Cell Membrane Sulfatide Promotes Sickle Cell Adhesion to Endothelium.

Sickle cell disease (SCD) affects millions of people worldwide, and is associated with significant morbidity and mortality. Although the clinical manifestations of the disease are very complex, much of the cause can be ascribed to occlusion of small vessels by the sickle red blood cells (RBCs). More than 30% of all deaths in SCD are due to the vasoocclusion, which results in ischemia, multiorgan failure and strokes. The proximate cause for vasoocclusion appears to be an increased adhesiveness of sickle cells to the vessel wall, and we postulate that the exposure of sulfatide on sickle cells accounts for their adhesive phenotype. Sulfatide binds with high affinity to many of the adhesion proteins known to be involved in cell adhesion to subendothelium and endothelium, including von Willebrand factor (VWF), thrombospondin (TSP), laminin and P-selectin. We therefore compared the expression and distribution of sulfatide in sickle cells to that in normal RBCs. When examined by flow cytometry using a previously described Alexa fluor-conjugated single-chain variable fragment (scFv) antibody, PA38, we found that sickle cells displayed more sulfatide on surface than normal RBCs (mean fluorescence 1.6±0.5 Vs. 0.9±0.3, p<0.05, n=6). When we examined sulfatide distribution by confocal microscopy using the labeled PA38, we found it to label more intensely in sickle cells than the normal RBCs and to be distributed heterogeneously, with areas of intense staining. The heterogeneous distribution suggested that the sulfatide might exist within membrane-microdomains/lipid rafts. We tested this possibility by sucrose density centrifugation of detergent lysates (1% Triton X-100) of erythrocyte ghosts from sickle and normal cells and found that sulfatide was distributed in raft fractions, as defined by being in the fractions containing the raft marker flotillin-1. Consistent with an important role for sulfatide in sickle vaso-occlusion, we found that both normal and sickle RBCs attached under flow to the surface of histamine-activated human umbilical vein endothelial cells (HUVEC). The sickle RBCs adhered more avidly, as they were able to rest higher shear stresses (1.86 and 2.5 dyne/cm²) than the normal RBCs before detaching Greater than 50% of the initial adhesion was inhibited by treatment with the anti-sulfatide scFv, PA38. We obtained similar results in terms of the greater shear resistance of sickle cells and the ability of PA38 to inhibit adhesion when we compared the adhesion of sickle and normal RBCs to surfaces coated with the adhesive ligands such as VWF (the ultra-large form) and laminin. Thus, our study elucidates an important role of red cell membrane sulfatide in sickle cell adhesion to the endothelium and to adhesive ligands, and suggests that this mechanism is important pathophysiologically in the development of sickle vaso-occlusion. Sulfatide distribution into lipid rafts may allow the formation of adhesive patches that facilitate adhesion.