Specific Aims: To continue to explore the mechanisms of von Willebrand factor (vWf) biosynthesis by further study of the cultures of endothelial cell (EC) obtained from individuals with von Willebrand's disease. To explore some specific aspects of vWf biosynthesis which may relate to formation of large multimers. To compare vWf biosynthesis in EC with that in HEL cells. To use the expression of our Vwf cDNA clone as a tool in these studies. Methods: Further studies will be conducted on the Type IIA von Willebrand's endothelial cells to try to determine the molecular nature of the defect and to more precisely localize the mechanism of its expression. Reasons for the increased level of vWf mRNA expression in these cells will be sought. We shall also attempt to develop the technique of direct cloning of a given segment from a patient's genomic DNA. A patient with circulating provWf will be the prototype for these experiments which we hope may prove to have more general applicability in the future. Certain aspects of vWf biosynthesis which we feel might be related to the formation of large vWf multimers will be explored in EC cultures. We will also compare vWf biosynthesis in HEL cells with the EC process. The metabolic labeling, immunoisolation and gel electrophoretic techniques which we have used successfully in the past will continue to be the mainstay of our experimental approach in these areas. In HEL cells, the vWf mRNA will be carefully searched for any differences from its EC counterpart which might result from alternative splicing. Finally, we will attempt to develop cell lines expressing our full length vWf cDNA clone. Such recombinant lines would be used as an adjunct to the studies described above, with a particular emphasis on factors responsible for the formation and maintenance of large vWf multimers.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL031311-08
Application #
3342405
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1984-07-01
Project End
1993-06-30
Budget Start
1991-07-01
Budget End
1993-06-30
Support Year
8
Fiscal Year
1991
Total Cost
Indirect Cost
Name
Dana-Farber Cancer Institute
Department
Type
DUNS #
149617367
City
Boston
State
MA
Country
United States
Zip Code
02215
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