Oxidative pathways in the sickle cell and beyond

Abstract

Polymerization of deoxy sickle cell hemoglobin (HbS) is well recognized as the primary event that triggers the classic cycles of sickling/unsickling of patients red blood cells (RBCs). RBCs are also subjected to continuous endogenous and exogenous oxidative onslaughts resulting in hemolytic rate increases which contribute to the evolution of vasculopathies associated with this disease. Compared to steady-state conditions, the occurrences of vaso-occlusive crises increase the levels of both RBC-derived microparticles as well as extracellular Hb in circulation. Common byproduct resulting from free Hb oxidation and from Hb-laden microparticles is heme (now recognized as damage associated molecular pattern (DAMP) molecule) which has been shown to initiate inflammatory responses. This review provides new insights into the interplay between microparticles, free Hb and heme focusing on Hb's pseudoperoxidative activity that drives RBC's cytosolic, membrane changes as well as oxidative toxicity towards the vascular system. Emerging antioxidative strategies that include the use of protein and heme scavengers in controlling Hb oxidative pathways are discussed.

Keywords: Ferryl hemoglobin; Heme oxidation; Microparticles; Pseudoperoxidase; Sickle cell hemoglobin.

Figure 1. Oxygenation and the pseudoperoxidase pathways in sickle cell hemoglobin

Hemoglobin (HbFe^II) reversibly binds oxygen (k_off/k_on), and spontaneously oxidizes at a slow rate (k_auto) to the non-functional ferric/metHb (HbFeIII) and superoxide (O2^·−) that dismutate (k_d) to give peroxide (H₂O₂). In the presence of this and/or exogenous H₂O₂, a catalytic cycle between the ferric (HbFe^III) and the ferryl (HbFe^IV) hemes is initiated, in which H₂O₂ is eliminated in a peroxidase-like manner. However, in the case of HbS the autoreduction of ferryl back to ferric heme is slower (dotted line) than that of normal HbA, leading to a longer lived and more damaging ferrylHb [19]. If H₂O₂ reacts with the ferric form of Hb a protein radical is produce (.Hb^IV). The radical escapes through βCys93 resulting in its irreversible oxidation and the collapse of the β subunits. These oxidative changes then lead to unfolding and denaturation of the protein and heme loss. Heme is recognized as damage associated molecular pattern (DAMP) molecule able to trigger inflammation.

Figure 2. Proposed erythrocyte internal oxidative changes that trigger membrane alternations and microparticles formation

Oxidative reactions within RBCs include the following pathways: (1) Ferrous hemoglobin (Fe^II-O₂) undergoes spontaneous oxidation (autooxidation) to form ferric (Fe^III) and ferryl forms (Fe^IV). Ferryl species has been shown to form in sickle cells where antioxidative mechanisms are compromised. Ferryl Hb once formed undergoes oxidative changes including irreversible oxidation of βCys93 and ubiquitination of lysine 96. Ferryl Hb can oxidatively target band 3 leading to clustering of band3 and microparticles formation (2) or denaturation and proteasome degradation (3) some ferryl Hb revert back to ferric by a process of autoreduction (4). Hydroxyurea has been shown to have an antioxidant effects by minimizing the consequences of these processes.