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.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL031311-02
Application #
3342400
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-07-01
Project End
1987-06-30
Budget Start
1985-07-01
Budget End
1986-06-30
Support Year
2
Fiscal Year
1985
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
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
Lynch, D C (1991) The fine structure of von Willebrand factor multimers and type IIA von Willebrand disease. Ann N Y Acad Sci 614:138-52
Carew, J A; Browning, P J; Lynch, D C (1990) Sulfation of von Willebrand factor. Blood 76:2530-9
Ngo, K Y; Glotz, V T; Koziol, J A et al. (1988) Homozygous and heterozygous deletions of the von Willebrand factor gene in patients and carriers of severe von Willebrand disease. Proc Natl Acad Sci U S A 85:2753-7
Levene, R B; Booyse, F M; Chediak, J et al. (1987) Expression of abnormal von Willebrand factor by endothelial cells from a patient with type IIA von Willebrand disease. Proc Natl Acad Sci U S A 84:6550-4
Collins, C J; Underdahl, J P; Levene, R B et al. (1987) Molecular cloning of the human gene for von Willebrand factor and identification of the transcription initiation site. Proc Natl Acad Sci U S A 84:4393-7
Nishino, K; Lynch, D C (1986) A polymorphism of the human von Willebrand factor (vWf) gene with BamHI. Nucleic Acids Res 14:4697
Lynch, D C; Zimmerman, T S; Ling, E H et al. (1986) An explanation for minor multimer species in endothelial cell-synthesized von Willebrand factor. J Clin Invest 77:2048-51
Lynch, D C; Zimmerman, T S; Collins, C J et al. (1985) Molecular cloning of cDNA for human von Willebrand factor: authentication by a new method. Cell 41:49-56