Almost every step in blood coagulation requires assembly of multiple proteins on membrane surfaces, yet protein-membrane interactions in blood clotting remain poorly understood at the molecular level. We are using new, high-resolution technologies including magic-angle spinning solid-state NMR (SSNMR) to probe the mechanisms by which blood clotting proteins interact with phospholipid surfaces, and how changes in membrane composition regulate clotting reactions. The primary focus of our studies is the membrane-bound complex of tissue factor and factor VIIa, the two-subunit enzyme responsible for triggering blood clotting in health and disease.
Aim 1 will delineate, at atomic resolution, the changes in structure and dynamics of PS- rich membrane domains in the presence of Ca2+, and will examine the roles of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine in configuring the membrane to support high affinity binding of clotting factors.
Aim 2 will investigate the role of phospholipid-phospholipid interactions in enhancing blood clotting reactions on membrane surfaces.
Aim 3 will delineate the lipid environments/conformations induced when clotting proteins bind to bilayers.
Aim 4 will solve the structure of tissue factor on the membrane surface and identify conformational changes in tissue factor when it interacts with ligands. Together these studies will provide valuable new insights into the role of the membrane surface in blood clotting, at atomic-scale resolution.

Public Health Relevance

Disorders of the blood clotting system represent the leading cause of disability and death in the United States, but we still have a very incomplete understanding of blood clotting reactions at the molecular level. These studies will shed new light on the regulation of the blood clotting system, with a particular focus on achieving a detailed understanding of how and why blood clotting reactions occur on membrane surfaces.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL103999-04
Application #
8450177
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Link, Rebecca P
Project Start
2010-07-06
Project End
2015-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
4
Fiscal Year
2013
Total Cost
$517,706
Indirect Cost
$178,220
Name
University of Illinois Urbana-Champaign
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
041544081
City
Champaign
State
IL
Country
United States
Zip Code
61820
Shi, Xiangyan; Rienstra, Chad M (2016) Site-Specific Internal Motions in GB1 Protein Microcrystals Revealed by 3D ²H-¹³C-¹³C Solid-State NMR Spectroscopy. J Am Chem Soc 138:4105-19
Hisao, Grant S; Harland, Michael A; Brown, Robert A et al. (2016) An efficient method and device for transfer of semisolid materials into solid-state NMR spectroscopy rotors. J Magn Reson 265:172-6
Gajsiewicz, Joshua M; Morrissey, James H (2015) Structure-Function Relationship of the Interaction between Tissue Factor and Factor VIIa. Semin Thromb Hemost 41:682-90
Courtney, Joseph M; Ye, Qing; Nesbitt, Anna E et al. (2015) Experimental Protein Structure Verification by Scoring with a Single, Unassigned NMR Spectrum. Structure 23:1958-66
Smith, Stephanie A; Travers, Richard J; Morrissey, James H (2015) How it all starts: Initiation of the clotting cascade. Crit Rev Biochem Mol Biol 50:326-36
Gajsiewicz, Joshua M; Nuzzio, Kristin M; Rienstra, Chad M et al. (2015) Tissue Factor Residues That Modulate Magnesium-Dependent Rate Enhancements of the Tissue Factor/Factor VIIa Complex. Biochemistry 54:4665-71
Ke, Ke; Yuan, Jian; Morrissey, James H (2014) Tissue factor residues that putatively interact with membrane phospholipids. PLoS One 9:e88675
Tavoosi, Narjes; Morrissey, James H (2014) Influence of membrane composition on the enhancement of factor VIIa/tissue factor activity by magnesium ions. Thromb Haemost 111:770-2
Watt, Eric D; Rienstra, Chad M (2014) Recent advances in solid-state nuclear magnetic resonance techniques to quantify biomolecular dynamics. Anal Chem 86:58-64
Sperling, Lindsay J; Tang, Ming; Berthold, Deborah A et al. (2013) Solid-state NMR study of a 41 kDa membrane protein complex DsbA/DsbB. J Phys Chem B 117:6052-60

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