Hemoglobin S Oxidation Promotes Plasma-Derived Microparticle Membrane Alterations and Toxicity
Authors: Jana Sirsendu , Strader Michael B , Kassa Tigist , Meng Fantao , Hicks Wayne , Tarandovskiy Ivan , De Paoli Silvia , Simak Jan , Miller Jeffery L , Mendelsohn Laurel , Nichols Jim , Thein Swee Lay , Belcher John D. , Vercellotti Gregory M , Alayash Abdu 
Published: October 2019

Abstract
Polymerization of sickle cell hemoglobin S (HbS) is recognized as a key event in the pathophysiology of sickle cell disease (SCD). Repeated HbS polymerization promotes an altered red blood cell (RBC) membrane, hemolysis, and microparticle (MP) formation, which have been shown to play significant roles in the interaction of RBCs with vascular endothelium and progression of vaso-occlusive events. Circulating RBC-derived MPs are elevated in SCD patients and they release a significant portion of their contents including oxidized HbS and heme to the cells of the vasculature. We have recently reported that free HbS oxidizes faster, remains locked in a highly oxidizing form (ferryl) longer, and loses heme faster than normal HbA (Kassa et al., J Biol Chem 290: 27939, 2015). The contributions of HbS higher oxidation states (ferric and ferryl heme) to MP formation, membrane alterations, and heme loss are poorly defined in SCD. RBC-derived MPs (ranging in size between 100-300 nm in diameter) generated by sheer stress or isolated by ultracentrifugation from the plasma (circulating) of SCD patients (N=6), ethnically matched control subjects (N=5), humanized transgenic sickle mice (Townes-SS, N=4), and control wild-type mice (Townes-AA, N=4) were identified by flow cytometry using CD235a glycophorin antibody and annexin V for externalized phosphatidylserine (PS). Time courses of Hb oxidation, obtained during 30 hour incubations of mouse or human MPs were biphasic. The initial levels of oxidized (ferric) Hb (30 to 45%) were slightly reduced within the first ~10 hours, likely due to the presence of RBC residual reductive enzymes within MPs. This was followed by a second phase in which Hb oxidation (ferric Hb) increased linearly and uncontrollably to 65 to 75% of total Hb. SCD MP's contained highly reactive ferryl Hb intermediates, carbonylated membrane proteins, and phosphorylated band 3 proteins. Quantitative proteomic analysis indicated a higher level of protein oxidation in MPs derived from SCD mice and patients. Five-fold higher levels of irreversibly oxidized βCys93 oxidation were found in untreated versus hydroxyurea-treated SCD patients. Intriguingly, HbS β subunits from SCD MPs were ubiquitinated and MPs isolated from untreated SCD patients had 25-fold higher ubiquitination levels than hydroxyurea-treated SCD patients that were comparable to normal controls. MP ubiquitination levels were correlated with HbS and an overall increase in MP oxidative stress, and inversely correlated with HbF. Compared to respective control MPs, incubation of either mice or human SCD MPs with human endothelial cells (HUVEC) activated apoptotic pathways and impacted cellular bioenergetic parameters by lowering mitochondrial oxygen consumption rates to a greater degree in a manner that was correlated with the redox state of Hb iron within MPs. Human endothelial cells incubated with SCD MPs showed greater intracellular reactive oxygen species production and heme oxygenase-1 induction. In summary, Hb transformation to higher oxidation forms is markedly increased in MPs generated from SCD mice and patients, which when incubated with endothelial cells, lead to mitochondrial dysfunction and apoptotic cell death. These mechanistic analyses of RBC-derived SCD microparticles suggest potential anti-oxidative reducing modalities that may interrupt MP heme-mediated pathophysiology in patients with SCD.

Disclosures
Belcher: Cydan/Imara: Research Funding; CSL-Behring: Research Funding. Vercellotti:CSL-Behring: Research Funding; Imara: Research Funding.