This project is designed to characterize and study the ability of IgG display and subclass on the platelet surface in mediating recognition, binding and destruction of the antibody coated platelets by Fc-receptor bearing cells. This project will have three major research efforts. In the first, various subclasses of antibody will be characterized in their ability to mediate Fc-receptor activity and correlation will be made with clinical observations. Normal platelets sensitized in vivo from patients with immune thrombocytopenia will be studied similarly. In each case, IgG and IgG subclasses on the platelet surface will be quantified using a monoclonal 125I anti-IgG assay developed in our laboratory. Secondly, subclass specific anti-platelet IgG antibodies will be purified from plasma derived from immune thrombocytopenic patients using immunoaffinity chromatography techniques. Subclass specific IgG fractions will be tested for anti-platelet activity. The platelet binding characteristics of these IgG subclass fractions and the Fc-receptor mediated interaction between effector cells and platelets coated with these various antibodies will be studied. Finally, the spleen has been implicated as a possible source of anti-platelet antibody production in human immune thrombocytopenia. Using techniques developed in our laboratory, studies will be carried out to identify, separate and culture subsets of splenic B-lymphocytes (from ITP patients) that are capable of producing specific anti-platelet antibody. These specific immune B-lymphocytes will subsequently be used to construct human-human hybridomas capable of continuous production of monoclonal anti-platelet antibodies of various subclass specificities as well as selective antigen binding characteristics. These monoclonal antibodies will be used for analysis of Fc-receptor interaction as well as isolation and characterization of the relevant platelet membrane antigens. These studies will, thus, provide new information as regards with molecular mechanism controlling human effector cell Fc-receptor interaction with antibody on the platelet and will have direct relevance to the pathogenesis of immune platelet destruction syndromes.
