Bioengineering a novel therapeutic protein complex to minimize the effects of medical device induced hemolysis

PROJECT SUMMARY
Cost estimates for CNS, pulmonary, cardiac, and renal complications following medical assist devices
requiring blood pumps, for example on pump cardiopulmonary bypass (CPB) is estimated at $80 million per
individual states in the US over a ten-year period. Because of the need for extended CPB (> 4hours) use in
complex surgeries, hemolysis, and the subsequent release of hemoglobin (Hb) into the circulation – which is
known to be multi-organ toxic and vasoactive – is a relevant contributor to more intensive management of
patients. To date, no effective strategy to remove toxic by-products of Hb are available. The burden caused
by kidney injury alone accounts for approximately $9 billion/year, 300,000 deaths/year and an average
increase in hospital stay of 3.5 days/patient This project focuses on understanding the contribution of
hemolysis, Hb and heme associated with secondary end organ injury following extended on pump
cardiopulmonary bypass (typically lasting up to 4 hours). To understand medical device related red blood cell
lysis in this setting, we propose a strategy that is focused on sequentially understanding the contributions of
Hb and heme and a therapeutic strategy to attenuate the end organ pathophysiology of these toxins. We will
prospectively determine the levels of Hb, heme and iron as well as the concentrations of each toxin in
plasma and urine of cardiac surgery patients. Simultaneously we will determine the concentrations of
Hb, heme and iron binding and clearance proteins, haptoglobin, hemopexin and transferrin, respectively.
Further we will determine plasma and urinary markers of end organ injury. We will use this data to construct
a biokinetic model that determines the limits of Hb toxin concentrations that associate with end organ injury
markers. The goal of this effort will be to define the need and timing for therapeutic interventions. To this
end we have bioengineered a novel Hb, heme and protein scavenger based on our extensive experience
with studying Hb toxicity. The novel protein construct is prepared by generating apo-Hb in multi-step process,
while simultaneously isolating haptoglobin from Cohn fractionation paste IV. Finally, the two proteins are
complexed and further purified to generate the apo-Hb-haptoglobin complex. This novel protein construct
binds heme in high heme exposure states, secures iron in the heme ligand and safely clears the complex to
monocytes and macrophages. Alternatively, in high Hb exposures apo-Hb exchanges binding sites on
haptoglobin clearing Hb dimers to monocytes and macrophages, while released apo-Hb dimers are degraded
by proteolysis and harmlessly cleared from circulation. To test the effect of the novel complex we have
planed a range of proof-of-concept studies in animal models of on pump CPB to define the ability of apo-
Hb-haptoglobin dosing to prevent end organ injury.

Grant Number: 5R01HL162120-04
NIH Institute/Center: NIH
Principal Investigator: Paul Buehler