Development of modified hemoglobin molecules with regulated nitric oxide quenching action and their application for cardiovascular system

长期以来，人们一直期待人工红细胞能在临床上用于治疗多种患者，因为它们不含病毒，可以输注给拒绝普通输血的人。为了减少基于血红蛋白（Hb）的氧载体的不良反应，如血压升高和血小板聚集增强，由其一氧化氮（NO）猝灭作用诱导，我们试图开发新的分子，将临床可用作人工红细胞。在1997 - 1999年的研究项目中，我们发现1。hb基分子通过内皮细胞间隙进入血管后才产生固氮作用，因此，hb基分子尺寸越大，固氮作用越小。2. hb基分子的大小也决定了大鼠心脏中NO的封存程度。3. 然后，我们尝试将Hb分子的β链进行s -亚硝基化，并能够制造纯度高达70%的s -亚硝基-Hb （SNO-Hb）。经静脉注射时，与无基质Hb相比，SNO-Hb对血压和血小板聚集的影响较小。此外,5。我们证明了下面列出的评估人工红细胞生物作用的方法的有效性；(1)通过连接到HPLC系统的微透析连续测量脑NO，(2)利用激光束散射进行离体血小板聚集测定，(3)利用近红外光谱评估脑氧合和细胞色素氧化酶的氧化还原行为。最后,6。我们新开发了一种长链聚乙二醇偶联Hb，并开始研究其生物活性。

Artificial red blood cells have long been awaited to be clinically available for the treatment of many kinds of patients because they are free of virus and can be infused to refusers of ordinal blood transfusion. In order to reduce adverse reactions of hemoglobin (Hb)-based oxygen carriers, such as elevation of blood pressure and potentiation of platelet aggregation, induced by their nitric oxide (NO) quenching action, we tried to develop new molecules that would be c1inically usable as artificial red blood cells.During 1997 to 1999 research project owing to the Grand-in-Aid, we found that 1. The NO sequestrating action of Hb-based molecules is brought about after they penetrate into blood vessels through the gaps between endothelial cells, thus, the bigger the size of Hb-based molecule is, the less the NO sequestration occurs. 2. The size of Hb-based molecules also decides the magnitude of NO sequestration in the rat heart. 3. We then tried to S-nitrosylate the β-chain of Hb molecule and became able to manufacture S-nitroso-Hb (SNO-Hb) of up to 70 % purity. When injected intravenously, SNO-Hb proved to have fewer effects on blood pressure and platelet aggregation compared with stroma-free Hb.Furthermore, 5. We proved the usefulness of the modalities listed below for the evaluation of biological actions of artificial red blood cells; (1) continuous brain NO measurement with microdialysis connected on line to an HPLC system, (2) ex vivo platelet aggrigometry using laser beam scatter, (3) assessment of cerebral oxygenation and redox behavior of cytochrome oxidase by near-infrared spectroscopy.Finally, 6. We newly developed a long chain polyethylene glycol-conjugated Hb and started investigating its biological activity.