This study will examine vitamin K-dependent proteins that display improved membrane affinity. The vitamin K-dependent plasma proteins function in both pro- and anti-coagulation pathways. Membrane contact is important for nearly all of the steps of these reactions. Calcium and membrane binding by vitamin K-dependent proteins depend on several gamma-carboxyglutamic acid (Gla) residues in amino acids 1-45 of these proteins. Gla is synthesized in the liver in a vitamin K-dependent reaction. Administration of vitamin K antagonists, which lower the level of Gla-containing proteins in the circulation, is a common biomedical application for persons subject to thrombosis. In addition, administration of intact proteins is important to treatment for some bleeding disorders. A major goal of this project is to establish the membrane contact mechanisms of the vitamin K-dependent proteins and to use that knowledge to generate proteins with improved function. This proposal presents a new hypothesis for membrane contact.
Seven specific aims i nclude: 1. Design mutants of protein C and factor VII that have improved membrane contact sites. 2. Determine the relationship between amino acids in the Gla domain and membrane binding affinity. 3. Characterize the relationship between membrane affinity and the ability of a zymogen protein to function as substrate for activation by other membrane-bound enzymes. 4. Determine the impact of membrane affinity (specific aim 1) on the enzyme activity of the activated forms of these proteins (factor VIIa and activated protein C, APC), including the impact of cofactor proteins, membrane content, and other properties. 5. Determine the mechanisms of membrane contact by vitamin K proteins. 6. Determine the role of the hydrophobic residues in the Gla domain. 7. Determine the structures responsible for greatest folding stability of the Gla domain. Methods used include expression of mutant proteins and their characterization by enzyme and coagulation assays, spectroscopic assays by fluorescence, and biophysical characterization with NMR and calorimetry. The use of carefully prepared membrane vesicles is required in most areas of this project. This study should reveal structure/function relationships of gamma-carboxyglutamic acid, and its ultimate function in creating a membrane-contact site. It will increase knowledge of the role that membranes play in enzymatic events of coagulation and in biological systems. It may ultimately provide new and/or improved materials for biomedical applications.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL060859-03
Application #
6184826
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1998-07-15
Project End
2003-06-30
Budget Start
2000-07-01
Budget End
2001-06-30
Support Year
3
Fiscal Year
2000
Total Cost
$404,474
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Stone, Matthew D; Harvey, Stephen B; Martinez, Michael B et al. (2005) Large enhancement of functional activity of active site-inhibited factor VIIa due to protein dimerization: insights into mechanism of assembly/disassembly from tissue factor. Biochemistry 44:6321-30
Stone, Matthew D; Nelsestuen, Gary L (2005) Efficacy of soluble phospholipids in the prothrombinase reaction. Biochemistry 44:4037-41
Henderson, Nicole; Key, Nigel S; Christie, Beverly et al. (2002) Response of factor VIII and IX-deficient blood to wild type and high membrane affinity mutant factor VIIa in an in vitro whole blood clotting assay: possible correlation to clinical outcome. Thromb Haemost 88:98-103
Stone, Matthew; Harvey, Stephen B; Kisiel, Walter et al. (2002) Unusual benefits of macromolecular shielding by polyethylene glycol for reactions at the diffusional limit: the case of factor VIIai and tissue factor. Biochemistry 41:15820-5
Martinez, M B; Flickinger, M; Higgins, L et al. (2001) Reduced outer membrane permeability of Escherichia coli O157:H7: suggested role of modified outer membrane porins and theoretical function in resistance to antimicrobial agents. Biochemistry 40:11965-74
Nelsestuen, G L; Stone, M; Martinez, M B et al. (2001) Elevated function of blood clotting factor VIIa mutants that have enhanced affinity for membranes. Behavior in a diffusion-limited reaction. J Biol Chem 276:39825-31
Nelsestuen, G L; Shah, A M; Harvey, S B (2000) Vitamin K-dependent proteins. Vitam Horm 58:355-89
Nelsestuen, G L; Ostrowski, B G (1999) Membrane association with multiple calcium ions: vitamin-K-dependent proteins, annexins and pentraxins. Curr Opin Struct Biol 9:433-7
Nelsestuen, G L (1999) Significance of reduced dimensionality in reaction kinetics: impact of multi-site particles. Chem Phys Lipids 101:37-44
Shen, L; Shah, A M; Dahlback, B et al. (1998) Enhancement of human protein C function by site-directed mutagenesis of the gamma-carboxyglutamic acid domain. J Biol Chem 273:31086-91