEc) is a naturally occurring extracellular hemoglobin (Hb) with high molecular weight (3.6MDa), low autoxidation rate, and limited nitric oxide (NO) dioxygenation activity. These properties make LtEc a potential candidate for use as red blood cell (RBC) substitute, i.e. Hb-based oxygen carrier (HBOC). Previous studies have shown that small amounts of LtEc can be safely transfused into mice, rats, and hamsters without eliciting major side effects. Therefore, this study was designed to understand oxygen (O2) transport to tissues and systemic/microvascular hemodynamics induced by LtEc during anemic conditions. Main methodsHamsters fitted with dorsal window chambers were hemodiluted to 18% hematocrit (Hct) using 6g/dL dextran 70kDa (Dex70). Hemodilution was then continued to 11% Hct using 10g/dL LtEc, 6g/dL Dex70 or 10g/dL human serum albumin (HSA). Blood pressure, heart rate, blood gas parameters, microvascular hemodynamics, microvascular blood flow, functional capillary density (FCD), intravascular pO2 and perivascular pO2 were studied. Key findingsLtEc maintained blood pressure without inducing vasoconstriction while increasing microvascular perfusion and FCD relative to Dex70 and HSA. LtEc increased blood O2 carrying capacity and maintained systemic and microvascular parameters without decreasing arteriolar diameter or increasing vascular resistance with during extreme anemia. LtEc increased O2 delivery compared to conventional plasma expanders. SignificanceLtEc or synthetic molecules that replicate the characteristics of LtEc could be effective O2 carriers with potential to be used in transfusion medicine to prevent tissue anoxia resulting from severe anemia.

Identification of Hemopexin as an Anti-Inflammatory Factor That Inhibits Synergy of Hemoglobin with HMGB1 in Sterile and Infectious Inflammation

Hemoglobin is released from lysed RBCs in numerous clinical settings. High mobility group box 1 (HMGB1) is a nuclear and cytosolic DNA-binding protein released from injured cells that has been shown to play an important role in inducing inflammation. Because both of these endogenous molecules are frequently present in sites of necrosis and inflammation, we studied their interaction on the activation of macrophages. We report in this article that hemoglobin and HMGB1 synergize to activate mouse macrophages to release significantly increased proinflammatory cytokines. Addition of microbial ligands that activate through TLR2 or TLR4 resulted in further significant increases, in a "three-way" synergy between endogenous and microbial ligands. The synergy was strongly suppressed by hemopexin (Hx), an endogenous heme-binding plasma protein. The findings suggest that hemoglobin may play an important role in sterile and infectious inflammation, and that endogenous Hx can modulate this response. Administration of Hx may be beneficial in clinical settings characterized by elevated extracellular hemoglobin and HMGB1.

α1-Acid Glycoprotein Up-regulates CD163 via TLR4/CD14 Protein Pathway: POSSIBLE PROTECTION AGAINST HEMOLYSIS-INDUCED OXIDATIVE STRESS

CD163, a scavenger receptor that is expressed at high levels in the monocyte-macrophage system, is a critical factor for the efficient extracellular hemoglobin (Hb) clearance during hemolysis. Because of the enormous detrimental effect of liberated Hb on our body by its ability to induce pro-inflammatory signals and tissue damage, an understanding of the molecular mechanisms associated with CD163 expression during the acute phase response is a central issue. We report here that α(1)-acid glycoprotein (AGP), an acute phase protein, the serum concentration of which is elevated under various inflammatory conditions, including hemolysis, up-regulates CD163 expression in both macrophage-like differentiated THP-1 (dTHP-1) cells and peripheral blood mononuclear cells in a time- and concentration-dependent manner. Moreover, the subsequent induction of Hb uptake was also observed in AGP-treated dTHP-1 cells. Among representative acute phase proteins such as AGP, α(1)-antitrypsin, C-reactive protein, and haptoglobin, only AGP increased CD163 expression, suggesting that AGP plays a specific role in the regulation of CD163. Consistently, the physiological concentrations of AGP induced CD163, and the subsequent induction of Hb uptake as well as the reduction of oxidative stress in plasma were observed in phenylhydrazine-induced hemolytic model mice, confirming the in vivo role of AGP. Finally, AGP signaling through the toll-like receptor-4 (TLR4) and CD14, the common innate immune receptor complex that normally recognizes bacterial components, was identified as a crucial stimulus that induces the autocrine regulatory loops of IL-6 and/or IL-10 via NF-κB, p38, and JNK pathways, which leads to an enhancement in CD163 expression. These findings provide possible insights into how AGP exerts anti-inflammatory properties against hemolysis-induced oxidative stress.

Microcirculation and NO-CO Studies of a Natural Extracellular Hemoglobin Developed for an Oxygen Therapeutic Carrier

Extracellular soluble hemoglobins (Hbs) have long been studied for their possible use as safe and effective alternatives to blood transfusion. While remarkable progress has been made in the use of cell-free Hb as artificial oxygen carrier, significant problems remain, including susceptibility to oxidative inactivation and propensity to induce vasoconstriction. Hemarina-M101 is a natural giant extracellular hemoglobin (3600 kDa) derived from marine invertebrate (polychaete annelid). Hemarina-M101 is a biopolymer composed of 156 globins and 44 non-globin linker chains and formulated in a product called HEMOXYCarrier®. Prior work has shown Hemarina-M101 to possess unique anti-oxidant activity and a high oxygen affinity. Topload experiment with this product into rats did not revealed any effect on heart rate (HR) and mean arterial pressure (MAP). A pilot study with the hamster dorsal skinfold window chamber model showed absence of microvascular vasoconstriction and no significant impact on mean arterial blood pressure. In vitro nitric oxide (NO) and carbon monoxide (CO) reaction kinetics measurements show that Hemarina-M101 has different binding rates as compared to human Hb. These results revealed for the first time that the presence of this marine hemoglobin appears to have no vasoactivity at the microvascular level in comparison to others hemoglobin based oxygen carriers (HBOCs) developed so far and merits further investigation.