We will examine the hypothesis, derived from our preliminary experiments, that thrombospondin (TSP) is the major plasma protein adhesogen responsible for the abnormal adherence of sickle RBC to vascular endothelial cells (EC), an event believed to be of critical pathogenetic importance in sickle disease. Studies are proposed under six broad categories of Specific Aims. [1] We will define the receptor complement on sickle RBC by FACS, determine if TSP binds to CD36 (glycoprotein IV) on sickle reticulocytes and assess the binding affinity using a novel microfluometric binding assay, explain why sickle retics have greater CD36 receptor density than HbAA retics, and isolate/characterize erythroid CD36. [2] Using adhesion assays and either peptides/antibodies or TSP fragments, we will define the domains of TSP that mediate RBC/EC interaction and whether TSP in plasma exerts its adhesogenic influence in conjunction with another protein. [3] Comparing large vessel EC (CD36 negative) to microvascular EC (CD36 positive) and large vessel EC that express CD36 (induced via infection with a retroviral vector containing CD36 CDNA), we will use various peptides and antibodies, as well as electron microscopic immunocytochemistry, to define the structure(s) on the endothelial surface that are receptive for TSP and promote RBC adherence, and we will measure TSP binding to endothelial cells. [4] The role of platelets in RBC/EC adhesion will be defined through electron microscopic immunocytochemistry and adherence assays in presence or absence of either resting platelets or those with activated surfaces. The degree to which plasma TSP is elevated and platelets are activated in vivo in sickle patients will be defined. Whether plasma TSP is derived from platelets will be determined. [5] Pathophysiologic relevance of TSP mediated RBC/EC adhesion will be evaluated by measuring strength of adhesion (including under flowing conditions) and the ability of cytokines to modulate the process. TSP will be directly compared to other proteins for ability to mediate RBC/EC adhesion. Ability of TSP or adherent RBC to mediate signal transduction and cause increased cytosolic calcium in EC will be defined. Instructive correlations will be sought by examining sickle patients, some longitudinally, for relevant parameters. [6] Since the long term goal of this work is to identify potential therapeutic options for possible use in sickle disease, the final stages of this project will involve design and evaluation of possibilities suggested by the results of our addressing the foregoing aims.
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