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

项目总结

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.