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

项目总结 医疗辅助装置后中枢神经系统、肺、心脏和肾脏并发症的成本估算 需要血泵，例如体外循环(CPB)上的泵，估计每台 在十年的时间里，美国的各个州。由于需要延长CPB(&gt；4小时)在 复杂的手术，溶血，以及随后的血红蛋白(Hb)释放到循环中-这是 已知的多器官毒性和血管活性-是更密集的治疗的相关因素 病人。到目前为止，还没有有效的策略来消除乙肝的有毒副产品。由此带来的负担 仅肾损伤每年就造成约90亿美元的死亡，30万人/年死亡，平均 住院天数增加3.5天/患者该项目侧重于了解 体外循环延长后继发性终末器官损伤与溶血、Hb、血红素的关系 体外循环(通常持续4小时)。了解与医疗器械相关的红细胞 在这种情况下，我们提出了一种策略，重点是顺序地理解 Hb和血红素以及减轻这些毒素的终末器官病理生理学的治疗策略。我们会 前瞻性测定Hb、血红素、铁的水平以及每种毒素的浓度 心脏手术患者的血浆和尿液。同时，我们将测定其浓度 Hb、血红素和铁结合和清除蛋白、结合珠蛋白、血凝素和转铁蛋白。 此外，我们还将测定终末期器官损伤的血和尿标志物。我们将使用这些数据来构建 确定与终末器官损伤相关的HB毒素浓度极限的生物动力学模型 记号笔。这项工作的目标将是确定治疗干预的需要和时机。对这件事 根据我们丰富的经验，我们设计出了一种新型的Hb、血红素和蛋白质清除剂 研究Hb的毒性。这种新型蛋白质结构是通过多步过程生成apo-Hb而制备的， 同时从Cohn分级糊IV中分离出结合珠蛋白。最后，这两种蛋白 络合并进一步纯化以产生apo-Hb-结合珠蛋白复合体。这种新的蛋白质结构 在高血红素暴露状态下结合血红素，确保血红素配体中的铁，并安全地清除络合物以 单核细胞和巨噬细胞。或者，在高Hb暴露中，apo-Hb交换结合位点 结合珠蛋白将Hb二聚体清除到单核细胞和巨噬细胞，而释放的apo-Hb二聚体被降解 通过蛋白质分解并无害地从循环中清除。为了测试这种新型复合体的效果，我们有 计划在体外循环动物模型中进行一系列概念验证研究，以确定脱机能力。 服用Hb-结合珠蛋白预防终末器官损伤。

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.