Abdu I. Alayash

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

Hemolysis 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.

Heme triggers TLR4 signaling leading to endothelial cell activation and vaso-occlusion in murine sickle cell disease

Treatment of sickle cell disease (SCD) is hampered by incomplete understanding of pathways linking hemolysis to vaso-occlusion. We investigated these pathways in transgenic sickle mice. Infusion of hemoglobin or heme triggered vaso-occlusion in sickle, but not normal, mice. Methemoglobin, but not heme-stabilized cyanomethemoglobin, induced vaso-occlusion, indicating heme liberation is necessary. In corroboration, hemoglobin-induced vaso-occlusion was blocked by the methemoglobin reducing agent methylene blue, haptoglobin, or the heme-binding protein hemopexin. Untreated HbSS mice, but not HbAA mice, exhibited ∼10% vaso-occlusion in steady state that was inhibited by haptoglobin or hemopexin infusion. Antibody blockade of adhesion molecules P-selectin, von Willebrand factor (VWF), E-selectin, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, platelet endothelial cell (EC) adhesion molecule 1, α4β1, or αVβ3 integrin prevented vaso-occlusion. Heme rapidly (5 minutes) mobilized Weibel-Palade body (WPB) P-selectin and VWF onto EC and vessel wall surfaces and activated EC nuclear factor κB (NF-κB). This was mediated by TLR4 as TAK-242 blocked WPB degranulation, NF-κB activation, vaso-occlusion, leukocyte rolling/adhesion, and heme lethality. TLR4(-/-) mice transplanted with TLR4(+/+) sickle bone marrow exhibited no heme-induced vaso-occlusion. The TLR4 agonist lipopolysaccharide (LPS) activated ECs and triggered vaso-occlusion that was inhibited by TAK-242, linking hemolysis- and infection-induced vaso-occlusive crises to TLR4 signaling. Heme and LPS failed to activate VWF and NF-κB in TLR4(-/-) ECs. Anti-LPS immunoglobulin G blocked LPS-induced, but not heme-induced, vaso-occlusion, illustrating LPS-independent TLR4 signaling by heme. Inhibition of protein kinase C, NADPH oxidase, or antioxidant treatment blocked heme-mediated stasis, WPB degranulation, and oxidant production. We conclude that intravascular hemolysis in SCD releases heme that activates endothelial TLR4 signaling leading to WPB degranulation, NF-κB activation, and vaso-occlusion.

Hemoglobin-based blood substitutes : oxygen carriers, pressor agents, or oxidants?

Hemoglobin-based blood substitutes are being developed as oxygen-carrying agents for the prevention of ischemic tissue damage and hypovolemic (low blood volume) shock. The ability of cell-free hemoglobin blood substitutes to affect vascular tone through the removal of nitric oxide has also prompted an evaluation of their usefulness for maintaining blood pressure in critically ill patients. Before the clinical potential of these substitutes can be fully realized, however, concerns remain as to the intrinsic toxicity of the hemoglobin molecule, particularly the interference of the heme prosthetic group with the tissue oxidant/antioxidant balance. This review provides some insights into the complex redox chemistry of hemoglobin and places an emphasis on how current knowledge may be exploited both to selectively enhance/suppress specific chemical reaction pathway(s) and to ultimately design safer hemoglobin-based therapeutics.

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.

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 Hbs 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.

Structural Basis of Peroxide-mediated Changes in Human Hemoglobin A NOVEL OXIDATIVE PATHWAY

Hydrogen peroxide (H2O2) triggers a redox cycle between ferric and ferryl hemoglobin (Hb) leading to the formation of a transient protein radical and a covalent hemeprotein cross-link. Addition of H2O2 to highly purified human hemoglobin (HbA0) induced structural changes that primarily resided within β subunits followed by the internalization of the heme moiety within α subunits. These modifications were observed when an equal molar concentration of H2O2 was added to HbA0 yet became more abundant with greater concentrations of H2O2. Mass spectrometric and amino acid analysis revealed for the first time that βCys-93 and βCys-112 were oxidized extensively and irreversibly to cysteic acid when HbA0 was treated with H2O2. Oxidation of further amino acids in HbA0 exclusive to the β-globin chain included modification of βTrp-15 to oxyindolyl and kynureninyl products as well as βMet-55 to methionine sulfoxide. These findings may therefore explain the premature collapse of the β subunits as a result of the H2O2 attack. Analysis of a tryptic digest of the main reversed phase-high pressure liquid chromatography fraction revealed two α-peptide fragments (α128 - α139) and a heme moiety with the loss of iron, cross-linked between αSer-138 and the porphyrin ring. The novel oxidative pathway of HbA0 modification detailed here may explain the diverse oxidative, toxic, and potentially immunogenic effects associated with the release of hemoglobin from red blood cells during hemolytic diseases and/or when cell-free Hb is used as a blood substitute.

Toxicities of hemoglobin solutions: in search of in‐vitro and in‐vivo model systems

Several hemoglobin‐based oxygen carriers (HBOCs) have been developed with a rationale focused on exploiting one or more physicochemical properties (e.g., oxygen affinity, molecular weight, viscosity, and colloid osmotic pressure) resulting from the chemical or recombinant modification of hemoglobin (Hb). Several chemically modified Hbs have reached late stages of clinical evaluation in the United States and Canada. These Hbs, in general, demonstrated mixed preclinical safety and efficacy, and reasonable safety in Phase I trials. However, as clinical development shifted into later stages, an undesirable safety and efficacy profile became clear in patient populations studied, and as a result some products were withdrawn from further clinical pursuit. Several questions still remain unanswered regarding the safety of Hb products for their proposed clinical indication(s). For example, 1) were preclinical studies predictive of clinical outcome? And, 2) were the most appropriate preclinical studies performed to predict clinical outcome?

Effects of polymerization on the hypertensive action of diaspirin cross-linked hemoglobin in rats

It is believed that the hypertensive effect of diaspirin crosslinked hemoglobin, a viable blood substitute, can be resolved by polymerization, which reduces the diffusion of this derivative into the interstitial space between nitric oxide-producing endothelium and the target vascular smooth muscle. We studied the systemic and renal responses to infusion of three cell-free human hemoglobins in anesthetized isovolemic rats: unmodified (HbA0), crosslinked (alpha-DBBF), and polymerized crosslinked (poly alpha-DBBF). HbA0 produced a significant increase in mean arterial blood pressure (MAP) throughout the 60-minute infusion. alpha-DBBF, on the other hand, produced a more marked and prolonged increase in MAP over 120 minutes. Only a moderate increase in MAP was observed in rats after a 30-minute infusion with poly alpha-DBBF. The extent of renal insufficiency produced by these proteins, as determined by the glomerular filtration rate, was in the following order: HbA0 > poly alpha-DBBF > alpha-DBBF. Infusion of poly alpha-DBBF, under hypovolemic but not isovolemic conditions in rats, produced an increase in heart rate, cardiac output, and stroke volume and a decrease in total peripheral resistance after 60 minutes. Chemical polymerization to increase the size of alpha-DBBF does not appear to improve its hemodynamic properties in rats, especially under partial exchange transfusion, a more clinically relevant indication for a hemoglobin-based blood substitute.

Ascorbate removes key precursors to oxidative damage by cell-free haemoglobin in vitro and in vivo

Haemoglobin initiates free radical chemistry. In particular, the interactions of peroxides with the ferric (met) species of haemoglobin generate two strong oxidants: ferryl iron and a protein-bound free radical. We have studied the endogenous defences to this reactive chemistry in a rabbit model following 20% exchange transfusion with cell-free haemoglobin stabilized in tetrameric form [via cross-linking with bis-(3,5-dibromosalicyl)fumarate]. The transfusate contained 95% oxyhaemoglobin, 5% methaemoglobin and 25 microM free iron. EPR spectroscopy revealed that the free iron in the transfusate was rendered redox inactive by rapid binding to transferrin. Methaemoglobin was reduced to oxyhaemoglobin by a slower process (t(1/2) = 1 h). No globin-bound free radicals were detected in the plasma. These redox defences could be fully attributed to a novel multifunctional role of plasma ascorbate in removing key precursors of oxidative damage. Ascorbate is able to effectively reduce plasma methaemoglobin, ferryl haemoglobin and globin radicals. The ascorbyl free radicals formed are efficiently re-reduced by the erythrocyte membrane-bound reductase (which itself uses intra-erythrocyte ascorbate as an electron donor). As well as relating to the toxicity of haemoglobin-based oxygen carriers, these findings have implications for situations where haem proteins exist outside the protective cell environment, e.g. haemolytic anaemias, subarachnoid haemorrhage, rhabdomyolysis.

Blood substitutes: why haven't we been more successful?

Persistent safety concerns have stalled the development of viable hemoglobin (Hb)-based oxygen carriers (HBOCs). HBOCs have several advantages over human blood, including availability, long-term storage, and lack of infectious risk. The basis of HBOC toxicity is poorly understood, however, several mechanisms have been suggested, including Hb extravasation across the blood vessel wall, scavenging of endothelial nitric oxide (NO), oversupply of oxygen, and heme-mediated oxidative side reactions. Although there are some in vitro and limited animal studies supporting these mechanisms, heme-mediated reactivity appears to provide an alternative path that can explain some of the observed pathophysiological changes. Moreover, recent mechanistic and animal studies support a role for globin and heme scavengers in controlling oxidative toxicity associated with Hb infusion.