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

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Program Projects (P01)
Project #
Application #
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Albert Einstein College of Medicine
United States
Zip Code
Esquerra, Raymond M; Bibi, Bushra M; Tipgunlakant, Pooncharas et al. (2016) Role of Heme Pocket Water in Allosteric Regulation of Ligand Reactivity in Human Hemoglobin. Biochemistry 55:4005-17
Sjodt, Megan; Macdonald, Ramsay; Spirig, Thomas et al. (2016) The PRE-Derived NMR Model of the 38.8-kDa Tri-Domain IsdH Protein from Staphylococcus aureus Suggests That It Adaptively Recognizes Human Hemoglobin. J Mol Biol 428:1107-29
Díaz-Trelles, Ramón; Scimia, Maria Cecilia; Bushway, Paul et al. (2016) Notch-independent RBPJ controls angiogenesis in the adult heart. Nat Commun 7:12088
Strader, Michael Brad; Kassa, Tigist; Meng, Fantao et al. (2016) Oxidative instability of hemoglobin E (β26 Glu→Lys) is increased in the presence of free α subunits and reversed by α-hemoglobin stabilizing protein (AHSP): Relevance to HbE/β-thalassemia. Redox Biol 8:363-74
Cordes, Thekla; Wallace, Martina; Michelucci, Alessandro et al. (2016) Immunoresponsive Gene 1 and Itaconate Inhibit Succinate Dehydrogenase to Modulate Intracellular Succinate Levels. J Biol Chem 291:14274-84
Buono, Michael J; Krippes, Taylor; Kolkhorst, Fred W et al. (2016) Increases in core temperature counterbalance effects of haemoconcentration on blood viscosity during prolonged exercise in the heat. Exp Physiol 101:332-42
Chintagari, Narendranath Reddy; Jana, Sirsendu; Alayash, Abdu I (2016) Oxidized Ferric and Ferryl Forms of Hemoglobin Trigger Mitochondrial Dysfunction and Injury in Alveolar Type I Cells. Am J Respir Cell Mol Biol 55:288-98
Rutardottir, Sigurbjörg; Karnaukhova, Elena; Nantasenamat, Chanin et al. (2016) Structural and biochemical characterization of two heme binding sites on α1-microglobulin using site directed mutagenesis and molecular simulation. Biochim Biophys Acta 1864:29-41
Zhang, Zhuo; Leong, Daniel J; Xu, Lin et al. (2016) Curcumin slows osteoarthritis progression and relieves osteoarthritis-associated pain symptoms in a post-traumatic osteoarthritis mouse model. Arthritis Res Ther 18:128
Shi, Patricia A; Choi, Erika; Chintagari, Narendranath R et al. (2016) Sustained treatment of sickle cell mice with haptoglobin increases HO-1 and H-ferritin expression and decreases iron deposition in the kidney without improvement in kidney function. Br J Haematol :

Showing the most recent 10 out of 78 publications