This project takes advantage of new understanding about membrane association by vitamin K-dependent proteins. Key proteins of the coagulation cascade (factors VII and IX) display low affinity for membranes. This affinity can be increased to that of high affinity vitamin K-dependent proteins by site directed mutations. In vitro assays show that these changes have a large impact on activity of factor VII and VIIa in vitro, using assays that mimic biological conditions. Enhancement is realized in both tissue factor dependent as well as tissue factor- independent systems.
Six specific aims i nclude: 1. Identify the site- directed mutant forms of human factor VIIa that have the highest activity, in vitro. Express these proteins in quantities suitable for studies in the mouse and rabbit. 2. Develop a reliable assay for bleeding challenge in quantities suitable for studies in the mouse and rabbit. 2. Develop a reliable assay for bleeding challenge in the hemophilic mouse, that provides the ability to test efficacy of factor VIIa mutants. 3. Test Efficacy of human factor VIIa mutants in the mouse model, using mutants identified in specific aim 1 and methods identified in specific aim 2. 4. Express site-directed mutants of mouse factor VIIa which correlate with the best human factor VIIa mutations. Test these in the mouse by procedures developed for the human protein molecules described in specific aims 2-3. 5. Test in vitro anti-coagulant properties of human active site-blocked factor VIIa molecules described in specific aim 1, to determine the best mutants to be tested in the rabbit model. 6. Obtain preliminary evidence for potential uses of other vitamin K-dependent proteins which have mutations in the Gla domain and increased affinity for membranes. These may include especially factors IX or X. The project uses site-directed protein mutation, protein expression, purification, and characterization by protein chemistry and numerous coagulation tests. In vivo testing will be carried out in mice and rabbits.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL065578-03
Application #
6642373
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
2002-08-01
Project End
2003-07-31
Budget Start
Budget End
Support Year
3
Fiscal Year
2002
Total Cost
$267,360
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Gharagozlou, Soheila; Sharifian, Ramazan A; Khoshnoodi, Jalal et al. (2009) Epitope specificity of anti-factor VIII antibodies from inhibitor positive acquired and congenital haemophilia A patients using synthetic peptides spanning A and C domains. Thromb Haemost 101:834-9
Hu, Genlin; Guo, Delan; Key, Nigel S et al. (2007) Cytokine production by CD4+ T cells specific for coagulation factor VIII in healthy subjects and haemophilia A patients. Thromb Haemost 97:788-94
Kren, Betsy T; Yin, Wenxan; Key, Nigel S et al. (2007) Blood outgrowth endothelial cells as a vehicle for transgene expression of hepatocyte-secreted proteins via Sleeping Beauty. Endothelium 14:97-104
Perez-Pujol, Silvia; Marker, Paul H; Key, Nigel S (2007) Platelet microparticles are heterogeneous and highly dependent on the activation mechanism: studies using a new digital flow cytometer. Cytometry A 71:38-45
Zhang, Yan; Wroblewski, Matthew; Hertz, Marshall I et al. (2006) Analysis of chronic lung transplant rejection by MALDI-TOF profiles of bronchoalveolar lavage fluid. Proteomics 6:1001-10
Wang, Jian-Guo; Mahmud, Shawn A; Thompson, Jacob A et al. (2006) The principal eosinophil peroxidase product, HOSCN, is a uniquely potent phagocyte oxidant inducer of endothelial cell tissue factor activity: a potential mechanism for thrombosis in eosinophilic inflammatory states. Blood 107:558-65
Bach, Ronald R (2006) Tissue factor encryption. Arterioscler Thromb Vasc Biol 26:456-61
Marsik, C; Endler, G; Halama, T et al. (2006) Polymorphism in the tissue factor region is associated with basal but not endotoxin-induced tissue factor-mRNA levels in leukocytes. J Thromb Haemost 4:745-9
Ohlfest, John R; Frandsen, Joel L; Fritz, Sabine et al. (2005) Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunotolerization and nonviral gene transfer using the Sleeping Beauty transposon system. Blood 105:2691-8
Park, Chang Won; Kren, Betsy T; Largaespada, David A et al. (2005) DNA methylation of Sleeping Beauty with transposition into the mouse genome. Genes Cells 10:763-76

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