B-CAM/LU is an adhesion molecule whose expression is increased in epithelial cancers and on red cells (RBC) of patients with sickle cell disease. We propose to investigate the hypothesis the B-CAM/LU mediates both adhesion to laminin as well as cell-cell adhesion in sickle cell disease, thus playing an important role in the vaso- occlusive process leading to pain and organ damage. We further propose to see the results of these investigations and to explore therapeutically applicable methods for reduction of SS RBC adhesion to endothelium and subendothelial matrix laminin by developing reagents capable of interfering with B-CAM/LU binding to its ligands. First, we will use a variety of methods to identify the surface molecules of RBC and endothelial cells that serve as ligands in B-CAM/LU-mediated adhesion processes. Second, we will determine the process(es) involved in activation of B-CAM/LU adhesive function on S RBC and in nucleated cells expressing recombinant forms of B-CAM/LU. Both phosphorylation and protein- protein interactions may play a role in this process. Specifically, we will study possible interaction of B-CAM/LU with integrins (especially alpha4beta1) and with CD44 based on our preliminary data. We will also investigate the effect of serine/threonine phosphorylation within the cytoplasmic domain of B-CAM/LU, and whether this occurs to a different degree in SS versus normal RBC, and in oxygenated versus deoxygenated RBC. We will further determine if the putative SH3 binding motif of B-CAM/LU associates with either tyrosine kinases or other signaling molecules with SH3 motifs. Third, to develop specific inhibitors that block B-CAM/LU- dependent adhesion, we will use various adhesion assays to study the effects of anti-B-CAM/LU monoclonal antibodies, as well as soluble recombinant B-CAM/LU and inhibitory peptides identified through screening of peptide display phage libraries. Overall, these studies will further define how B-CAM/LU contributes to adhesion and vaso-occlusion and will identify avenues by which its interactions might be abrogated in a therapeutic setting.
