Red Blood Cell Adhesion in Sickle Cell Disease

描述（由申请人提供）：血管闭塞是镰状细胞病（SS-D）发病和死亡的主要原因。我们假设镰状红细胞（RBC）对血管内皮的粘附增强涉及镰状红细胞膜内的粘附分子，最终导致SS-D的微血管闭塞。然而，设计合理的方法来治疗镰状细胞病的能力依赖于对镰状细胞病中主要病理细胞相互作用的更全面的理解。因此，本项目的中心目标是进一步阐明红细胞粘附在SS-D微血管闭塞中的作用。因此，本项目的具体目标是：1)鉴定和表征镰状红细胞膜内粘附表位；2)研究镰状红细胞粘附调节剂对转基因镰状小鼠血管闭塞进化的影响；3)检测转基因镰状小鼠脑血管微循环中的镰状红细胞粘附。我们将利用重组噬菌体展示技术开发单克隆抗体来鉴定和表征镰状红细胞膜内的粘附表位。为了测试特异性黏附表位的作用，我们将采用体外流动黏附试验，在控制剪切力的条件下，洗涤红细胞与纯化的黏附蛋白或培养的内皮细胞相互作用。我们还将使用专门表达镰状血红蛋白的转基因小鼠，在更复杂的SS-D体内模型中进一步研究红细胞粘附在急性血管闭塞发展中的作用。最后，我们将使用这种转基因SS-D小鼠模型来专门解决红细胞粘附在大脑独特的内皮细胞上的问题。我们预计这些研究将导致鉴定镰状红细胞膜内的特定粘附成分，增强镰状红细胞对血管壁的粘附，包括脑血管内皮。这些研究还将提供抑制镰状红细胞粘附到血管壁的机制，防止内皮损伤，从而防止SS-D体内随后的血管阻塞。因此，我们期望从该建议中获得的信息将导致治疗SS-D血管闭塞的新方法。

DESCRIPTION (provided by applicant): Vaso-occlusion is the major cause of morbidity and mortality in sickle cell disease (SS-D). We hypothesize that enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium involves adhesive molecules within the sickle RBC membrane, ultimately leading to microvascular occlusion in SS-D. However, the ability to design rational approaches to treat sickle cell disease is dependent upon a more complete understanding of the predominant pathologic cellular interactions operative in sickle cell disease. Therefore, the central goal of this project is to further elucidate role of RBC adhesion in microvascular occlusion in SS-D. Consequently, the specific aims for this project are to: 1) Identify and characterize adhesive epitopes within the sickle RBC membrane, 2) Investigate the effect of modulators of sickle RBC adhesion on the evolution of vaso-occlusion in transgenic Sickle Mice, and 3) Examine sickle RBC adhesion in the cerebrovascular microcirculation of transgenic Sickle Mice. We will use recombinant phage display technology to develop monoclonal antibodies to identify and characterize adhesive epitopes within the sickle RBC membrane. To test the contribution of specific adhesive epitopes, we will employ an in vitro flow adhesion assay where washed RBCs interact with purified adhesive proteins or cultured endothelial cells under conditions of controlled shear force. We will also use transgenic mice that exclusively express sickle Hb to further examine the role of RBC adhesion in the development of acute vaso-occlusion in the more complex setting of an in vivo model of SS-D. Finally, we will use this transgenic mouse model of SS-D to specifically address RBC adhesion to the unique endothelium of the brain. We anticipate that these studies will lead to the identification of specific adhesive components within the sickle RBC membrane that augment the adhesion of sickle RBCs to the vessel wall, including cerebrovascular endothelium. These studies will also provide insight as to mechanisms that will inhibit the adhesion of the sickle RBC to the vessel wall, prevent endothelial injury and, therefore, subsequent vascular obstruction in vivo in SS-D. Thus, we expect that information derived from this proposal will lead to new therapeutic approaches for the treatment of vaso-occlusion in SS-D.