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.
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.
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