Novel Functions of Red Cell Proteins Lu and LW

描述（由申请人提供）：红细胞粘附蛋白Lu和LW（现称为ICAM-4）是定义明确的血型，但对其膜功能知之甚少。在红细胞生成过程中，成红细胞在巨噬细胞周围的成红细胞岛内分化。我们假设ICAM-4通过ICAM-4/α 4 β 1结合介导成红细胞之间的相互作用，并通过ICAM-4/α V结合调节成红细胞与巨噬细胞的粘附。与ICAM-4区域相对应的肽与α V和β 1相互作用，抑制成红细胞岛的形成。此外，我们确定了分泌的ICAM-4亚型，这可能会调节结合。我们和其他人已经表明ICAM-4也结合存在于内皮细胞、中性粒细胞和血小板上的整联蛋白。因此，我们将探讨ICAM-4对镰状细胞病血管病理学的贡献。Lu以高亲和力结合含有α 5链的层粘连蛋白（层粘连蛋白10/11）。重要的是，培养的成红细胞从第6天开始越来越多地结合层粘连蛋白10/11，并且结合水平增加Lu的表达。我们假设Lu-层粘连蛋白粘附在去核和/或骨髓排出过程中起作用，因为α 5层粘连蛋白定位于骨髓窦状隙的内皮下基底膜。为了验证我们的假设，我们建议：1）通过使用定点突变和体外结合试验鉴定ICAM-4参与α 4 β 1结合的区域来检查ICAM-4的功能;表征阻断剂对成红细胞岛的形成和解离的影响;使用微量移液管技术评估在存在和不存在阻断剂的情况下岛内细胞之间的相互作用;通过动态力光谱测量单个粘附结合强度;并研究ICAM-4敲除小鼠中的成红细胞岛。2)通过鉴定Lu上的层粘连蛋白结合区域，确定Lu-层粘连蛋白受体复合物的功能;开发封闭抗体和肽，并在体外层粘连蛋白10/11中测试其对核挤出和网织红细胞生成的影响;并通过分析Lu敲除小鼠中的细胞凋亡、去核和网织红细胞增多症。3)通过研究向转基因/敲除镰状小鼠输注针对ICAM-4的肽和抗体（其阻断镰状红细胞与内皮细胞的粘附）对血管血流的影响，探索ICAM-4对镰状细胞病血管病理学的贡献。这些目标的成功实现将进一步促进我们对正常红细胞生成机制和镰状细胞病病理生理学的理解，这可能导致新的治疗方式 .

DESCRIPTION (provided by applicant): Erythrocyte adhesion proteins Lu and LW (now termed ICAM-4) are well-defined blood groups, but little is known regarding their membrane function. During erythropoiesis, erythroblasts differentiate within erythroblastic islands surrounding a macrophage. We hypothesize that ICAM-4 mediates interactions between erythroblasts via ICAM-4/alpha4beta1 binding and regulates adhesion of erythroblasts to macrophages via ICAM-4/alphaV binding. Peptides corresponding to areas of ICAM-4 that interact with alphaV and beta1 inhibit erythroblastic island formation. Additionally, we identified a secreted isoform of ICAM-4, which may modulate binding. We and others have shown that ICAM-4 also binds integrins present on endothelial cells, neutrophils and platelets. Hence, we will explore the contribution of ICAM-4 to vascular pathology of sickle cell disease. Lu binds laminins containing the alpha5 chain (laminins 10/11) with high affinity. Importantly, cultured erythroblasts increasingly bind laminin 10/11 from day 6 onwards and the level of binding paralleled increasing expression of Lu. We hypothesize that Lu-laminin adhesion functions during enucleation and/or marrow egress, since alpha5 laminin localizes to subendothelial basement membranes of bone marrow sinusoids. To test our hypotheses we propose to: 1) Examine ICAM-4 function by identifying regions of ICAM-4 involved in alpha4beta1 binding employing site directed mutagenesis and in vitro binding assays; characterize the effect of blocking reagents on formation and dissociation of erythroblastic islands; assess interactions between cells within islands in the presence and absence of blocking reagents using micropipette techniques; measure single adhesion bond strength by dynamic force spectroscopy; and study erythroblastic islands in ICAM-4 knockout mice. 2) Determine function of the Lu-laminin receptor complex by identifying the laminin binding region on Lu; developing blocking antibodies and peptides and testing their effects on nuclear extrusion and reticulocyte generation in vitro laminin 10/11; and by analyzing apoptosis, enucleation, and reticulocytosis in Lu knockout mice. 3) Explore contributions of ICAM-4 to vascular pathology in sickle cell disease by studying effects on vascular blood flow of infusing transgenic/knockout sickle mice with peptides and antibodies directed against ICAM-4 which block adhesion of sickle red cells to endothelial cells. Successful accomplishment of these aims will further our goals of developing a mechanistic understanding of normal erythropoiesis and the pathophysiology of sickle cell disease which could lead to novel therapeutic modalities .