The Nature of the P-Selectin Ligands on Sickle Cells.
Authors: Embury Stephen H. , Baran Christine E. , Hefner Colleen A. , Seto Christi K. , Matsui Neil M. 
Published: October 2019

Abstract
Elucidation of the adhesive interactions that effect microvascular occlusion in sickle cell disease both increases our understanding of the pathophysiology of vasoocclusion and identifies molecular targets for the development of therapeutic interventions. Work from our laboratory has established that sickle RBC adhere to P-selectin on thrombin-activated endothelial cells and to immobilized, recombinant P-selectin in vitro (Matsui et al. Blood 98:1955, 2001) and that this adhesion can be inhibited by agents that block P-selectin (Matsui et al. Blood 100:3790, 2002). Based on these findings, we established that sickle RBC adherence to endothelial P-selectin has a substantial influence on microvascular blood flow in vivo and that blocking P-selectin enhances microvascular flow (Embury et al. Blood In Press). We reasoned that characterization of the cognate ligands for P-selectin ligand on sickle RBC could identify additional targets for therapeutic intervention. We had determined that that sickle RBC did not express the P-selectin ligand, P-selectin glycoprotein-1, but that membrane sialic acid is required for sickle RBC binding to P-selectin. Here we describe further characterization of the P-selectin binding determinants on sickle RBC membranes. We assessed the expression of sialyl Lewis X (sLeX) on sickle RBC using flow cytometry and the importance of sLeX expression to the rolling adhesion of sickle RBC to P-selectin in vitro. Using the monoclonal antibodies (mAb) HECA-452 and CSLEX-1 in flow cytometry we detected significant expression of sLeX on sickle RBC (p < 0.003 and p < 0.02, respectively) but not on non-sickle RBC (p < 0.07 and p < 0.3, respectively). Treatment of sickle RBC with sialidase caused a partial, dose dependant reduction of the level of detectable sLeX and of rolling adhesion to immobilized P-selectin (approximately 40% and 85%, respectively), which correlated positively. To assess the possible selective contribution of reticulocytes as a subset of higher sLeX expressing sickle RBC we employed dual label flow cytometry to determine whether sLeX and the transferrin receptor (CD71) are co-expressed. Using mAb YDJ1.2.2 for the transferrin receptor as a reticulocyte marker and CSLEX-1 showed that sLeX was expressed both on sickle reticulocytes and on older sickle RBC. Treatment of sickle RBC with O-sialoglycoprotein endopeptidase, which cleaves sialylated O-glycans, also reduced both their sLeX expression and rolling adhesion on P-selectin (approximately 30% and 65%, respectively). Treatment of sickle RBC with N-glycosidase F did not reduce sLeX or adhesion levels, trypsin treatment produced inconsistent effects, and phosphatidylinositol-specific phospholipase C caused a significant decrease in adhesion but not a significant reduction in sLex expression. These findings suggest that sickle RBC possess more than one type of glycoprotein as a ligand for P-selectin. We also used a solid-phase binding assay to detect a significant level of P-selectin binding to membrane lipids extracted from sickle RBC. Thus, the P-selectin binding determinants on sickle RBC include sialic acid, sLeX, O-linked glycans, PI-linked glycoproteins, and glycolipids. Each of these P-selectin ligands represents a potential target of new adhesion blocking drugs for the treatment of sickle cell disease.