Vaso-occlusion is the major cause of morbidity and mortality in sickle cell disease (SS-D). We hypothesize that enhanced adhesion of sickle red blood cells (RBCs) to the vascular endothelium involves adhesive molecules within the sickle RBC membrane, ultimately leading to microvascular occlusion in SS-D. However, the ability to design rational approaches to treat sickle cell disease is dependent upon a more complete understanding of the predominant pathologic cellular interactions operative in sickle cell disease. Therefore, the central goal of this project is to further elucidate role of RBC adhesion in microvascular occlusion in SS-D. Consequently, the specific aims for this project are to: 1) Identify and characterize adhesive epitopes within the sickle RBC membrane, 2) Investigate the effect of modulators of sickle RBC adhesion on the evolution of vaso-occlusion in transgenic Sickle Mice, and 3) Examine sickle RBC adhesion in the cerebrovascular microcirculation of transgenic Sickle Mice. We will use recombinant phage display technology to develop monoclonal antibodies to identify and characterize adhesive epitopes within the sickle RBC membrane. To test the contribution of specific adhesive epitopes, we will employ an in vitro flow adhesion assay where washed RBCs interact with purified adhesive proteins or cultured endothelial cells under conditions of controlled shear force. We will also use transgenic mice that exclusively express sickle Hb to further examine the role of RBC adhesion in the development of acute vaso-occlusion in the more complex setting of an in vivo model of SS-D. Finally, we will use this transgenic mouse model of SS-D to specifically address RBC adhesion to the unique endothelium of the brain. We anticipate that these studies will lead to the identification of specific adhesive components within the sickle RBC membrane that augment the adhesion of sickle RBCs to the vessel wall, including cerebrovascular endothelium. These studies will also provide insight as to mechanisms that will inhibit the adhesion of the sickle RBC to the vessel wall, prevent endothelial injury and, therefore, subsequent vascular obstruction in vivo in SS-D. Thus, we expect that information derived from this proposal will lead to new therapeutic approaches for the treatment of vaso-occlusion in SS-D.
