The overall aim of this project is to achieve an enhanced understanding of the elements involved in transcription and post-translational processing of the vitamin K-dependent clotting factors. Using human Factor X as a paradigm for study, experiments proposed here will (1) determine cis and trans regulatory elements required for the expression of Factor X. Using footprinting assays, gel shift, and mutagenesis followed by transfection assays with reporter genes, the location of functional transcription factor (TF) binding sites within the F.X promoter will be determined. Binding sites for several liver-specific TF's (HNF-3 and C/EBP) will be sought and co-transfection assays used to determine the effects of these TF's on the F.X promoter. These findings will be used to explore two other phenomena, the pathophysiology of hemophilia B Leyden, and the pattern of clotting factor expression in HepG2 cells. (2) We shall explore the function of the signal sequences in these proteins. The signal sequences of secreted proteins function to target these proteins to the endoplasmic reticulum. We have described a Factor X variant (Factor X Santo Domingo) in which a mutation in the signal sequence results in severe Factor X deficiency. We propose to carry out further experiments to determine the intracellular fate of this mutant protein. In the course of characterizing this variant, we have established for the first time the signal peptidase (SP) cleavage site in F.X. We propose to determine the SP cleavage sites for Factors VII and II as well. This will, as a consequence, define the start sites of the propeptides, which are required for the posttranslational carboxylation of the vitamin K-dependent proteins. (3) We shall determine the role of specific Gla residues in Factor X through the study of naturally occurring and recombinant Factor X variants with mutations in the Gla domain. Using site directed mutagenesis, Gla(16) to Asp and Gla(20) to Asp variants will be constructed. These, along with a naturally occurring Gla(26) to Asp variant, will be characterized by physicochemical means and by coagulation assays, to define the roles of specific residues in maintaining conformation and effecting phospholipid binding.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL048322-04
Application #
2224395
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1992-08-17
Project End
1996-03-31
Budget Start
1994-04-01
Budget End
1995-03-31
Support Year
4
Fiscal Year
1994
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Whinna, H C; Lesesky, E B; Monroe, D M et al. (2004) Role of the gamma-carboxyglutamic acid domain of activated factor X in the presence of calcium during inhibition by antithrombin-heparin. J Thromb Haemost 2:1127-34
Arruda, V R; Hagstrom, J N; Deitch, J et al. (2001) Posttranslational modifications of recombinant myotube-synthesized human factor IX. Blood 97:130-8
Camire, R M; Larson, P J; Stafford, D W et al. (2000) Enhanced gamma-carboxylation of recombinant factor X using a chimeric construct containing the prothrombin propeptide. Biochemistry 39:14322-9
Larson, P J; Camire, R M; Wong, D et al. (1998) Structure/function analyses of recombinant variants of human factor Xa: factor Xa incorporation into prothrombinase on the thrombin-activated platelet surface is not mimicked by synthetic phospholipid vesicles. Biochemistry 37:5029-38
Carew, J A; Pollak, E S; High, K A et al. (1998) Severe factor VII deficiency due to a mutation disrupting an Sp1 binding site in the factor VII promoter. Blood 92:1639-45
Wu, S M; Stafford, D W; Frazier, L D et al. (1997) Genomic sequence and transcription start site for the human gamma-glutamyl carboxylase. Blood 89:4058-62
Ambrosini, G; Plescia, J; Chu, K C et al. (1997) Activation-dependent exposure of the inter-EGF sequence Leu83-Leu88 in factor Xa mediates ligand binding to effector cell protease receptor-1. J Biol Chem 272:8340-5
Larson, P J; Stanfield-Oakley, S A; VanDusen, W J et al. (1996) Structural integrity of the gamma-carboxyglutamic acid domain of human blood coagulation factor IXa Is required for its binding to cofactor VIIIa. J Biol Chem 271:3869-76
Pollak, E S; Hung, H L; Godin, W et al. (1996) Functional characterization of the human factor VII 5'-flanking region. J Biol Chem 271:1738-47
Chu, K; Wu, S M; Stanley, T et al. (1996) A mutation in the propeptide of Factor IX leads to warfarin sensitivity by a novel mechanism. J Clin Invest 98:1619-25

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