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      Crystal Structure of Thrombin in Complex with S-Variegin: Insights of a Novel Mechanism of Inhibition and Design of Tunable Thrombin Inhibitors

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          Abstract

          The inhibition of thrombin is one of the important treatments of pathological blood clot formation. Variegin, isolated from the tropical bont tick, is a novel molecule exhibiting a unique ‘two-modes’ inhibitory property on thrombin active site (competitive before cleavage, noncompetitive after cleavage). For the better understanding of its function, we have determined the crystal structure of the human α-thrombin:synthetic-variegin complex at 2.4 Å resolution. The structure reveals a new mechanism of thrombin inhibition by disrupting the charge relay system. Based on the structure, we have designed 17 variegin variants, differing in potency, kinetics and mechanism of inhibition. The most active variant is about 70 times more potent than the FDA-approved peptidic thrombin inhibitor, hirulog-1/bivalirudin. In vivo antithrombotic effects of the variegin variants correlate well with their in vitro affinities for thrombin. Our results encourage that variegin and the variants show strong potential for the development of tunable anticoagulants.

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          Structural basis of substrate specificity in the serine proteases.

          John Perona, D. C. Craik (1995)
          Structure-based mutational analysis of serine protease specificity has produced a large database of information useful in addressing biological function and in establishing a basis for targeted design efforts. Critical issues examined include the function of water molecules in providing strength and specificity of binding, the extent to which binding subsites are interdependent, and the roles of polypeptide chain flexibility and distal structural elements in contributing to specificity profiles. The studies also provide a foundation for exploring why specificity modification can be either straightforward or complex, depending on the particular system.
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            Molecular recognition mechanisms of thrombin.

            James Huntington (2005)
            Thrombin is the final protease generated in the blood coagulation cascade, and is the only factor capable of cleaving fibrinogen to create a fibrin clot. Unlike every other coagulation protease, thrombin is composed solely of its serine protease domain, so that once formed it can diffuse freely to encounter a large number of potential substrates. Thus thrombin serves many functions in hemostasis through the specific cleavage of at least a dozen substrates. The solution of the crystal structure of thrombin some 15 years ago revealed a deep active site cleft and two adjacent basic exosites, and it was clear that thrombin must utilize these unique features in recognizing its substrates. Just how this occurs is still being investigated, but recent data from thrombin mutant libraries and crystal structures combine to paint the clearest picture to date of the molecular determinants of substrate recognition by thrombin. In almost all cases, both thrombin exosites are involved, either through direct interaction with the substrate protein or through indirect interaction with a third cofactor molecule. The purpose of this article is to summarize recent biochemical and structural data in order to provide insight into the thrombin molecular recognition events at the heart of hemostasis.
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              Directing thrombin.

              James Huntington, Helen Philippou, Jon Lane (2005)
              Following initiation of coagulation as part of the hemostatic response to injury, thrombin is generated from its inactive precursor prothrombin by factor Xa as part of the prothrombinase complex. Thrombin then has multiple roles. The way in which thrombin interacts with its many substrates has been carefully scrutinized in the past decades, but until recently there has been little consideration of how its many functions are coordinated or directed. Any understanding of how it is directed requires knowledge of its structure, how it interacts with its substrates, and the role of any cofactors for its interaction with substrates. Recently, many of the interactions of thrombin have been clarified by crystal structure and site-directed mutagenesis analyses. These analyses have revealed common residues used for recognition of some substrates and overlapping surface exosites used for recognition by cofactors. As many of its downstream reactions are cofactor driven, competition between cofactors for exosites must be a dominant mechanism that determines the fate of thrombin. This review draws together much recent work that has helped clarify structure function relationships of thrombin. It then attempts to provide a cogent proposal to explain how thrombin activity is directed during the hemostatic response.
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                Author and article information

                Contributors
                : Role: Editor
                Journal
                Journal ID (nlm-ta): PLoS One
                Journal ID (publisher-id): plos
                Journal ID (pmc): plosone
                Title: PLoS ONE
                Publisher: Public Library of Science (San Francisco, USA )
                ISSN (Electronic): 1932-6203
                Publication date Collection: 2011
                Publication date (Electronic): 28 October 2011
                Volume: 6
                Issue: 10
                Electronic Location Identifier: e26367
                Affiliations
                [1 ]Department of Biological Sciences, National University of Singapore, Singapore, Singapore
                [2 ]Institute of Zoology, Slovak Academy of Sciences, Bratislava, Slovakia
                [3 ]NERC Centre for Ecology and Hydrology, Oxford, United Kingdom
                [4 ]Department of Biological Sciences, University of North Texas, Denton, Texas, United States of America
                [5 ]The Chemo-Sero-Therapeutic Research Institute, Kumamoto, Japan
                [6 ]Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University, Richmond, Virginia, United States of America
                Aligarh Muslim University, India
                Author notes

                Conceived and designed the experiments: CYK PJ KS RMK. Performed the experiments: CYK S. Kumar UPR S. Kim KS. Analyzed the data: CYK S. Kumar MK PAN KS RMK. Contributed reagents/materials/analysis tools: TI JM SI. Wrote the paper: CYK MK PAN KS RMK. Designed and synthesized variegin peptides, carried out thrombin inhibition assays, crystallized thrombin: s-variegin complex, analyzed data and wrote the manuscript: CYK. Determined the structure: S. Kumar. Analyzed data and wrote the manuscript: MK PAN. Designed and carried out zebrafish larvae venous thrombosis model experiments: UPR S. Kim PJ. Prepared and provided thrombin: TI JM SI. Collected crystal diffraction data, determined the structure, analyzed data and wrote the manuscript: KS. Conceptualized, designed the study, analyzed data and wrote the manuscript: RMK.

                [¤]

                Current address: X-ray Crystallographic Unit, Biopolis Shared Facility, Singapore, Singapore

                Article
                Publisher ID: PONE-D-11-14107
                DOI: 10.1371/journal.pone.0026367
                PMC ID: 3203879
                PubMed ID: 22053189
                SO-VID: 31fa305a-2e7a-4717-b803-09697f77ec9c
                Copyright © Koh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                Date received : 21 July 2011
                Date accepted : 25 September 2011
                Page count
                Pages: 16
                Categories
                Subject: Research Article
                Subject: Biology
                Subject: Biochemistry
                Subject: Enzymes
                Subject: Enzyme Kinetics
                Subject: Enzyme Structure
                Subject: Proteins
                Subject: Coagulation Factors
                Subject: Protein Chemistry
                Subject: Protein Interactions
                Subject: Protein Structure
                Subject: Biomacromolecule-Ligand Interactions
                Subject: Blood Chemistry
                Subject: Drug Discovery
                Subject: Biophysics
                Subject: Protein Chemistry
                Subject: Medicine
                Subject: Cardiovascular

                ScienceOpen disciplines: Uncategorized
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                ScienceOpen disciplines: Uncategorized

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