MECHANISMS OF RBC ADHESION AND ENDOTHELIAL INJURY IN SICKLE CELL DISEASE

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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.