The present proposal represents the cotinuation of this laboratory's long-term commitment to the study of platelet interactions with von Willebrand factor (vWF) in health and disease. Investigations focusing upon the abnormal platelets from patients with platelet-type von Willebrand Disease (PT-vWD) have accrued evidence that the normality of these platelets is associated with the glycoprotein (GP) IB/IX receptor complex. Complementing this work on PT-VWD is the recent discovery in this laboratory a family having an autosomal dominant, qualitative disorder of the GPIB/IX receptor complex, termed """"""""variant"""""""" Bernard-Soulier Disease (BSD). The first goal of the opposed studies is to obtain and to amplify the DNA coding for this receptor in these patient groups, in patients with autosomal recessive, classic BSD, and in normal controls. Due to the nearly intron-less nature of the genes for GPlba and Ib,6, much of this work should be able to be accomplished using genomic DNA from circulating leukocytes. MRNA from platelets will also be obtained and CDNA synthesized, an effort to determine possible clinical abnormalities at the gene expression well. The RNAse a protection assay will be used to localize mutations or polymorphisms manageable regions of a gene. This method is capable of detecting relatively subtle cleotide mismatches affecting only one or a few base pairs. This localization 11 then permit sequencing efforts to be focused on a relatively small portion of patient genomic DNA or CDNA. On selected target regions of the DNA the Taq I polymerase ain reaction (PCR) will be used to produce amplifications of approximately 400 bp, which will be cloned into plasmids. Double stranded sequencing of the inserts will then be performed by the dideoxy method, using primers for both the T7 and the SP6 omoters contained within the plasmid vector. Alternatively, PCR ampification products will be directly sequenced without cloning. The sequence analysis should permit the identification of mutational and polymorphic sites existing at the gene and expression levels. Following the identification of such sites, monoclonal antibodies will be produced that recognize the corresponding peptide sequences. Such antibodies should prove useful in subsequent studies for the clinical identification of affected patients. The proposed investigations should provide significant new knowledge that potentially may lead to the development of improved diagnosis and therapy for hemorrhagic and thrombotic disorders involving the GPIB/IX receptor complex.
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