Abstract

We propose to use recombinant DNA technology to exchange several putative functional domains between human factor IX, X and VII. These domains include, the """"""""gla"""""""" region, exon C, the epidermal growth factor regions and the heavy chain and first 40 amino acids of the heavy chain. Each construction will be expressed in a transient expression system in the cos cell line. These altered factor IX molecules will all have the factor IX activation peptide. A monoclonal antibody specific for the threonine dimorphic form of the factor IX molecule (and which does not react with bovine factor IX) will be used to purify the proteins produced from the recombinant constructions. Each purified altered factor IX molecule will be assayed for its ability to function in coagulation in the one stage assay in the throboplastin time. The molecules will also be tested to determine if they can be activated, if they bind normally to factor VIII (or bind to the cofactor for factor X or VII) and tested to determine if they bind to endothelial cells.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL038973-05
Application #
3355487
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1987-07-01
Project End
1992-06-30
Budget Start
1991-07-01
Budget End
1992-06-30
Support Year
5
Fiscal Year
1991
Total Cost
Indirect Cost

  Institution

Name
University of North Carolina Chapel Hill
Department
Type
Schools of Arts and Sciences
DUNS #
078861598
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599

  Publications

Jin, J; Chang, J; Stafford, D W et al. (2001) Residues Y179 and H101 of a hydrophobic patch of factor VII are involved in activation by factor Xa. Biochemistry 40:11405-10
Jin, J; Perera, L; Stafford, D et al. (2001) Four loops of the catalytic domain of factor viia mediate the effect of the first EGF-like domain substitution on factor viia catalytic activity. J Mol Biol 307:1503-17
Chang, J; Jin, J; Lollar, P et al. (1998) Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity. J Biol Chem 273:12089-94
Chang, J Y; Monroe, D M; Stafford, D W et al. (1997) Replacing the first epidermal growth factor-like domain of factor IX with that of factor VII enhances activity in vitro and in canine hemophilia B. J Clin Invest 100:886-92
Wolberg, A S; Morris, D P; Stafford, D W (1997) Factor IX activation by factor XIa proceeds without release of a free intermediate. Biochemistry 36:4074-9
Cheung, W F; Stafford, D W; Sugo, T (1996) Localization of a calcium-dependent epitope to the amino terminal region of the Gla domain of human factor IX. Thromb Res 81:65-73
Wolberg, A S; Li, L; Cheung, W F et al. (1996) Characterization of gamma-carboxyglutamic acid residue 21 of human factor IX. Biochemistry 35:10321-7
Chang, J Y (1996) Factor VIIa-tissue factor interactions: an evaluation using factor VII-factor IX chimeras. Haemostasis 26 Suppl 1:35-9
Cheung, W F; Stafford, D W (1995) Localization of an epitope of a calcium-dependent monoclonal antibody to the N-terminal region of the Gla domain of human factor VII. Thromb Res 79:199-206
Kuo, W L; Stafford, D W; Cruces, J et al. (1995) Chromosomal localization of the gamma-glutamyl carboxylase gene at 2p12. Genomics 25:746-8

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