The goal of this project is to characterize the genetic regulation of two membrane glycoproteins (GPIIb-IIIa) which are critical for normal platelet aggregation. We plan to clone the cDNAs for these glycoproteins using leukemia cells (HELL-cells) shown to contain GPIIb-IIIa. First, we will enrich the HEl-cell population making GPIIb-IIIa using fluorescent-activated cell sorting techniques, then subculture positive cells. Next, we will stimulate synthesis of mRNA specific for GPIIb-IIIa, using 5-azacytidine and DMSO treatment of HEL-cells. We will conduct [35S]-methionine pulse-chase experiments with HEL-cells in order to determine whether a large precursor molecule is synthesized and then cleaved, or whether GPIIb-IIIa are synthesized independently. Poly(A+) mRNA will be purified, then used to construct a cDNA library, in lambda phage GT 11. The library will be screened with polyclonal antibodies against SDS-denatured GPIIb-IIIa. Oligonucleotide probes will also be used for screening. We have cyanogen bromide-treated GPIIb-IIIa to obtain fragments for amino acid sequencing. Fragments will be purified from SDS-polyacrylamide gels. Based on amino acid sequences with limited codon degeneracy, 16-mer nucleotide probes will be constructed. If cloning is unsuccessful using lambda phage, then a cDNA library will be made with plasmid PBR322 and screened with the oligonucleotide probes. The cloned cDNAs for GPIIb-IIIa will be characterized by restriction enzyme mapping and hybrid-selected in vitro translation. If the cloned DNA is incomplete, full-length DNA will be constructed using a nucleotide restriction fragment as a primer for extension with purified poly(A+) mRNA and reverse transcriptase. Fragments of the full-length cDNA will be cloned in M13 phage and overlapping DNA segments sequenced. Also, genomic material from leukocytes from normal individuals and patients whose platelets lack GPIIb-IIIa (Glanzmann's thrombasthenia) will be analyzed with 32P-cDNA probes and Southern blotting. Using several restriction enzymes, we should be able to determine the extent of normal polymorphism in these genes. In addition, we may obtain information on the nature of the genetic defect(s) in thrombasthenia. Ultimately, the information obtained from these studies should advance our understanding of normal platelet function as well as the mechanisms which regulate synthesis of integral membrane proteins.
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