MECHANISMS OF RBC ADHESION AND ENDOTHELIAL INJURY IN SICKLE CELL DISEASE

血管闭塞导致疼痛危象发作和血管损伤 各种终末器官代表了最重要的病理生理过程 镰状细胞性贫血（SCA） 镰状红血异常粘连 细胞到血管内皮细胞似乎在这一过程中起着重要作用。 过程 我们的研究表明，年轻镰状红细胞的异常粘附 对培养的内皮细胞的作用被高分子量 RGD（冯-维勒布兰德因子）的多聚体，并通过加入 合成的含有RGD（arg-gly-asp）的肽。 另外我们 观察到粘附内皮细胞和单核细胞的致密 脱氧RSC和ISC，其中磷脂酰丝氨酸（PS）存在于 红细胞双层的外叶，可被PS抑制 脂质体。 我们建议测试三个假设有关的粘附在 SCA血管闭塞的发病机制。 第一， 年轻的SS RBC是由存在于SS RBC上的RGD受体介导的;其次， 致密SS RBC的粘附由存在的假定“PS”受体介导 三是镰状红细胞的异常粘附 导致内皮细胞损伤，由脂氧合酶代谢产物（15- HETE; 15-羟基二十碳四烯酸），导致 内皮细胞单层通过影响表面表达的 内皮细胞-细胞粘附分子（EC-CAM）参与连接 形成和伴随的血管活性分子的释放。 这些假设将通过以下方法进行检验：（1）分离、纯化和 来自SS RBC的RGD粘附受体的功能表征;（2） 内皮细胞中推定“PS”受体的鉴定 单核细胞膜，利用PS的新型酰基硅烷衍生物作为 光亲和探针;这些受体的结构信息将 通过部分氨基酸序列分析和克隆完整的 （3）SS红细胞15-HETE的测定及其与DNA的关系 对生物合成、表面表达和mRNA转录水平的影响 利用对EC-CAM特异的抗体和cDNA探针;和 (4)测定SS RBC与内皮细胞或15-HETE的粘附 影响PDGF-β（血小板）的蛋白质和特异性mRNA转录物 衍生生长因子）、内皮素-1和利用特异性 抗体和cDNA探针，这可能有助于血管内膜 SCA的血管增生、血管痉挛和高凝状态。 综合起来，这些研究应该可以让我们更好地了解 镰状细胞粘附血管内皮的分子机制 以及伴随的内皮损伤，从而提供了对 镰状细胞性贫血的血管阻塞过程。

Vascular occlusion leading to episodes of painful crises and damage to various end organs represents the most important pathophysiologic process in sickle cell anemia (SCA). The abnormal adhesion of sickle red blood cells to vascular endothelium appears to play an important role in this process. Our studies show that the aberrant adhesion of young sickle RBC to cultured endothelial cells is potentiated by high molecular weight multimers of RGD (von-Willebrand factor) and blocked by the addition of synthetic RGD (arg-gly-asp) containing peptides. Additionally, we observed that the adherence to endothelial cells and monocytes of dense deoxygenated RSC and ISC, in which phosphatidylserine (PS) is present in the external leaflet of the bilayer of RBC, could be inhibited by PS liposomes. We propose to test three hypothesis concerning adhesion in the pathogenesis of vasocclusion in SCA. First, the abnormal adhesion of young SS RBC is mediated by RGD receptors present on SS RBC; second, the adherence of dense SS RBC is mediated by putative "PS" receptors present on endothelial cell; and third, the abnormal adherence of sickle RBC causes endothelial cell injury, mediated by lipoxygenase metabolite (15- HETE; 15-hydroxy eicosa-tetraenoic acid), resulting in disruption of the endothelial cell monolayer by affecting the surface expression of the endothelial cell-cell adhesion molecular (EC-CAM) involved in junction formation and by concomitant release of vasoactive molecules. These hypothesis will be tested by; (1) isolation, purification and functional characterization of the RGD adhesion receptor from SS RBC; (2) identification of putative "PS" receptors in endothelial cell and monocyte membranes utilizing a novel acylsilane derivative of PS as a photoaffinity probe; the structural information of these receptors will be obtained by partial amino acid sequence analysis and cloning of full length corresponding CDNA; (3) determination of 15-HETE in SS RBC and its effect on the biosynthesis, surface expression and MRNA transcript levels of EC-CAM, utilizing antibodies and CDNA probes specific for EC-CAM; and (4) determine whether adhesion of SS RBC to endothelial cells or 15-HETE affects the protein and specific MRNA transcripts of PDGF-beta (platelet derived growth factor), endothelin-1 and tissue factor utilizing specific antibodies and CDNA probes, which could contribute to vascular intimal hyperplasia, vasospasm and hypercoagulability, respectively, in SCA. Taken together, these studies should allow us to better understand the molecular mechanism of adherence of sickle cells to vascular endothelium and concomitant injury to endothelium, thus providing new insight into the process of vasocclusion in sickle cell anemia.