The goal of this project is to characterize the synthesis and structure of the fibrinogen receptor on the plasma membrane of platelets. Platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa) exist as a heterodimeric complex in the platelet membrane and function as the receptor for fibrinogen. As a first step in accomplishing these goals, the cDNA and GPIIb and GPIIIa will be cloned and sequenced. Several cDNA libraries will be screened with polyclonal antibodies to GPIIb and GPIIIa. One library will be constructed in Lambda gtll using RNA from a human erythroleukemia (HEL) cell line known to express GPIIb and GPIIIa. In addition, a human umbilical vein endothelial (HUVE) cell cDNA library in Lambda gtll has been obtained and clones have already been identified. These clones are being analyzed on Southern blots with oligonucleotide probes synthesized form known amino acid sequence of GPIIb and GPIIIa. Antibody-identified clones which hybridize with our oligonucleotide probes will be subcloned into plasmid vector pSP64 in order to obtain milligram quantities of cDNA. In addition, this cDNA will be inserted into the bacteriophage M13 vector and the DNA sequence will be obtained. Following proof of identity, these clones will be used to probe either the HUVE or the HEL cell libraries and obtain the full-length cDNA for both GPIIb and GPIIIa. Once the cDNA has been obtained, the complete amino acid sequence will be determined and structural domains of the molecules deduced. Using a fluorescence-activated cell sorter (FACS), individual chromosomes from normal human cell lines will be sorted onto nitrocellulose filters. Incubating these filters with 32P-labelled cDNA of GPIIb and GPIIIa will allow identification of the chromosome(s) on which these genes reside. The molecular defect in Glanzmann's thrombasthenia will also be studied. Using peripheral blood mononuclear cells from the patients, DNA will be extracted, digested with restriction endonucleases, electrophoresed, and transfered to nitrocellulose filters. By """"""""probing"""""""" these filters with 32P-labelled cDNA clones, deletions and/or mutations in one or both genes may be identified. Polymorphisms that serve as markers for the disease will be sought. Errors in transcription will be studied by analysis of bone marrow RNA - by both Northern blotting and in situ hybridization with cDNA probes. Lastly, the cDNA and GPIIb and GPIIIa will be expressed in frog ooctes which will serve as a model system for studying fibrinogen binding. By genetically engineering deletions in the cDNA, we hope to localize the binding site on one and/or both glycoproteins.
