The long-term goal of this new R29 proposal is to understand the antigenic determinants and immunologic requirements involved in alloimmunization. This proposal focuses on human platelet alloantigen 1 (HPA-1). HPA-1 is a biallelic system; a single nucleotide difference between alleles creates a polymorphism at position 33 of GP IIIa, a component of the fibrinogen receptor. Homozygotes for HPA-1a encode a leucine at position 33; homozygotes for HPA-1b encode proline. HPA-1 is the most common cause of two bleeding disorders, neonatal alloimmune thrombocytopenia and posttransfusion purpura. In both disorders, anti-HPA-1a antibodies are made against HPA-1a GPIIIa, leading to thrombocytopenia. In this proposal, experiments are designed to further explore the HPA-1a and test the feasibility of making a murine model. In pursuing this goal, important questions about the HPA-1 system will be addressed.
The specific aims are 1) to further define the structural requirements for HPA-1a antigenicity, 2) to determine if the structural requirements for HPA-1a antigenicity can be recreated in murine GPIIIa, and 3) to test the feasibility of making an animal model of HPA-1a alloimmunization by first creating a murine cell line expressing an HPA-1a fibrinogen receptor. By manipulating recombinant proteins containing human GPIIIa domains, the role of the polymorphism, the three N-terminal disulfide bonds, and the long range Cys5 Cys435 disulfide bond in HPA-1a antigenicity will be evaluated. This information will be used to recreate an HPA-1a-like antigen in murine GPIIIa and the criteria for eliciting an anti-HPA-1a response in mice will be determined. The ability of HPA-1a murine GPIIIa to assemble into a fibrinogen receptor and bind ligand in transfected megakaryoblastic cells will also be tested.
| Barron-Casella, E A; Nebbia, G; Rogers, O C et al. (1999) Construction of a human platelet alloantigen-1a epitope(s) within murine glycoprotein IIIa: identification of residues critical to the conformation of the antibody binding site(s). Blood 93:2959-67 |
