Immune thrombocytopenia is often due to autoantibodies or alloantibodies against platelet glycoproteins (GP), particularly GPIIb/IIIa. We will focus on two areas in immune thrombocytopenia: evaluation of the antigenic spectrum and determination of antibody characteristics. Antigen localization. We will determine the epitope specificity of both platelet- associated and plasma antibodies concentrating initially on antibodies to GPIIb/IIIa, since these are the most common. We will: (1) Show whether antiplatelet antibodies are complex-specific or protein-specific using direct binding and inhibition assays. (20 Test for antibody binding to a series of peptides which span the glycoprotein molecule. These peptides will be produced as fusion proteins with maltose binding protein in E. Coli by insertion of vectors containing CDNA fragments generated by the polymerase chain reaction. The fusion proteins will be purified by passage through an amylose column. Once the epitope is localized to one of the large peptides, further localization will be achieved by determining the antibody binding pattern to smaller peptides within this region. (3) Determine the incidence in chronic ITP of patients with platelet-associated autoantibody reactive with extracellular portions of GPIIIa and plasma autoantibody specific for the cytoplasmic part of GPIIIa. The pathogenetic role in vivo (if any) of the anti-cytoplasmic antibodies will be studied in baboons. Autoantibody characteristics. We will determine whether human antiplatelet antibody, which mimics patient antibody, can be synthesized in vitro by E. Coli after introduction of vectors containing PCR-generated heavy and light chain combinatorial libraries produced from patient RNA. Antibody specificity of positive clones will be compared with patient antibody. We will also study the ability of patient platelet-associated and plasma antibody to fix complement and bind to megakaryocytes. The results of the above studies will be correlated with the patients' clinical course to determine which of the parameters influence disease severity and response to therapy.
Showing the most recent 10 out of 18 publications
