PEGylation of Apohemoglobin Haptoglobin Complex reduces Associated Free Stroma Hemoglobin Toxicities
Authors: Munoz Carlos , Lucas Daniela , Muller Cynthia R. , Gopal Srila , Cabrales Pedro 
Published: May 2023

Apohemglobin (ApoHb) haptoglobin (Hp) complex (ApoHb-Hp) is a hemoglobin (Hb) and heme scavenging protein that leverages Hp acellular free Hb scavenging capacity and apoHb free circulating heme scavenging capacity. This protein complex provides substantial relief against hemolytic anemia such as sickle cell, thalassemia, and IMHA (immune-mediated hemolytic anemia). Unfortunately, apoHb is highly unstable at physiological temperature, forming protein aggregates and precipitates. These characteristics diminish the potential benefits of using the heme scavenger in vivo. To address the issues associated with ApoHb, the protein was conjugated with polyethylene glycol (PEG). By modifying the surface of apoHb, the protein increases in size and increases its circulatory half-life. However, its effectiveness does decrease as the surface modification sterically hinders active sites.The aim of the present study was to evaluate the i n vivo effects of PEG-apoHb-Hp complex by challenging it against a bolus injection (10% of the animal’s blood volume) of human Hb utilizing intravital microscopy on male Golden Syrian hamsters instrumented with dorsal window chamber. This study evaluated four experimental treatment groups saline, PEG-apoHb only, Hp only, and PEG-apoHb-Hp under the hHb challenge. The microvascular hemodynamics (flow) in arterioles < 50 μm, arterioles > 50 μm and venules 20<x<100 μm in diameter were recorded at baseline, 20 minutes after treatment, and 20 minutes after hHb challenge. Systemic parameters, blood gases, blood, and multiorgan damage/ inflammation were recorded[crm1]. At baseline and treatment, the animals recorded similar systemic values, blood gases, and microcirculatory health indicators. However, following the hHb challenge there was a significant drop in microcirculatory health when treated with saline and a progressive improvement when using PEG-apoHb only, Hp only, and PEG-apoHb-Hp. Specifically, the arterioles < 50 μm had a significantly greater effects with the varying treatments. PEG-apoHb-Hp complex had better results preventing vasoconstriction and improving flow rates. Furthermore, the functional capillary density presented improved results when treated with PEG-apohb only, Hp only, and PEG-apoHb-Hp. Finally, the cardiac and kidney inflammation and function showed significantly lower inflammatory markers and improved kidney function when treated apoHb only, Hp only, and PEG-apoHb-Hp.The results suggested that PEG-apoHb or Hp alone does not prevent the toxicities associated with acellular Hb, however, the PEG-apoHb-Hp complex showed significantly ameliorated microhemodynamics and systemic parameters. Furthermore, the organ inflammation was diminished as observed by decreased levels of inflammatory markers in the heart, liver, kidney, and spleen. This study suggests that PEG-apoHb-Hp does offer a therapeutic effect against acellular Hb challenge.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.