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221 Xanthine Oxidase Has a Protective Role during Heme Crisis By Binding and Degrading Heme

Program: Oral and Poster Abstracts
Type: Oral
Session: 102. Regulation of Iron Metabolism
Hematology Disease Topics & Pathways:
Biological Processes, microenvironment
Saturday, December 5, 2020: 2:30 PM

Heidi M Schmidt1,2, Scott Hahn, MS3*, Gowtham K Annarapu, PhD4, Mara Carreño4*, Francisco Schopfer4*, Sruti Shiva, PhD1*, Dario A. Vitturi, PhD5*, Eric Kelley, PhD6* and Adam C Straub, PhD1,7*

1Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA
2Pittsburgh Heart, Lung and Blood Vascular Medicine Institute, Department of Medicine, Univeristy of Pittsburgh, Pittsburgh, PA
3Pittsburgh Heart, Lung and Blood Vascular Medicine Institute, Department of Medicine, University of Pittsburgh, Pittsburgh, PA
4University of Pittsburgh, Pittsburgh, PA
5Heart, Lung, Blood and Vascular Medicine Institute, Department of Medicine; Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, PA
6West Virginia University, Morgantown, WV
7Heart, Lung, Blood and Vascular Medicine Institute, Department of Medicine, University of Pittsburgh, Pittsburgh, PA

Xanthine oxidase (XO) is a key enzyme in the purine degradation pathway, catalyzing the catabolism of hypoxanthine to xanthine and xanthine to uric acid. A byproduct of these reactions is the generation of the reactive oxygen species (ROS), hydrogen peroxide and superoxide. XO is produced primarily in the liver; however, following hepatic stressors such as inflammation, hypoxia, or ischemia, XO is released from the liver and enters the circulation. XO can then bind distal endothelium via electrostatic interactions with glycosaminoglycans (GAGs). Current dogma believes that XO plays a harmful role in pathologies due to the increase in ROS production that can alter signaling pathways and damage endothelial cells. XO activity has been shown to be elevated in a number of hemolytic conditions including, sickle cell disease, malaria, and sepsis; however, the involvement of XO in these pathological conditions has not been fully elucidated. These conditions result in increased hemolysis, releasing free heme and hemoglobin into the circulation, inducing an inflammatory response, and damaging endothelial cells. Identifying the involvement of XO during heme crisis could improve our understanding of the pathologies associated with hemolytic conditions and lead to the identification or development of more effective treatment options.

We hypothesized that XO has damaging properties under basal conditions; however, the presence of XO is crucial and protective during heme crisis by serving as a secondary mechanism of heme degradation when canonical heme degradation pathways are saturated.

In order to explore the role of XO in heme crisis, we developed a novel heme crisis model in which we pre-treated mice with the clinically relevant dose of 10 mg/kg/day febuxostat, an FDA approved XO inhibitor, for five days in drinking water. Following inhibition of XO, the mice were challenged with two identical doses of hemin one hour apart and monitored for 24 hours. We observed a 20-fold increase in XO activity in the mice treated with 50 μmol/kg hemin. This increase was completely inhibited in the mice treated with febuxostat. Surprisingly, the febuxostat treated mice had worsened survival compared to the untreated mice. This suggests a protective role for XO during heme crisis. We hypothesized that XO has a protective role by preventing platelet activation and degrading excess free heme. To investigate this hypothesis, we used flow cytometry to quantify heme-induced platelet activation. Healthy human platelets were isolated and treated with 2.5 μM hemin, 10 mU/mL XO, 200 μM hypoxanthine, and 20 μM febuxostat. We observed 72% activation with heme alone, while incubation with XO and hypoxanthine resulted in almost complete prevention of platelet activation (16%). We were also able to partially restore platelet activation (45%) when febuxostat was added. Based on these results, we hypothesize that XO binds GAGs on the platelet surface and degrades heme in order to protect platelets from heme-induced activation. To assess the ability of XO to degrade heme, we tested whether XO binds heme. We performed computational modeling in which we identified a potential heme binding site in the FAD domain of XO with a kd = 128 nM. We confirmed heme-XO binding by performing a heme binding assay. We incubated heme (25 μM) alone, heme + XO (50 μM), and heme + XO + hypoxanthine (100 μM) for 20 minutes. Heme binding was assessed by dot blotting in nitrocellulose followed by chemiluminescent detection and dot density quantification. We observed a 2-fold increase in dot density when heme and XO were incubated and a 4-fold increase with heme, XO, and hypoxanthine. These results support a potential heme-XO interaction that is amplified when the enzyme is active. Lastly, we measured XO’s ability to degrade heme using UV visible spectrophotometry. We incubated hemin, XO, and hypoxanthine together and measured the absorbance over 20 minutes. We observed a decrease in absorbance at 618 nm, indicative of heme degradation.

In conclusion, contrary to the current dogma, we have identified a potential protective role for XO during hemolytic crisis. We found that febuxostat treatment worsened survival in heme challenged mice, XO prevented heme-induced platelet activation, identified a potential heme-XO binding site, and observed XO-induced heme degradation. XO may have a protective role in hemolytic conditions by serving as a secondary mechanism of heme degradation.

Disclosures: No relevant conflicts of interest to declare.

*signifies non-member of ASH