Platelet-specific alloantigens have been implicated in the pathogenesis of two well-characterized thrombocytopenic disorders: neonatal alloimmune thrombocytopenic purpura and post-transfusion purpura. The overall goal of this proposal is to investigate the biochemical properties of platelet membrane glycoproteins (GP) IIb and IIIa in order to determine the structural features that are responsible for conferring platelet-specific alloantigenicity. To address this issue, four related studies will be conducted. The first study will examine the role of carbohydrate moieties in the structure and antigenicity of the Bak and Lek alloantigen systems. Oligosaccharides will be systematically removed from GPIIb and GPIIIa and the deglycosylated proteins assayed for their retention of specific antigenic determinants. The second study will entail isolation and characterization of small, immunologically reactive fragments derived from GPIIb and GPIIIa that contain the P1-A1, Lek and Bak antigenic determinants. Amino acid composition and sequence analysis will be performed to determine the specific structural heterogeneity between the alleles in these three alloantigens systems. The third study will involve production of monoclonal antibodies directed against the P1-A1 and Bak epitopes to be used as both research reagents for further characterization of the structural and functional regions of GPIIb and GPIIIa involved in alloantigenicity, and also as extremely useful typing reagents for consistent, reproducible clinical detection of these alloantigens on patient platelets. Finally, several endothelial cell cDNA clones that appear to code for GPIIb and GPIIIa analogs will be characterized. Cloning of the glycoproteins that bear platelet alloantigens will permit a detailed investigation of both the structure and regulation of expression of these allelic epitopes. Together, these four interrelated projects comprise a research program that should yield valuable, timely new information about the detailed structure and mode of inheritance of human platelet alloantigens. This knowledge will contribute to our understanding of the specific morphological features of these glycoproteins that are responsible for eliciting an alloimmune response, and will provide further insights regarding overall platelet membrane protein structure and function. This should, in turn, lead to improvements in transfusion therapy, platelet storage, and our understanding and management of congenital platelet functional disorders.
