MECHANISM OF SICKLE ERYTHROCYTE/ENDOTHELIAL ADHESION

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We propose to test the hypothesis that sickle red cell adhesion to endothelial cells initiates or contributes to the localized vaso- occlusive crises characteristic of sickle cell disease. Specifically, we suggest that adhesion varies between endothelial cells from different vascular beds, that adhesion will increase during crisis periods, and that endothelial cell agonists will increase adhesion. We have previously reported that high molecular weight von Willebrand factor (vWF) multimers increase sickle red cell adhesion to umbilical vein endothelial cells. We suggest that endothelial cell stimulation occurs periodically due to chemical or mechanical stresses and leads to localized release of high molecular weight vWF multimers. Preliminary data suggest that adhesion is increased when endothelial cells are stimulated with endotoxin or thrombin. We hypothesize that these multimers bind to sickle erythrocytes and endothelial cells in venules in vivo, leading to vascular obstruction. Recent data indicates that sickle red cells suspended in autologous plasma are more adhesive to microvascular endothelial cells than to umbilical vein endothelial cells. This suggests that the mechanism or red cell adhesion is dependent upon the location of the endothelium. Based on these results we propose that sickle red cell adhesion in vivo is related to complex changes in the erythrocyte surface, the endothelium, and the intravascular milieu. The following parameters are regarded as important determinants of sickle erythrocyte adherence: (i) endothelial cell origin (large or small vessel); (ii) patient clinical condition (crisis or steady-state); (iii) red cell age; (iv) local wall shear stress; (v) the time of exposure of endothelial cells to agonists; and (vi) the duration of contact between sickle erythrocytes, stimulated endothelial cells, and secreted vWF multimers. The influence of these parameters on sickle red cell adhesion to endothelial cells will be investigated in parallel-plate flow chamber adhesion assays. Epi-fluorescence videomicroscopy and digital image processing will be employed to quantify sickle red cell adhesion to stimulated and unstimulated endothelial cells under physiological flow conditions. Development of a red cell recirculation system is proposed to allow red cells, agonists, and endothelial cells continuous contact under flow. Antibody studies will be implemented to determine adhesive receptors on endothelial cells and red cells. Characterization of alterations in the endothelium, plasma, and erythrocytes responsible for increased adhesion in vitro will identify factors not related to hemoglobin S polymerization and red cell sickling which could contribute to or participate in vaso-occlusion in vivo.