Haptoglobin Treatment for Aneurysmal Subarachnoid Hemorrhage: Review and Expert Consensus on Clinical Translation.

Aneurysmal subarachnoid hemorrhage (aSAH) is a devastating form of stroke frequently affecting young to middle-aged adults, with an unmet need to improve outcome. This special report focusses on the development of intrathecal haptoglobin supplementation as a treatment by reviewing current knowledge and progress, arriving at a Delphi-based global consensus regarding the pathophysiological role of extracellular hemoglobin and research priorities for clinical translation of hemoglobin-scavenging therapeutics. After aneurysmal subarachnoid hemorrhage, erythrocyte lysis generates cell-free hemoglobin in the cerebrospinal fluid, which is a strong determinant of secondary brain injury and long-term clinical outcome. Haptoglobin is the body's first-line defense against cell-free hemoglobin by binding it irreversibly, preventing translocation of hemoglobin into the brain parenchyma and nitric oxide-sensitive functional compartments of cerebral arteries. In mouse and sheep models, intraventricular administration of haptoglobin reversed hemoglobin-induced clinical, histological, and biochemical features of human aneurysmal subarachnoid hemorrhage. Clinical translation of this strategy imposes unique challenges set by the novel mode of action and the anticipated need for intrathecal drug administration, necessitating early input from stakeholders. Practising clinicians (n=72) and scientific experts (n=28) from 5 continents participated in the Delphi study. Inflammation, microvascular spasm, initial intracranial pressure increase, and disruption of nitric oxide signaling were deemed the most important pathophysiological pathways determining outcome. Cell-free hemoglobin was thought to play an important role mostly in pathways related to iron toxicity, oxidative stress, nitric oxide, and inflammation. While useful, there was consensus that further preclinical work was not a priority, with most believing the field was ready for an early phase trial. The highest research priorities were related to confirming haptoglobin's anticipated safety, individualized versus standard dosing, timing of treatment, pharmacokinetics, pharmacodynamics, and outcome measure selection. These results highlight the need for early phase trials of intracranial haptoglobin for aneurysmal subarachnoid hemorrhage, and the value of early input from clinical disciplines on a global scale during the early stages of clinical translation.

Hemolysis, free hemoglobin toxicity, and scavenger protein therapeutics.

During hemolysis, erythrophagocytes dispose damaged red blood cells. This prevents the extracellular release of hemoglobin, detoxifies heme, and recycles iron in a linked metabolic pathway. Complementary to this process, haptoglobin and hemopexin scavenge and shuttle the red blood cell toxins hemoglobin and heme to cellular clearance. Pathological hemolysis outpaces macrophage capacity and scavenger synthesis across a diversity of diseases. This imbalance leads to hemoglobin-driven disease progression. To meet a void in treatment options, scavenger protein-based therapeutics are in clinical development.

Heme-stress activated NRF2 skews fate trajectories of bone marrow cells from dendritic cells towards red pulp-like macrophages in hemolytic anemia.

Heme is an erythrocyte-derived toxin that drives disease progression in hemolytic anemias, such as sickle cell disease. During hemolysis, specialized bone marrow-derived macrophages with a high heme-metabolism capacity orchestrate disease adaptation by removing damaged erythrocytes and heme-protein complexes from the blood and supporting iron recycling for erythropoiesis. Since chronic heme-stress is noxious for macrophages, erythrophagocytes in the spleen are continuously replenished from bone marrow-derived progenitors. Here, we hypothesized that adaptation to heme stress progressively shifts differentiation trajectories of bone marrow progenitors to expand the capacity of heme-handling monocyte-derived macrophages at the expense of the homeostatic generation of dendritic cells, which emerge from shared myeloid precursors. This heme-induced redirection of differentiation trajectories may contribute to hemolysis-induced secondary immunodeficiency. We performed single-cell RNA-sequencing with directional RNA velocity analysis of GM-CSF-supplemented mouse bone marrow cultures to assess myeloid differentiation under heme stress. We found that heme-activated NRF2 signaling shifted the differentiation of bone marrow cells towards antioxidant, iron-recycling macrophages, suppressing the generation of dendritic cells in heme-exposed bone marrow cultures. Heme eliminated the capacity of GM-CSF-supplemented bone marrow cultures to activate antigen-specific CD4 T cells. The generation of functionally competent dendritic cells was restored by NRF2 loss. The heme-induced phenotype of macrophage expansion with concurrent dendritic cell depletion was reproduced in hemolytic mice with sickle cell disease and spherocytosis and associated with reduced dendritic cell functions in the spleen. Our data provide a novel mechanistic underpinning of hemolytic stress as a driver of hyposplenism-related secondary immunodeficiency.

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.