von Willebrand factor (vWF) is a large adhesive glycoprotein that mediates the interaction of platelets to damaged endothelial surfaces. The primary translation product is a precursor (pro vWF) with a MW of about 240,000. This proprotein then dimerizes, and undergoes carbohydrate processing, sulfation and proteolytic cleavage prior to its release from the cell. The cleavage products are the pro-vWF segment (vWF AgII) and mature vWF; the provWF prescursor is not normally found in the circulation. A patient has been identified with circulating pro- vWF, giving us the unique opportunity to describe the molecular defect leading to altered proteolysis. vWF shares amino acid homology with other propolypeptides in the region of the cleavage site, and a defect in this basic amino acid region is postulated to cause defective cleavage, as has been demonstrated for some of these other proproteins. Using genomic clones of vWFI will identify the area of interest by generating restriction digests, transferring these to Southern blots and probing with synthetic oligonucleotides. I will then isolate total genomic DNA from the patient's neutrophils, and identify the cleavage region on genomic blots with these oligonucleotide probes. This area will then be cloned utilizing the technique of Nicholls, and the two alleles identified an the region sequenced by subcloning into an M13 vector. Further studies during Phase 2 of this award may include oligonucleotide directed site specific mutagenesis to define the boundaries and specificity of the cleavage recognition site, as well as to define other functional domains of the vWF protein.
