Genetic defects in Glanzmann thrombasthenia, a hereditary bleeding disorder, have not yet been identified. Platelets from patients with this disease do not bind fibrinogen and therefore, cannot participate in normal blood clotting. An understanding of the molecular pathology of Glanzmann thrombasthenia should enhance our understanding of the biogenesis of the normal platelet fibrinogen receptor. Hence, the goals of this proposed research are: (1) to characterize the molecular defects in 7 different kindreds with Glanzmann thrombasthenia - with particular emphasis on the isolation of genomic sequences controlling the normal expression of the genes for platelet membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa), and (2) to analyze the cellular consequences of these defects. Several approaches will be used to clone and sequence the molecular defects in the patients with Glanzmann thrombasthenia. DNA will be analyzed by Southern blotting, and the abnormal alleles will be isolated from genomic DNA libraries. Defects in coding sequences will be sought by analysis of platelet RNA using Northern blotting and the polymerase chain reaction (PCR). In the latter case, non- overlapping segments of RNA will be reverse transcribed and the resulting cDNA amplified. Abnormal segments will be identified by denaturing gradient gel electrophoresis and ribonuclease cleavage. When DNA and RNA analyses are normal, defects in the controlling elements for GPIIb and GPIIIa will be sought. This first requires isolation and cloning of the normal promotors and enhancers for these genes, Using the cDNA probes we have previously isolated, chromosome 17 genomic libraries will be screened to obtain 5' and 3' sequences for each gene. These sequences will be engineered into the pRSV cat plasmid, a vector which has been used successfully to express chloramphenicol acetyltransferase (CAT) in mammalian cells. This vector will be transfected into HEL cells which contain the necessary machinery to synthesize GPIIb and GPIIIa. CAT activity will then be assayed to determine which sequences contain the control elements. Once the normal promotors and enhancers have been isolated, those of the patients can be cloned either by PCR amplification of their genomic DNA or by probing a genomic DNA library constructed from patient DNA. The various molecular defects will be analyzed in several expression systems to determine their physiologic effects. Qualitatively abnormal RNA will be expressed (l) in Xenopus oocytes, to examine protein synthesis and receptor assembly, and (2) in HEL cells to examine effects on transcription. The previously identified abnormal cassette of cDNA will be ligated into the normal cDNA in order to perform these assays. Finally, apparent defects in transcriptional controlling elements will be studied both in CAT assays and in assays that detect alterations in mRNA level and stability.
