Inhibiting Binding of Sickle Red Cell ICAM-4 to Endothelial Cell αVβ3 Integrin Decreases Red Cell Adhesion and Vaso-Occlusion.
Authors: Kaul Dhananjay , Liu Xiao-du , Zhang Xiaoqin , Mankelow Tosti , Parsons Stephen , Spring Frances , An Xiuli , Mohandas Narla , Anstee David , Chasis Joel Anne 
Published: October 2019

Abstract
Convincing evidence is accumulating that in sickle cell disease adhesion molecules on sickle red cells (RBCs) contribute to increased interactions with vascular endothelial cells leading to vaso-occlusion. Erythrocyte ICAM-4, a member of the immunoglobulin superfamily, binds multiple integrins including αVβ3. Earlier investigations showed that antibodies to αVβ3 inhibit human sickle RBC adherence to rat endothelium and improve microvascular dynamics, suggesting that αVβ3 may be an endothelial cell integrin functioning in vaso-occlusion. More recent in vitro studies revealed that epinephrine can act via a PKA-dependent pathway to activate ICAM-4 mediated sickle RBC adhesion to endothelial cell αVβ3. We are exploring the contribution of ICAM-4/αV interactions to vaso-occlusion utilizing a well established ex vivo mesocecum vasculature model that enables intravital microscopic observation and quantitation of sickle RBC adhesion to endothelial cells in the microcirculation, as well as hemodynamic measurements. A molecular model of ICAM-4 presents the extracellular region as two Ig-like domains comprised of A,B,C,D,E,F, and G strands. Earlier, we identified a patch or footprint on ICAM-4 comprised of three A strand residues and five G strand residues, that mediates adhesion to αV. In the current studies we quantitated sickle RBC adhesion to endothelial cells in the presence of two synthetic peptides that block ICAM-4/αV adhesion, peptides FWV and ATSR which correspond to sequences of the A and G strands of ICAM-4 domain 1, respectively. Sickle RBCs were obtained from stable HbSS patients who had not been transfused for at least 4 months and who were not receiving hydroxyurea. Isolated rat mesocecum was perfused with platelet-activating factor (PAF), that enhances sickle RBC adhesion. Then peptide FWV or ATSR (250 μMol/L) was infused, and following a 30 minute incubation, a bolus of washed, oxygenated sickle RBCs (Hct 30%) was perfused at a pressure of 60 mm Hg. Peptides were each tested with cells obtained from the same patients. Quantitation of adherent sickle RBCs per 100 μm2 in the affected venules was determined by direct microscopic observations and video analysis. In preparations treated with PAF alone, adhesion of sickle RBCs showed a strong inverse correlation with venular diameter. Adhesion was maximal in small-diameter venules, frequently resulting in vessel blockage. In preparations treated with peptide FWV, sickle RBC adhesion was distinctly decreased in venules of all diameters. ATSR resulted in even more pronounced inhibition of RBC adhesion, particularly in small-diameter venules. Comparison of regression lines revealed significantly lower Y-intercepts for both FWV and ATSR as compared with PAF-treated controls (p<0.00001). With both FWV and ATSR no blockage was observed in postcapillary venules. In marked contrast, a strand F control peptide, AWSS, had minimal effect. Our data greatly strengthen the thesis that N-terminal domain 1 of ICAM-4 on sickle RBCs binds endothelial cells via αVβ3 and that this interaction significantly contributes to vaso-occlusion in sickle cell disease. These findings suggest that peptides or small molecule mimetics of ICAM-4 may have potential as therapeutic agents.