Single Molecule Analysis of Erythrocyte Adhesion in Sickle Cell Disease

镰状红细胞之间的黏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通过镰状红细胞表面黏附分子的异常表达和/或功能来调节的。最近对模型膜和完整生物系统的研究已经开始阐明黏附分子的生化和生物物理性质，这是稳定黏附所必需的；这些 性质包括黏附分子的表达、大小、侧向迁移率、表面密度、表面分布和对分子同源配体的亲和力。尽管已鉴定出与镰状红细胞与血管内皮细胞和T淋巴细胞黏附有关的许多分子相互作用，但这些分子相互作用对稳定黏附的重要性质仍有待鉴定。我们开发了一套独特的生物物理和成像技术，在单个黏附分子水平上研究细胞与细胞黏附所涉及的分子相互作用。这些方法包括荧光法 光漂白恢复、偏振荧光耗尽、单粒子跟踪、激光光钳、玻璃支撑的平面双层膜、荧光共振能量转移以及动态体外和体内(活体)黏附试验。我们建议用这些方法来研究(1)镰状红细胞膜蛋白I和脂质动力学，(2)镰状红细胞与激活的血管内皮细胞之间的黏附相互作用，(3)镰状红细胞与激活的T淋巴细胞之间的黏附相互作用。我们将使用这些方法来研究镰状红细胞上的黏附分子VLA-4(α4β1整合素)、CD36和CD2，VCAM-1和α-V，激活的血管内皮细胞上的β3整合素，激活的T淋巴细胞上的CD58和黏附的血浆蛋白凝血酶反应蛋白的黏附相互作用。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的分子和细胞事件的定量了解，理想情况下，还将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.