A study of the biosynthesis and molecular biology of von Willebrand factor is proposed. An effort will be made to discover the details of provWf cleavage and vWf oligomer assembly. Metabolic labeling of human endothelial cell (EC) cultures will be employed and immunoisolated, endogenously labeled vWf analyzed by SDS agarose and acrylamide gel electrophoreses. A search for the putative enzyme(s) responsible for provWf conversion to vWf is also proposed. The presence of large vWf oligomers appears necessary for normal vWf mediated platelet aggregation in man. Specifically, derangements in oligomer formation are associated with Type II von Willebrand disease (vWd). Conversion of provWf to vWf has also been demonstrated to be correlated with formation of such large vWf species. In addition to EC, vWf is also produced in megakaryo-cytes and 25% of whole blood vWf is associated with the platelet. A comparison of the biosynthetic pathway of vWf in megakaryocytes to that in FC will be developed. Studies will be attempted using both a continuous line of promega-karyoblasts and isolated marrow megakaryocytes. An attempt will also be made to generate a vWf cDNA clone. This would be used to determine a predicted amino acid sequence and to begin analysis of the vWf genomic structure in normals and vWd individuals. Toward this aim, a large scale human EC culture capability has been developed and adequate amounts of human EC mRNA prepared. Construction of a cDNA library will be attempted by standard techniques. It is proposed to screen the library with synthetic oligonucleotide probes constructed based upon known vWf amino acid sequence, and/or with probes generated from immunoisolated polysome-derived vWf mRNA. Isolation of DNA and EC cultures from vWd individuals will also be carried out, to develop a source of material for detailed analysis of the vWf biosynthetic pathway, vWf mRNA(s), and vWf gene structure in various types of vWd.
