Paul W. Buehler

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.

Hemoglobin-driven pathophysiology is an in vivo consequence of the red blood cell storage lesion that can be attenuated in guinea pigs by haptoglobin therapy

Massive transfusion of blood can lead to clinical complications, including multiorgan dysfunction and even death. Such severe clinical outcomes have been associated with longer red blood cell (rbc) storage times. Collectively referred to as the rbc storage lesion, rbc storage results in multiple biochemical changes that impact intracellular processes as well as membrane and cytoskeletal properties, resulting in cellular injury in vitro. However, how the rbc storage lesion triggers pathophysiology in vivo remains poorly defined. In this study, we developed a guinea pig transfusion model with blood stored under standard blood banking conditions for 2 (new), 21 (intermediate), or 28 days (old blood). Transfusion with old but not new blood led to intravascular hemolysis, acute hypertension, vascular injury, and kidney dysfunction associated with pathophysiology driven by hemoglobin (Hb). These adverse effects were dramatically attenuated when the high-affinity Hb scavenger haptoglobin (Hp) was administered at the time of transfusion with old blood. Pathologies observed after transfusion with old blood, together with the favorable response to Hp supplementation, allowed us to define the in vivo consequences of the rbc storage lesion as storage-related posttransfusion hemolysis producing Hb-driven pathophysiology. Hb sequestration by Hp might therefore be a therapeutic modality for enhancing transfusion safety in severely ill or massively transfused patients.

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?

Hemoglobin-based oxygen carriers: From mechanisms of toxicity and clearance to rational drug design.

Hemoglobin-based oxygen carriers (HBOCs) have been developed to support blood oxygen transport capacity during hemorrhagic shock, hemolysis and ischemic insult. Existing product candidates have demonstrated considerable efficacy in experimental animal models and in clinical trial subjects; however, severe adverse safety signals that appeared in recent phase II and phase III clinical trials involving certain HBOCs have in part hindered further development and licensing. Emerging insights into hemoglobin (Hb) toxicity as well as physiologic Hb scavengers such as haptoglobin and CD163 that are capable of detoxifying extracellular Hb in vivo suggest that alternative product candidates could be designed. Together with novel animal models and biomarkers tailored to monitor the effects of extracellular Hb, a new generation of HBOCs can be envisioned.

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.

Haptoglobin, hemopexin, and related defense pathways—basic science, clinical perspectives, and drug development

Hemolysis, which occurs in many disease states, can trigger a diverse pathophysiologic cascade that is related to the specific biochemical activities of free Hb and its porphyrin component heme. Normal erythropoiesis and concomitant removal of senescent red blood cells (RBC) from the circulation occurs at rates of approximately 2 × 106 RBCs/second. Within this physiologic range of RBC turnover, a small fraction of hemoglobin (Hb) is released into plasma as free extracellular Hb. In humans, there is an efficient multicomponent system of Hb sequestration, oxidative neutralization and clearance. Haptoglobin (Hp) is the primary Hb-binding protein in human plasma, which attenuates the adverse biochemical and physiologic effects of extracellular Hb. The cellular receptor target of Hp is the monocyte/macrophage scavenger receptor, CD163. Following Hb-Hp binding to CD163, cellular internalization of the complex leads to globin and heme metabolism, which is followed by adaptive changes in antioxidant and iron metabolism pathways and macrophage phenotype polarization. When Hb is released from RBCs within the physiologic range of Hp, the potential deleterious effects of Hb are prevented. However, during hyper-hemolytic conditions or with chronic hemolysis, Hp is depleted and Hb readily distributes to tissues where it might be exposed to oxidative conditions. In such conditions, heme can be released from ferric Hb. The free heme can then accelerate tissue damage by promoting peroxidative reactions and activation of inflammatory cascades. Hemopexin (Hx) is another plasma glycoprotein able to bind heme with high affinity. Hx sequesters heme in an inert, non-toxic form and transports it to the liver for catabolism and excretion. In the present review we discuss the components of physiologic Hb/heme detoxification and their potential therapeutic application in a wide range of hemolytic conditions.

Toxicological Consequences of Extracellular Hemoglobin: Biochemical and Physiological Perspectives

Under normal physiology, human red blood cells (RBCs) demonstrate a circulating lifespan of approximately 100-120 days with efficient removal of senescent RBCs taking place via the reticuloendothelial system, spleen, and bone marrow phagocytosis. Within this time frame, hemoglobin (Hb) is effectively protected by efficient RBC enzymatic systems designed to allow for interaction between Hb and diffusible ligands while preventing direct contact between Hb and the external environment. Under normal resting conditions, the concentration of extracellular Hb in circulation is therefore minimal and controlled by specific plasma and cellular (monocyte/macrophage) binding proteins (haptoglobin) and receptors (CD163), respectively. However, during pathological conditions leading to hemolysis, extracellular Hb concentrations exceed normal plasma and cellular binding capacities, allowing Hb to become a biologically relevant vasoactive and redox active protein within the circulation and at extravascular sites. Under conditions of genetic, drug-induced, and autoimmune hemolytic anemias, large quantities of Hb are introduced into the circulation and often lead to acute renal failure and vascular dysfunction. Interestingly, the study of chemically modified Hb for use as oxygen therapeutics has allowed for some basic understanding of extracellular Hb toxicity, particularly in the absence of functional clearance mechanisms and in circulatory antioxidant depleted states.