The proposed research project addresses how the zymogen prothrombin is converted to the mature protease thrombin. The project is aimed at gaining a mechanistic understanding of this key reaction of the blood coagulation cascade using a combination of x-ray structural biology, kinetics, site-directed mutagenesis and fluorescence measurements at the single molecule level. Specifically, the project builds on our recent success in the crystallization of prothrombin and the discovery of its conformational flexibility due to the linkers connecting the Gla domain, kringles and protease domain and will address the following basic questions: What is the role of prothrombin flexibility in the function of the zymogen? How does the conformation of prothrombin change upon binding to the prothrombinase complex and its components? What is the role of the linkers in controlling the conformation of the zymogen, as well as the rate and pathway of activation? What are the regions of prothrombin important for the conversion to the mature enzyme thrombin? The project consists of the following specific aims: 1. Elucidate the role of conformational flexibilit in the structure and function of prothrombin; 2. Identify the epitopes that control the rate and pathway of prothrombin activation.
In specific aim 1, we will build on recent breakthrough structures of prothrombin to attach fluorescence dyes at various positions across the length of the zymogen to enable single molecule measurements of the conformations in solution when free and bound to the prothrombinase complex and its components. We will endeavor to elucidate the role of flexible linkers connecting the Gla domain to kringle-1 (Lnk1), kringle-1 to kringle-2 (Lnk2), and kringle-2 to the A chain (Lnk3) in controlling the conformation of prothrombin. We hypothesize that the linkers, and especially Lnk2, play a dominant role in controlling the rate and pathway of prothrombin activation by changing their conformation upon interaction with the prothrombinase complex and its components. Developments under this specific aim will provide unprecedented details on the structural changes that accompany prothrombin activation.
In specific aim 2, we will complement the studies under specific aim 1 with an Ala scanning mutagenesis mapping of residues controlling the rate and pathway of prothrombin activation. The scan will be guided by available structural information and will target charged and aromatic residues >70% exposed to solvent, as well as residues whose naturally occurring mutations are associated with bleeding phenotypes. We hypothesize that the linkers orchestrate the spatial arrangement of specific domains of prothrombin to optimize the rate of activation and dictate selection of the pathway by prothrombinase. Developments under this specific aim will identify epitopes responsible for prothrombin activation that will offer targets or therapeutic intervention and afford a molecular interpretation of the bleeding phenotypes associated with many naturally occurring mutations.

Public Health Relevance

Recent statistics indicate that cardiovascular disease and its thrombotic complications will remain the leading cause of death and disability and will represent a major burden to productivity in the US and worldwide well into the year 2020. Because of its involvement in thrombotic deaths, thrombin and its precursor prothrombin remain major targets of antithrombotic and anticoagulant therapies. Progress in the understanding of how prothrombin is activated to thrombin will benefit our knowledge of a key reaction of the blood coagulation cascade and inform new strategies of therapeutic intervention that could influence the life-style and life expectancy of millions of people in the US and worldwide.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL049413-21
Application #
9205523
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Warren, Ronald Q
Project Start
1994-12-01
Project End
2020-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
21
Fiscal Year
2017
Total Cost
$378,750
Indirect Cost
$128,750
Name
Saint Louis University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
050220722
City
Saint Louis
State
MO
Country
United States
Zip Code
63103
Wu, Xiaobin; Kim, Heejeong; Seravalli, Javier et al. (2016) Potassium and the K+/H+ Exchanger Kha1p Promote Binding of Copper to ApoFet3p Multi-copper Ferroxidase. J Biol Chem 291:9796-806
Pozzi, Nicola; Bystranowska, Dominika; Zuo, Xiaobing et al. (2016) Structural Architecture of Prothrombin in Solution Revealed by Single Molecule Spectroscopy. J Biol Chem 291:18107-16
Pozzi, Nicola; Chen, Zhiwei; Di Cera, Enrico (2016) How the Linker Connecting the Two Kringles Influences Activation and Conformational Plasticity of Prothrombin. J Biol Chem 291:6071-82
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Pozzi, Nicola; Zerbetto, Mirco; Acquasaliente, Laura et al. (2016) Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin. Biochemistry 55:3984-94
Pozzi, N; Di Cera, E (2016) Dual effect of histone H4 on prothrombin activation. J Thromb Haemost 14:1814-8
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Wood, D C; Pelc, L A; Pozzi, N et al. (2015) WEDGE: an anticoagulant thrombin mutant produced by autoactivation. J Thromb Haemost 13:111-4
Vogt, Austin D; Chakraborty, Pradipta; Di Cera, Enrico (2015) Kinetic dissection of the pre-existing conformational equilibrium in the trypsin fold. J Biol Chem 290:22435-45

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