Single Molecule Analysis of Erythrocyte Adhesion in Sickle Cell Disease

涉及镰状红细胞的粘附相互作用在血管闭塞危象、溶血性贫血和镰状细胞病的其他临床表现的病理生理学中至关重要。这些相互作用似乎是由镰状红细胞表面粘附分子的异常表达和/或功能介导的。最近在模型膜和完整生物系统中的研究已经开始阐明粘附分子的生物化学和生物物理特性，这些特性对于稳定的粘附是必要的; 这些特性包括粘附分子的表达、大小、横向移动性、表面密度、表面分布和对分子的同源配体的亲和力。尽管已经确定了许多参与镰状红细胞粘附于血管内皮细胞和T淋巴细胞的分子相互作用，但这些对稳定粘附很重要的分子相互作用的性质仍有待表征。我们已经开发了一套独特的生物物理和成像技术来研究，在单个粘附分子的水平上，参与细胞-细胞粘附的分子相互作用。这些方法包括荧光 光漂白恢复、偏振荧光耗尽、单粒子跟踪、激光光镊、玻璃支撑的平面双层膜、荧光共振能量转移和动态体外和体内（活体内）粘附测定。本文拟将这些方法应用于研究（1）镰状红细胞膜蛋白和脂质动力学，（2）镰状红细胞与活化血管内皮细胞之间的粘附相互作用，（3）镰状红细胞与活化T淋巴细胞之间的粘附相互作用。我们将使用这些方法来研究粘附分子VLA-4（α 4 β 1整合素），CD 36和CD 2对镰状红细胞，VCAM-1和α-v，β 3整合素对活化血管内皮细胞，CD 58对活化T淋巴细胞和粘附血浆蛋白血小板反应蛋白的粘附相互作用。这些研究的结果有望导致对镰状细胞病病理生理学中重要分子和细胞事件的定量理解，并且理想地，为中断这些分子和细胞事件的最关键方面的靶向治疗指明方向。

Adhesive interactions involving sickle erythrocytes are critically important in the pathophysiology of vaso-occlusive crisis, hemolytic anemia, and other clinical manifestations of sickle cell disease. These interactions appear to be mediated by the abnormal expression and/or function of adhesion molecules on the surface of sickle erythrocytes. Recent studies in model membranes and intact biological systems have begun to elucidate the biochemical and biophysical properties of adhesion molecules that are necessary for stable adhesion; these properties include adhesion molecule expression, size, lateral mobility, surface density, surface distribution, and affinity for the molecule's cognate ligand. Although a number of molecular interactions involved in sickle lerythrocyte adhesion to vascular endothelial cells and T lymphocytes have been identified, the properties of these molecular interactions that are important for stable adhesion remain to be characterized. We have developed a unique set of biophysical and imaging techniques to study, at the level of individual adhesion molecules, the molecular interactions involved in cell-cell adhesion. The methods include fluorescence photobleaching recovery, polarized fluorescence depletion, single particle tracking, laser optical tweezers, glass-supported planar bilayer membranes, fluorescence resonance energy transfer, and dynamic in vitro and in vivo (intravital) adhesion assays. Here we propose to apply these methods to the study of (1) membrane protein iand lipid dynamics in sickle erythrocytes, (2) adhesive interactions between sickle erythrocytes and activated vascular endotheliai cells, and (3) adhesive interactions between sickle erythrocytes and activated T lymphocytes. We shall use these methods to study adhesive interactions involving the adhesion molecules VLA-4 (alpha4beta1integrin), CD36, and CD2 on sickle erythrocytes, VCAM-1 and alpha-v, beta3 integrin on activated vascular endothelial cells, CD58 on activated T lymphocytes, and the adhesive plasma protein thrombospondin. Results from these studies are expected to lead to a quantitative understanding of important molecular and cellular events in the pathophysiology of sickle cell disease, and, ideally, to point the way to targeted therapies that interrupt the most critical aspects of these molecular and cellular events.