von Willebrand disease (vWD-the second most common inherited bleeding disorder) is associated with quantitative and qualitative abnormalities in von Willebrand factor (vWF). Bleeding in many vWD patients may often be effectively treated with the drug desmopressin (DDAVP), which appears to promote release of vWF from tissue stores. However, some vWD patients require infusion of vWF-containing, plasma-derived cryoprecipitate. Other patients, with apparently normal vWF synthetic capability, may sometimes require vWF supplementation in situations such as uremia or after prolonged cardiac by-pass surgery. Many will respond to DDAVP, but others require cryoprecipitate. We propose an investigation into the feasibility of producing a biologically active, recombinant von Willebrand factor (vWF) product. Such a product might be useful as a substitute for cryoprecipitate in treatment of DDAVP resistant bleeding. It would, in principle, offer patients the advantages of a virus-free product with a reproducible theraputic effect. Since there are a number of significant scientific questions to be answered before the feasibility of such a product could be established, and since the prospective market size is not large enough to attract a private company, our understanding is that commercial development of recombinant vWF for theraputic purposes is not planned at present. Nevertheless, the need for such a product is significant.
Our Specific Aims are to develop a recombinant expression system capable of producing large amounts of biologically active vWF using our existing full-length vWF cDNA clone. We propose to explore the feasibility of using immortalized human endothelial cells and bovine papilloma virus transfected cells for such a purpose. In the cell lines to be studied, the distribution of vWF multimers will be examined for the presence of the high molecular weight species necessary for optimal biologic activity and the specific biologic activity of the recombinant vWF will be determined. Ristocetin-induced platelet agglutination will be utilized as a functional assay to help select appropriate cell lines producing active recombinant vWF. Methods for enhancing production of total vWF and, particularly, high molecular weight vWF multimers in these cells will be evaluated. Finally, the stability of the product and methods for its purification from culture medium will be investigated.
|Stoddart Jr, J H; Andersen, J; Lynch, D C (1996) Clearance of normal and type 2A von Willebrand factor in the rat. Blood 88:1692-9|