PROJECT 2: ENGINEERING HbA TO EVALUATE TOXICITY AND CLEARANCE DESCRIPTION: ABSTRACT: Toxicities due to acellular hemoglobin (Hb) have been observed for hemolytic disease, transfusions with old blood, and administration of extracellular hemoglobin-based oxygen carriers (HBOCs) and include: (a) interference with endothelial and smooth muscle signaling due to dioxygenation of NO;(b) self-destructive oxidization of the globin itself, nearby proteins, and lipids;(c) slow clearance and inflammation due to saturation of the haptoglobin receptor system and macrophage activation;(d) iron overioad symptoms due to heme loss, globin denaturation, and iron accumulation;and (e), in the case of HBOCs, pathological auto-regulation of capillary flow and decreased tissue perfusion due to eariy O2 release in arteries and arterioles. Our overall goal is to test the relative importance of each of these toxicity mechanisms using genetically engineered recombinant HbA molecules in cell, organ, and animal model systems in direct collaboration with Dr. Abdu Alayash, who will act as a co-investigator and director of Core D, in the in vivo capillary systems designed to evaluate oxygen perfusion and nitric oxide scavenging in Project 1, and in the nitrite reduction and nanoparticle NO-releasing experiments described in Project 3.
The specific aims of Project 2 are to: (1) differentially modulate NO dioxygenation and 0? binding in order to determine the relative importance of NO scavenging versus auto-regulation of O? delivery in causing hypertension and vasculature dysfunction;(2) determine the mechanisms that cause oxidative degradation of acellular HbA, protein radical generation, and oxidative damage of surrounding tissues, membranes, and plasma proteins;(3) evaluate the role of hemin loss and unfolding on the toxicity of acellular Hb in vivo;(4) examine the functional and physiological effects of haptoglobin binding to wild-type HbA and crosslinked rHb tetramers on inhibition of autooxidation, ferryl radical formation, hemin loss, and denaturation of the bound aiPi dimers and on HbA clearance from the blood stream. PUBLIC HEALTH: Identification of the mechanisms causing acellular Hb toxicity will allow the design of therapies to mitigate problems associated with hemolytic diseases, whole blood transfusions, and the use of HBOCs. For example, administration with ascoriaate or nitrite/NO releasing agents could be used to counteract autooxidation or NO scavenging side effects, respectively. The protein engineering strategies obtained can also be used to optimize the safety and efficiency of recombinant Hb-based O2 carriers. PERFOMANCE

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
1P01HL110900-01A1
Application #
8396976
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2012-08-01
Budget End
2013-07-31
Support Year
1
Fiscal Year
2012
Total Cost
$338,196
Indirect Cost
$16,750
Name
Albert Einstein College of Medicine
Department
Type
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
Kettisen, Karin; Strader, Michael Brad; Wood, Francine et al. (2018) Site-directed mutagenesis of cysteine residues alters oxidative stability of fetal hemoglobin. Redox Biol 19:218-225
Alayash, Abdu I (2018) Oxidative pathways in the sickle cell and beyond. Blood Cells Mol Dis 70:78-86
Wallace, Martina; Green, Courtney R; Roberts, Lindsay S et al. (2018) Enzyme promiscuity drives branched-chain fatty acid synthesis in adipose tissues. Nat Chem Biol 14:1021-1031
Sjodt, Megan; Macdonald, Ramsay; Marshall, Joanna D et al. (2018) Energetics underlying hemin extraction from human hemoglobin by Staphylococcus aureus. J Biol Chem 293:6942-6957
Jana, Sirsendu; Strader, Michael Brad; Meng, Fantao et al. (2018) Hemoglobin oxidation-dependent reactions promote interactions with band 3 and oxidative changes in sickle cell-derived microparticles. JCI Insight 3:
Alayash, Abdu I (2017) Hemoglobin-Based Blood Substitutes and the Treatment of Sickle Cell Disease: More Harm than Help? Biomolecules 7:
Meng, Fantao; Alayash, Abdu I (2017) Determination of extinction coefficients of human hemoglobin in various redox states. Anal Biochem 521:11-19
Hirsch, Rhoda Elison; Sibmooh, Nathawut; Fucharoen, Suthat et al. (2017) HbE/?-Thalassemia and Oxidative Stress: The Key to Pathophysiological Mechanisms and Novel Therapeutics. Antioxid Redox Signal 26:794-813
Strader, Michael Brad; Bangle, Rachel; Parker Siburt, Claire J et al. (2017) Engineering oxidative stability in human hemoglobin based on the Hb providence (?K82D) mutation and genetic cross-linking. Biochem J 474:4171-4192
Jani, Vivek P; Mailo, Shawn; Athar, Ali et al. (2017) Blood Quality Diagnostic Device Detects Storage Differences Between Donors. IEEE Trans Biomed Circuits Syst 11:1400-1405

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