There are three related objectives of this proposal. First, we will characterize structure-function relationships of the platelet cytoadhesive receptor, glycoproteins IIb and IIIa (GPIIb-IIIa). The heterodimer is the primary receptor for fibrinogen, and is critical for normal blood clotting. By better understanding the basis for receptor activity, it may be possible to alter function and thereby develop new approaches to prevent thromboses. We will first precisely localize functional portions of each molecule. Thus, we will determine sites necessary for binding intact fibrinogen as well as its PGDS and dodecapeptide components. We will also determine intramolecular structures necessary for formation of the GPIIb-IIIa complex. Site-directed mutagenesis and deletional mutants of GPIIb and GPIIIa cDNA will be used to transcribe mutant mRNAs using a SP6 promoter. Regions of each cDNA to be mutated will be selected by two techniques: Computer predictions based on primary structure will be made regarding ligand binding sites and intramolecular binding sites necessary for GPIIb-IIIa complex formation. In addition, antibodies will be raised against either recombinant GPIIb and IIIa peptides or synthesized peptides. Once peptides are identified which elicit functionally inhibitory antibodies, the nucleotides on the corresponding cDNA will be mutated. Normal and mutant mRNA will be microinjected into frog oocytes or COS cells. The effects of each mutation on assembly of the GPIIb-IIIa complex and on 125I- RGDS, 125I-gamma chain dodecapeptide and 125I-fibrinogen binding to oocytes or COS cells will be determined. Formation of the GPIIb-IIIa complex will be detected using complex-specific antibodies, as well as crossed immunoelectrophoresis. We will also study patients with the hereditary disorder, Glanzmann's thrombasthenia, to determine underlying genetic defects responsible for decreased GPIIb-IIIa on platelets. Deletions will be sought by Southern hybridization of DNA from peripheral blood leukocytes with 32P-cDNA probes. In addition, possible abnormalities in mRNA will be analyzed in platelets and megakaryocytes by: i) the polymerase chain reaction using Taq polymerase and oligonucleotide primers based on known GPIIb-IIIa cDNA sequence, ii) Northern hybridization, and iii) in situ hybridization with 35S-cDNA probes on bone marrow megakaryocytes. A long-range goal of this project will be to determine the organization of the genes for GPIIb and IIIa by cloning of genomic DNA from a human cosmid library. Based on our observation that the GPIIb and IIIa genes are in proximity on chromosome 17, we will map these genes, determine whether they are linked and whether there are common regulatory elements. Promoter and enhancer elements of both genes will be identified using chloramphenicol acetyltransferase (CAT) assays. CAT constructs will be transfected into HEL cells and assayed following stimulation of GPIIb-IIIa synthesis by DMSO.
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