We propose to leverage recent advances in computer technology and algorithm development, together with the growing database of three-dimensional (3D) structures of Vitamin K-dependent (VKD) proteins and their complexes, to build reliable 3D complexes in electrically neutral solvent and with structural water molecules in place. It is our hypothesis that theoretical techniques are now at a level of sophistication and accuracy to warrant the judicious application to key coagulation systems: the prothrombin-prothombinase complex, which provides the penultimate step in the cascade (formation of thrombin) and the extrinsic tenase complex, which initiates the extrinsic blood coagulation cascade. We propose to provide solvent-equilibrated models for these systems. The plan:
AIM I : To model the prothrombinase-prothrombin complex, including the apparently flexible central fragment involving the kringle domains of prothrombin?the goal being to understand the molecular details of the multiple cleavages;
AIM : To complete a consensus model of TF/FVIIa/FXa followed by model studies of TFalone, TF as a dimer, and TF and its putative complexes with protein disulfide isomerase (PDI) (also to be modeled), glutathione and NO?the goal being to understand more completely the issues surrounding cryptic vs procoagulant TF;
AIM III : To complete a model study with solvation of the Vitamin K cycle reactions (enzymes: vitamin K carboxylase and vitamin K epoxide reductase) using quantum mechanics.
The final aim recognizes the need to not only develop all-atom, solvated, 3D structures, but also to provide insight into the quantum mechanical bond-breaking and bond-forming mechanisms that regulate coagulation. Underlying our applied work is the continual development of new techniques?modeling the interaction of protein domains to membrane surfaces( e.g. Gla domains to phospholipid surfaces rich in phosphatidylserine), improving protein-protein docking algorithms, modeling conformational changes in proteins (e.g. the conformational change of the C2 domain of Factor Va when binding to a membrane surface, FVIIa when binding TF, the Gla domain when binding calcium ions) and improving molecular dynamics methods. The developed complex structures will be made available through the internet and these will maintain value as a base for systematic improvement even as new experimental structures are solved.

Public Health Relevance

The study of molecular processes in the blood coagulation cascade (here: prothrombin, factor Vila/Tissue factor, Vitamin K cycle) have high relevance to public health. These molecular processes have impact in many blood diseases, including hemophilia, stroke and plaque in the vascular system. Our research is aimed directly at understanding the molecular processes of normal and abnormal blood coagulation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Program Projects (P01)
Project #
5P01HL006350-51
Application #
8375949
Study Section
Heart, Lung, and Blood Initial Review Group (HLBP)
Project Start
Project End
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
51
Fiscal Year
2012
Total Cost
$192,110
Indirect Cost
$62,306
Name
University of North Carolina Chapel Hill
Department
Type
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Boulaftali, Yacine; Owens 3rd, A Phillip; Beale, Ashley et al. (2016) CalDAG-GEFI Deficiency Reduces Atherosclerotic Lesion Development in Mice. Arterioscler Thromb Vasc Biol 36:792-9
Bode, Michael F; Mackman, Nigel (2016) A combined deficiency of tissue factor and PAR-4 is associated with fatal pulmonary hemorrhage in mice. Thromb Res 146:46-50
Owens 3rd, A Phillip; Edwards, Todd L; Antoniak, Silvio et al. (2015) Platelet Inhibitors Reduce Rupture in a Mouse Model of Established Abdominal Aortic Aneurysm. Arterioscler Thromb Vasc Biol 35:2032-2041
Wu, Sangwook; Lee, Chang Jun; Pedersen, Lee G (2014) Analysis on long-range residue-residue communication using molecular dynamics. Proteins 82:2896-2901
Bode, Michael; Mackman, Nigel (2014) Regulation of tissue factor gene expression in monocytes and endothelial cells: Thromboxane A2 as a new player. Vascul Pharmacol 62:57-62
Perera, Lalith; Beard, William A; Pedersen, Lee G et al. (2014) Applications of quantum mechanical/molecular mechanical methods to the chemical insertion step of DNA and RNA polymerization. Adv Protein Chem Struct Biol 97:83-113
Parker, Christine H; Morgan, Christopher R; Rand, Kasper D et al. (2014) A conformational investigation of propeptide binding to the integral membrane protein ?-glutamyl carboxylase using nanodisc hydrogen exchange mass spectrometry. Biochemistry 53:1511-20
Boulaftali, Yacine; Hess, Paul R; Getz, Todd M et al. (2013) Platelet ITAM signaling is critical for vascular integrity in inflammation. J Clin Invest 123:908-16
Mutoh, Shingo; Sobhany, Mack; Moore, Rick et al. (2013) Phenobarbital indirectly activates the constitutive active androstane receptor (CAR) by inhibition of epidermal growth factor receptor signaling. Sci Signal 6:ra31
Feng, D; Stafford, K A; Broze, G J et al. (2013) Evidence of clinically significant extravascular stores of factor IX. J Thromb Haemost 11:2176-8

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