We will characterize the structure, binding sites for multiple ligands, function in platelet-neutrophil interactions, and novel ligands for the leukocyte integrins, Mac-1 (CD11b/CD18) and p150,95 (CD11c/CD18). We will characterize a ligand for L-selectin on neutrophils that cooperates with Mac-1 in homotypic aggregation and in adhesion in shear flow. Mac-1 and the closely related integrin p150,95 bind multiple ligands, including iC3b, fibrinogen, ICAM-1, and heparan sulfate. Genetic deficiency of these receptors in leukocyte adhesion deficiency-I (LAD-I) results in bacterial infections that are often fatal in childhood. Anti-adhesion therapy with mAb to the Mac-1alpha subunit (CD11b) and the leukocyte integrin beta2 subunit (CD18) is efficacious in a wide range of animal models of disease; thus, determination of the structure of Mac-1 may advance development of anti-inflammatory therapeutic agents. Selectins and their ligands are also critical in leukocyte emigration in vivo and are important pharmacologic targets. We will test the hypothesis that multiple Mac-1 ligands bind to overlapping but distinct sites by testing mutations around the metal ion- dependent adhesion site (MIDAS) in the I domain and other regions including the seven N-terminal repeats and a postulated MIDAS in the beta subunit. We will test mutants that may lock in one of the conformational states in the I domain, and test the hypothesis that interactions between C-terminal segments of the P150,95 alpha and beta subunits regulate ligand binding. Alpha subunits, and alpha-beta complexes that are truncated to remove the """"""""stalks,"""""""" will be expressed and purified to determine the structure of the globular ligand-binding region. We will characterize Mac- 1- and P-selectin-dependent neutrophil interactions with platelets. We will identify a Mac-1 ligand on platelets and characterize its relationship to fibrinogen, characterize adhesion molecules required for trans-platelet chemotaxis, and characterize the chemoattractant(s) important in activation by platelets of neutrophil integrin adhesiveness, both in shear flow and stasis. We will characterize novel Mac-1 ligands on endothelial cells and neutrophils, and their relationship to heparan sulfate. Finally, we will functionally and structurally characterize a ligand on neutrophils required for shear-dependent interactions with L- selectin.
Showing the most recent 10 out of 61 publications
