This study addresses critical questions in the immunobiology of ITP, with a particular emphasis upon autoantibodies directed against epitopes within the glycoprotein (GP) Ib/IX complex. Improved methodologies for the detection of these antibodies will be developed, through the use of native mammalian glycoprotein chains recombinantly expressed on the surface of heterologous cells. Antibodies from multiple patients will be studied with respect to commonality of epitope determinants within the complex. In view of the likelihood that many of these will be nonlinear, conformational epitopes, a major effort will be made to discover mimotope sequences recognized by multiple autoantibodies. The possible neutralizing B cell lines will be derived from patients producing anti-GPIb antibodies. The possible neutralizing effect of such mimotopes will be explored, as a basis for future therapeutic efforts for ITP patients. Immortalized B cell lines will be derived from patients producing anti-GPIb antibodies. The immortalized B cell clones will identify the repertoire of anti-GPIb antibodies in ITP patients, in order to identify the molecular and functional characteristics of these antibodies which are critical to disease-causing antibodies. The GPIb epitopes recognized by these antibodies, and relevant idiotope(s) which define their pathogenicity, will be identified. As an additional approach for the identification of anti-GPIb antibody (ScFv) clones with similar specificities by the technique of competitive elution. Such ScFv clones will then be characterized with respect to immunoglobulin chain origins and CDR3 composition by DNA sequencing. These ScFv will also be studied in binding assays to determine if anti-idiotype antibodies have similar effects upon the ScFv as upon the original anti-platelet antibodies; if so, the ScFv will become a valuable substitute for the precious natural antibody for future studies. Finally, the mechanism of recovery in acute ITP will be examined to identify the possible role of anti-idiotypic antibodies in IVIG. If anti-idiotype antibodies can in fact be identified, their ability to produce similar effects upon anti-platelet antibodies from other patients will be examined in order to identify a cross-reactive ITP-associated idiotype.
