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 overload 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 early 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 O2 binding in order to determine the relative importance of NO scavenging versus auto-regulation of O2 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 ?1?1 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 ascorbate 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.
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