MECHANISMS OF RBC ADHESION AND ENDOTHELIAL INJURY IN SICKLE CELL DISEASE

血管闭塞导致阵发性疼痛危象和损伤 各种终末器官代表着最重要的病理生理过程 镰状细胞性贫血(SCA)。镰刀状红血的异常黏附 细胞到血管内皮细胞似乎在这方面起着重要作用 进程。我们的研究表明，年轻的镰状红细胞的异常黏附 高分子量对培养内皮细胞的促进作用 RGD(von-Willebrand因子)的多聚体，并通过添加 合成RGD(精氨酸-甘氨酸-天冬氨酸)含有多肽。此外，我们 观察到与内皮细胞和单核细胞的黏附致密 脱氧RSC和ISC，其中磷脂酰丝氨酸(PS)存在于 PS可抑制RBC双层膜的外叶 脂质体。 我们建议检验关于粘附性的三个假说 SCA血管闭塞的发病机制。首先，粘附性异常。 年轻的SS红细胞是由SS红细胞上存在的RGD受体介导的；第二， 致密SS红细胞的黏附是由可能存在的“PS”受体介导的 对内皮细胞的影响；第三，镰状红细胞的异常黏附 脂氧合酶代谢产物(15-)介导的内皮细胞损伤 HETE；15-羟基二十碳五烯酸)，导致 影响血管内皮细胞表面表达的内皮细胞单层 内皮细胞-细胞黏附分子(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式进行检验：(1)分离、纯化和 SS红细胞RGD黏附受体的功能鉴定(2) 内皮细胞和血管内皮细胞中可能的PS受体的鉴定 利用PS的新型酰基硅烷衍生物作为单核细胞膜 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全长克隆获得 相应长度的CDNA；(3)SS RBC中15-HETE的测定及其 对生物合成、表面表达和mRNA转录水平的影响 利用针对EC-CAM的抗体和CDNA探针；以及 (4)测定SS RBC与内皮细胞或15-HETE的粘附性 影响血小板衍生生长因子-β(PDGF-β)的蛋白和特异性mRNA转录 衍生生长因子)、内皮素-1和组织因子利用特异性 抗体和cDNA探针，这可能有助于血管内膜 SCA分别表现为增生、血管痉挛和高凝状态。 综上所述，这些研究应该能让我们更好地理解 镰状细胞与血管内皮细胞黏附的分子机制 和伴随的内皮损伤，从而提供了新的洞察力 镰状细胞性贫血的血管闭塞过程。

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.