Effects of Nitric Oxide in Sickle Cell Blood

描述（由申请人提供）：我们的总体目标是阐明镰状细胞血液中一氧化氮（NO）的生化和生物物理方面。使用天然产生的一氧化氮，正被认为是治疗镰状细胞病的一种方法。它可能使患者受益于其作为血管扩张剂的能力，减少红细胞镰状细胞，减少镰状细胞粘附，并改善氧运输。然而，如果不彻底了解一氧化氮在血液中的作用（主要是通过其与血红蛋白的相互作用），就无法正确评估一氧化氮对患者的潜在益处或危害。镰状细胞血液，甚至正常血液中一氧化氮的大部分生物学特性尚不清楚，以前关于这一主题的研究有时报告了相互矛盾的结果。我们将重点关注一氧化氮在全血中的作用，或者至少在尽可能模拟体内条件下的作用。我们将把镰状血的研究结果与我们对正常血液、分离的正常红细胞和镰状红细胞的制备以及在各种条件下制备的纯化血红蛋白的研究结果进行比较。我们将使用各种光谱和其他生物物理工具，其中一些将作为该项目的一部分开发。这些方法包括显微镜、分光光度法、超离心、停止和淬火混合、激光光解和衍射、化学发光、电子自旋共振、核磁共振和使用积分球检测器的吸收光谱。我们将使用这些工具来评估一氧化氮对红细胞镰状细胞动力学和平衡状态的影响。我们还将研究NO如何影响镰状细胞聚合物熔化导致红细胞脱镰的机制和动力学。我们将研究NO对镰状血红蛋白和正常血红蛋白全血氧亲和力的影响，并研究NO对氧结合和解离动力学的影响。此外，我们将研究NO如何影响镰状红细胞的膜损伤，并研究由此产生的后果，因为它们与红细胞变形性有关。最后，我们将利用静态光谱和时间分辨光谱研究生理条件下NO与脱氧和含氧HbS的主要反应。我们的研究将帮助我们了解与NO在镰状细胞病和一般人群中的作用相关的重要问题。

DESCRIPTION (provided by applicant): Our overall goal is to elucidate biochemical and biophysical aspects of nitric oxide (NO) in sickle cell blood. The use of NO, which is produced naturally, is being considered as a treatment for sickle cell disease. It may benefit patients in its ability to act as a vasodllator, to decrease red blood cell sickling, decrease sickle cell adherence, and to improve oxygen transport. However, the potential of NO to benefit or harm patients cannot be properly assessed without a thorough understanding of its effects in blood, primarily through its interaction with hemoglobin. Much of nitric oxide biology in sickle cell blood, and even in normal blood, is not understood and previous studies on this subject have sometimes reported contradictory results. We will focus on the effects of NO in whole blood or, at least, under conditions that mimic those in vivo as much as possible. We will compare our results from sickle blood to those from studies we conduct on normal blood, on preparations of isolated normal and sickle red blood cells, and on purified hemoglobins prepared under various conditions. We will use a variety of spectroscopic and other biophysical tools, some of which will be developed as part of this project. These include microscopy, ektacytometry, ultracentrifugation, stopped and quench-flow mixing, laser photolysis and diffraction, chemiluminescence, electron spin resonance, nuclear magnetic resonance, and absorption spectroscopy using an integrating sphere detector. We will use these tools to assess nitric oxide's effects on both the kinetics and equilibrium state of red blood cell sickling. We will also study how NO affects the mechanism and kinetics of red blood cell unsickling due to sickle cell polymer melting. We will study the effects of NO on the oxygen affinity of sickle and normal hemoglobin in whole blood and examine the effects of NO on the kinetics of oxygen binding and dissociation. In addition, we will examine how NO effects membrane damage in sickle red blood cells and study the resulting consequences as they relate to red blood cell deformability. Finally, we will study the primary reactions of NO with deoxy and oxy HbS using static and time resolved spectroscopies under physiological conditions. Our studies will help us understand important issues related to NO's role in sickle cell disease and in the general population.