Hemoglobin oxidation–dependent reactions promote interactions with band 3 and oxidative changes in sickle cell–derived microparticles
Sirsendu Jana, Michael Brad Strader, Fantao Meng, Wayne Hicks, Tigist Kassa, Ivan Tarandovskiy, Silvia De Paoli, Jan Simak, Michael R. Heaven, John D. Belcher, Gregory M. Vercellotti, Abdu I. Alayash
Sirsendu Jana, Michael Brad Strader, Fantao Meng, Wayne Hicks, Tigist Kassa, Ivan Tarandovskiy, Silvia De Paoli, Jan Simak, Michael R. Heaven, John D. Belcher, Gregory M. Vercellotti, Abdu I. Alayash
View: Text | PDF
Research Article Hematology Vascular biology

Hemoglobin oxidation–dependent reactions promote interactions with band 3 and oxidative changes in sickle cell–derived microparticles

Abstract

The contribution of intracellular hemoglobin (Hb) oxidation to RBC-derived microparticle (MP) formation is poorly defined in sickle cell disease (SCD). Here we report that sickle Hb (HbS) oxidation, coupled with changes in cytosolic antioxidative proteins, is associated with membrane alterations and MP formation in homozygous Townes–sickle cell (Townes-SS) mice. Photometric and proteomic analyses confirmed the presence of high levels of Hb oxidation intermediates (ferric/ferryl) and consequent β-globin posttranslational modifications, including the irreversible oxidation of βCys93 and the ubiquitination of βLys96 and βLys145. This is the first report to our knowledge to link the UPS (via ubiquitinated Hb and other proteins) to oxidative stress. Ferryl Hb also induced complex formation with band 3 and RBC membrane proteins. Incubation of Townes-SS MPs with human endothelial cells caused greater loss of monolayer integrity, apoptotic activation, heme oxygenase-1 induction, and concomitant bioenergetic imbalance compared with control Townes-AA MPs. MPs obtained from Townes-SS mice treated with hydroxyurea produced fewer posttranslational Hb modifications. In vitro, hydroxyurea reduced the levels of ferryl Hb and shielded its target residue, βCys93, by a process of S-nitrosylation. These mechanistic analyses suggest potential antioxidative therapeutic modalities that may interrupt MP heme-mediated pathophysiology in SCD patients.

Authors

Sirsendu Jana, Michael Brad Strader, Fantao Meng, Wayne Hicks, Tigist Kassa, Ivan Tarandovskiy, Silvia De Paoli, Jan Simak, Michael R. Heaven, John D. Belcher, Gregory M. Vercellotti, Abdu I. Alayash

×

Figure 2

Hemoglobin S within microparticles undergoes oxidation and oxidative changes.

Options: View larger image (or click on image) Download as PowerPoint
Hemoglobin S within microparticles undergoes oxidation and oxidative cha...
Kinetic absorbance spectra of HbA control (A) and RBC MPs prepared from Townes-SS mice (B) (note: ferryl Hb spectrum is recognized by 2 new peaks at 545 and 584 nm and a flattened region between 500 and 700 nm). The samples were incubated for the indicated times in PBS at 37°C. Reverse-phase HPLC analyses of RBC MPs (AA and SS) before and after 36 hours of incubation (autoxidation) (C). The flow rate was 1 ml/min at 25°C. The eluate was monitored at 280 nm (for globin chains) and 405 nm (for heme). (D) Time course kinetics of metHb formation during autoxidation of HbA and HbS inside MPs prepared from AA and SS mice compared with free HbA samples, as determined spectrophotometrically during the 30-hour incubation. (E) Carbonylated protein content and (F) total lipid hydroperoxide content were measured in RBC MPs (n = 4). Upper horizontal line in box plots represents 75th percentile, lower horizontal line represents 25th percentile, and horizontal line within box represents mean value. Vertical error bars represent 95% confidence interval. Student’s t test, 2-tailed, *P < 0.05.