Apohemoglobin-haptoglobin complex attenuates the pathobiology of circulating acellular hemoglobin and heme.

Haptoglobin (Hp) is the plasma protein that binds and clears cell-free hemoglobin (Hb), whereas apohemoglobin (apoHb, i.e., Hb devoid of heme) can bind heme. Therefore, the apoHb-Hp protein complex should facilitate holoHb-apoHb αβ-dimer exchange and apoHb-heme intercalation. Thus, we hypothesized that apoHb-Hp could facilitate both Hb and heme clearance, which, if not alleviated, could have severe microcirculatory consequences. In this study, we characterized apoHb-Hp and Hb/heme ligand interactions and assessed their in vivo consequences. Hb exchange and heme binding with the apoHb-Hp complex was studied with transfer assays using size-exclusion high-performance liquid chromatography coupled with UV-visible spectrophotometry. Exchange/transfer experiments were conducted in guinea pigs dosed with Hb or heme-albumin followed by a challenge with equimolar amounts of apoHb-Hp. Finally, systemic and microcirculatory parameters were studied in hamsters instrumented with a dorsal window chamber via intravital microscopy. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. Dosing with the apoHb-Hp complex reversed Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, reduced microvascular blood flow, and diminished functional capillary density. Therefore, this study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure. This study highlights the apoHb-Hp complex as a novel therapeutic strategy to attenuate the adverse systemic and microvascular responses to intravascular Hb and heme exposure. In vitro and in vivo Hb exchange and heme transfer experiments demonstrated proof-of-concept Hb/heme ligand transfer to apoHb-Hp. The apoHb-Hp complex reverses Hb- and heme-induced systemic hypertension and microvascular vasoconstriction, preserves microvascular blood flow, and functional capillary density. In summary, the unique properties of the apoHb-Hp complex prevent adverse systemic and microvascular responses to Hb and heme-albumin exposure and introduce a novel therapeutic approach to facilitate simultaneous removal of extracellular Hb and heme.