The red blood cell in sickle cell anemia is characterized not only by abnormal hemoglobin but also by many membrane alterations. During the lifespan of the sickle cell, membrane is lost in the form of spectrin- free microvesicles. The role of these vesicles in the pathophysiology of sickle cell disease remains completely unknown. Further, the geste that membrane proteins having a glycosyl-phosphatidylinositol (GPI) anchor are enriched relative to other membrane proteins in the vesicles releases from red cells during in vitro vesiculation. A number of GPI- anchored proteins serve a critical role in protecting red cells from lysis by activated complement regulatory proteins, decay-accelerating factor (DAF, CD55) and membrane inhibitor of reactive lysis (MIRL,CD59), in sickle cell anemia affect the susceptibility of sickle cells to complement-dependent injury and play an important role in the pathophysiology of sickle cell disease. Characterization of these proteins in cells and vesicles obtained from the blood of sickle cell patients will provide a means of probing the role of vesiculation in the disease process. Specifically, sickle cells will be examined for density-dependent differences in the quantity and function of DAF and MIRL. Structural alterations in these molecules detected by mass spectrometry will be correlated with functional changes, and the role of other cellular abnormalities characteristic of dense SS red cells in the induction of increased sensitivity to complement-dependent lysis will be determined. Patient samples will be examined before, during, and after states in which complement activation is likely to occur for evidence of complement-mediated erythrocyte destruction by quantitating C5b-9 neoantigen levels in red cells, plasma, and in circulating red cell ghosts. The samples also will be examined for the presence of vesicles enriched in DAF and MIRL. An assay for the quantification of circulating membrane vesicles will be established using immunofluorescence analysis of whole blood incubated with fluorescently labeled anti-glycophorin A. Correlations will be made with clinical parameters such as the presence of infection or vasoocclusive crisis and with laboratory parameters including the percentage of irreversibly sickled cells, the hemoglobin distribution width, and the degree of complement activation. Finally, in vitro models will be established to examine the relationship between the hemoglobin oxidation state of SS red cells and their sensitivity to complement-mediated injury, to see if membrane vesicles play a role in regulating complement activation, and to see if their are differences in the susceptibility of AA vs. SS red cells to vesiculation induced by C5b- 9 membrane attack complexes. These studies will provide new insights into the role of complement dysregulation and vesiculation in the pathophysiology of sickle cell disease.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Comprehensive Center (P60)
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