Bioengineering a Dual Function Protein Construct to Detoxify Heme and Hemoglobin

摘要 在以广泛溶血为特征的病理生理条件下（例如，获得性和遗传性 溶血性疾病），游离血红素和无细胞血红蛋白（Hb）被释放到血流中，并引起 各种不良反应，即：血管收缩、高血压和终末器官损害。因此，治疗 溶血条件将受益于游离血红素和无细胞Hb的清除剂，如血红素结合蛋白（Hpx）和 结合珠蛋白（Hp）。Hpx的一种可能的功能替代物是脱辅基血红蛋白（apoHb）。ApoHb是 通过从Hb中去除血红素而衍生，并且其空的血红素结合口袋对血红素具有高亲和力。因此，我们认为， apoHb可替代Hpx作为一种新型的体内血红素清除剂。 然而，使用apoHb作为体内血红素清除剂的主要潜在问题是其低的热稳定性。 在生理温度下稳定，循环半衰期短（类似于Hb，5分钟）。幸运的是，前 研究表明，与Hb类似，apoHb可与Hp结合形成高度稳定的复合物。apoHb-Hp 复合物保留其结合血红素的能力，并且与游离apoHb相比在生理温度下更稳定。 apoHb-Hp复合物除了能结合游离血红素外，还能通过交换Hp而结合游离Hb Hb α β二聚体的结合apoHb α β二聚体。因此，我们假设apoHb-Hp复合物将具有 结合和解毒溶血状态下产生的游离血红素和血红蛋白的双重能力。的 产生的Hb-Hp复合物比游离血红素或无细胞Hb毒性小得多，并且容易从循环中清除 通过CD 163介导的单核细胞/巨噬细胞摄取。为了验证这一假设，我们提出了以下具体的 目标。 具体目的1：apoHb-Hp复合物的生物物理和生物化学表征及其对 在体外结合血红素和血红蛋白。 具体目的2：体内测定血红素和Hb转移和结合至apoHb-Hp。 具体目标3：apoHb-Hp的系统性临床前评价，以预防和/或阻止 血管内溶血（Hb/heme）引起肺性心血管疾病。 具体目标4：apoHb-Hp的微血管评价，以防止血管闭塞并减少血管 炎症

Abstract During pathophysiological conditions characterized by extensive hemolysis (e.g. acquired and genetic hemolytic diseases), free heme and cell-free hemoglobin (Hb) are released into the blood stream and elicit a variety of adverse effects, namely: vasoconstriction, hypertension, and end organ damage. Thus treatment of hemolytic conditions would benefit from scavengers of free heme and cell-free Hb such as hemopexin (Hpx) and haptoglobin (Hp), respectively. A possible functional alternative to Hpx is apohemoglobin (apoHb). ApoHb is derived by removing heme from Hb, and its vacant heme-binding pockets have a high affinity for heme. Hence, apoHb could serve as a novel in vivo heme scavenger instead of Hpx. However, major potential issues with the use of apoHb as an in vivo heme scavenger are its low thermal stability at physiological temperature, and short circulatory half-life (similar to Hb, 5 min). Fortuitously, previous studies have shown that, similar to Hb, apoHb can bind to Hp forming a highly stable complex. The apoHb-Hp complex retains its ability to bind heme, and is more stable at physiological temperature compared to free apoHb. In addition to being able to bind free heme, the apoHb-Hp complex can scavenge free Hb by exchanging Hp bound apoHb αβ dimers for Hb αβ dimers. Therefore, we hypothesize that the apoHb-Hp complex will have the dual ability to bind and detoxify free heme and Hb that are produced during states of hemolysis. The resulting Hb-Hp complex is much less toxic than free heme or cell-free Hb and is readily cleared from circulation via CD163 mediated monocyte/macrophage uptake. To test this hypothesis, we propose the following specific aims. Specific Aim 1: Biophysical and biochemical characterization of the apoHb-Hp complex and its ability to bind heme and Hb in vitro. Specific Aim 2: In vivo determination of heme and Hb transfer and binding to apoHb-Hp. Specific Aim 3: Systematic pre-clinical evaluation of apoHb-Hp to prevent and/or halt the progression of intravascular hemolysis (Hb/heme)-induced pulmonary cardiovascular disease. Specific Aim 4: Microvascular evaluation of apoHb-Hp to prevent vaso-occlusion and reduce vascular inflammation.