DOCSLIB.ORG

Regulation of the Innate Immune Response by the Blood Coagulation

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Regulation of the Innate Immune Response by the Blood Coagulation REGULATION OF THE INNATE IMMUNE RESPONSE BY THE BLOOD COAGULATION CASCADE By Laura Day Healy A DISSERTATION Presented to the Department of Cell & Developmental Biology Of the Oregon Health & Science University School of Medicine In partial fulfillment of the requirements for the degree of Doctor of Philosophy In Cell & Developmental Biology June 2017 © Laura Healy All Rights Reserved School of Medicine Oregon Health & Science University ________________________________________________________________________ Certificate of Approval ______________________________________ This is to certify that the PhD Dissertation of Laura Healy “Regulation of the innate immune response by the blood coagulation cascade” Has been approved ______________________________________ Mentor: Owen J.T. McCarty, Ph.D. ______________________________________ Member/Chair: Eric D. Cambronne, Ph.D. ______________________________________ Member: Jeffrey A. Gold, M.D. ______________________________________ Member: Linda Susan Musil, Ph.D. ______________________________________ Member: Philip J.S. Stork, M.D. ______________________________________ Member: Abhinav Nellore, Ph.D. TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................................................ i List of Figures and Tables ......................................................................................................................... iv List of Abbreviations ................................................................................................................................. vi Acknowledgements .................................................................................................................................... ix Abstract ................................................................................................................................................1 Chapter 1. Introduction to neutrophil activation and thrombus formation .....................................4 1.1 Roles for the innate immune response and blood coagulation in infectious disease ............4 1.2 Innate immunity ....................................................................................................................6 1.2.1 Overview of innate immunity and neutrophils .......................................................................6 1.2.2 Neutrophil adhesive proteins and neutrophil function ..........................................................8 1.2.3 Neutrophil extracellular traps .............................................................................................11 1.3 Vascular cells in hemostasis and thrombosis ......................................................................12 1.3.1 Overview of vascular cells ...................................................................................................12 1.3.2 Protease activated receptors ................................................................................................16 1.4 Blood coagulation proteins ..................................................................................................17 1.4.1 Extrinsic coagulation ...........................................................................................................17 1.4.2 Intrinsic coagulation ............................................................................................................19 1.5 Crosstalk between the anticoagulant protein C pathway and inflammation .......................20 1.5.1 Overview of protein C structure and activation ..................................................................20 1.5.2 Anticoagulant functions .......................................................................................................22 1.5.3 Anti-inflammatory functions ................................................................................................22 1.6 Immunothrombosis in relation to disease states ..................................................................25 1.7 Thesis Overview ..................................................................................................................26 Chapter 2. Materials & Methods .........................................................................................................28 2.1 Ethical Considerations .........................................................................................................28 2.2 Common Reagents ..............................................................................................................28 2.3 Blood cell collection and neutrophil isolation .....................................................................28 2.3.1 Blood collection ...................................................................................................................28 i 2.3.2 Neutrophil purification ........................................................................................................29 2.4 Immunofluorescence microscopy ........................................................................................29 2.4.1 General protocol ..................................................................................................................29 2.4.2 Image analysis .....................................................................................................................30 Chapter 3. Regulation of thrombus formation through coagulation factor localization to NETs 31 3.1 Abstract ...............................................................................................................................31 3.2 Introduction .........................................................................................................................32 3.3 Background .........................................................................................................................32 3.4 Materials and Methods ........................................................................................................33 3.4.1 Reagents ...............................................................................................................................33 3.4.2 Preparation of human neutrophils .......................................................................................34 3.4.3 Immunofluorescence microscopy .........................................................................................35 3.4.4 Preparation of endothelial cells ...........................................................................................36 3.4.5 Image Analysis .....................................................................................................................36 3.5 Results .................................................................................................................................37 3.5.1 Quantification of coagulation factor binding to neutrophils during NETosis in a purified system ..................................................................................................................................37 3.5.2 Quantification of coagulation factor binding to neutrophils during NETosis in plasma ....45 3.5.3 Quantification of coagulation factor binding to DNA and phospholipids comprising NETs .............................................................................................................................................48 3.6 Discussion ...........................................................................................................................50 Chapter 4. Regulation of neutrophil function by coagulation enzymes. ..........................................53 4.1 Abstract ...............................................................................................................................53 4.2 Introduction .........................................................................................................................54 4.3 Background .........................................................................................................................54 4.4 Material and Methods ..........................................................................................................56 4.4.1 Reagents ...............................................................................................................................56 4.4.2 Preparation of leukocytes ....................................................................................................57 4.4.3 Platelet isolation and secretome preparation ......................................................................58 4.4.4 Immunofluorescence microscopy .........................................................................................58 4.4.5 Neutrophil static binding assay ...........................................................................................59 4.4.6 APC binding assay ...............................................................................................................60 ii 4.4.7 NETs formation assays ........................................................................................................60 4.4.8 Platelet adhesion assay to leukocytes and NETs .................................................................61 4.4.9 Image analysis .....................................................................................................................61 4.4.10 LDH activity assay ...............................................................................................................62
Load more

Recommended publications
  • Downregulation of the Clotting Cascade by the Protein C
    Downregulation of the clotting cascade by the protein C F. Stavenuiter, E.A.M. Bouwens, L.O. Mosnier I Simposio Conjunto EHA - SAH Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA, USA HEMATOLOGÍA, Vol.17 Número Extraordinario XXI CONGRESO E-mail: [email protected] Octubre 2013 Abstract APC mutants, which provide unique insights into The protein C pathway provides important biologi- the relative contributions of APC’s anticoagulant or cal activities to maintain the fluidity of the circula- cytoprotective activities to the beneficial effects of tion, prevent thrombosis, and protect the integrity APC in various murine injury and disease models. of the vasculature in response to injury. Activated Because of its multiple physiological and pharmaco- protein C (APC), in concert with its co-factors and logical activities, the anticoagulant and cytoprotec- cell receptors, assembles in specific macromolecular tive protein C pathway have important implications complexes to provide efficient proteolysis of multiple for the (patho)physiology of vascular disease and substrates that result in anticoagulant and cytopro- for translational research exploring novel therapeu- tective activities. Numerous studies on APC’s struc- tic strategies to combat complex medical disorders ture-function relation with its co-factors, cell recep- such as thrombosis, inflammation, ischemic stroke tors, and substrates provide valuable insights into the and neurodegenerative disease. molecular mechanisms and presumed assembly of Learning goals the macromolecular complexes that are responsible At the conclusion of this activity, participants should for APC’s activities. These insights allow for molecu- know that: lar engineering approaches specifically targeting the - the protein C pathway provides multiple im- interaction of APC with one of its substrates or co- portant functions to maintain a regulated bal- factors.
    [Show full text]
  • Probing Prothrombin Structure by Limited Proteolysis Laura Acquasaliente, Leslie A
    www.nature.com/scientificreports OPEN Probing prothrombin structure by limited proteolysis Laura Acquasaliente, Leslie A. Pelc & Enrico Di Cera Prothrombin, or coagulation factor II, is a multidomain zymogen precursor of thrombin that undergoes Received: 29 November 2018 an allosteric equilibrium between two alternative conformations, open and closed, that react diferently Accepted: 2 April 2019 with the physiological activator prothrombinase. Specifcally, the dominant closed form promotes Published: xx xx xxxx cleavage at R320 and initiates activation along the meizothrombin pathway, whilst the open form promotes cleavage at R271 and initiates activation along the alternative prethrombin-2 pathway. Here we report how key structural features of prothrombin can be monitored by limited proteolysis with chymotrypsin that attacks W468 in the fexible autolysis loop of the protease domain in the open but not the closed form. Perturbation of prothrombin by selective removal of its constituent Gla domain, kringles and linkers reveals their long-range communication and supports a scenario where stabilization of the open form switches the pathway of activation from meizothrombin to prethrombin-2. We also identify R296 in the A chain of the protease domain as a critical link between the allosteric open-closed equilibrium and exposure of the sites of cleavage at R271 and R320. These fndings reveal important new details on the molecular basis of prothrombin function. Te response of the body to vascular injury entails activation of a cascade of proteolytic events where zymo- gens are converted into active proteases1. In the penultimate step of this cascade, the zymogen prothrombin is converted to the active protease thrombin in a reaction catalyzed by the prothrombinase complex composed of the enzyme factor Xa, cofactor Va, Ca2+ and phospholipids.
    [Show full text]
  • Insights Into Vitamin K-Dependent Carboxylation: Home Field Advantage Francis Ayombil 1 and Rodney M
    Editorials 15. Iyer S, Uren RT, Dengler MA, et al. Robust autoactivation for apop - guide clinical decision making in acute myeloid leukemia: a pilot tosis by BAK but not BAX highlights BAK as an important therapeu - study. Leuk Res. 2018;64:34-41. tic target. Cell Death Dis. 2020;11(4):268. 18. Zelenetz AD, Salles G, Mason KD, et al. Venetoclax plus R- or G- 16. Matulis SM, Gupta VA, Neri P, et al. Functional profiling of veneto - CHOP in non-Hodgkin lymphoma: results from the CAVALLI phase clax sensitivity can predict clinical response in multiple myeloma. 1b trial. Blood. 2019;133(18):1964-1976. Leukemia. 2019;33(5):1291-1296. 19. Adams CM, Clark-Garvey S, Porcu P, Eischen CM. Targeting the 17. Swords RT, Azzam D, Al-Ali H, et al. Ex-vivo sensitivity profiling to BCL2 family in B cell lymphoma. Front Oncol. 2019;8:636. Insights into vitamin K-dependent carboxylation: home field advantage Francis Ayombil 1 and Rodney M. Camire 1,2 1Division of Hematology and the Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia and 2Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA E-mail: RODNEY M. CAMIRE - [email protected] doi:10.3324/haematol.2020.253690 itamin K-dependent (VKD) proteins play critical recognized that the propeptide sequence is critical for VKD roles in blood coagulation, bone metabolism, and protein carboxylation. 6 This insight led to the development Vother physiologic processes. These proteins under - of GGCX substrates that contained a propeptide sequence go a specific post-translational modification called and portions of the Gla domain which are superior when 7,8 gamma ( γ)-carboxylation which is critical to their biologic compared to FLEEL alone.
    [Show full text]
  • Interview Anouk Gentier in Medicines
    Dutch Medicines Days Marysa van den Berg important in the functioning of the peptide.’ Patient material was being used to test the peptide. Gentier: ‘We get both healthy and diseased smooth muscle cells taken from pa- tients that underwent surgery for their lesions in the University Hospital of Maastricht. These cells are then cultured, put upon a plate, and incubated with a medium enriched by either calcium, phosphate or both. We then treat them with either protein S Gla, bound to an imaging agent, in various con- centrations or a negative control. Then we use a colorimetric assay to measure the num- ber of microcalcifications formed.’ Promising vesicles Not only is the protein S Gla domain capa- ble of detecting microcalcifications in the MAASTRICHT UNIVERSITY body. The peptide can also block the forma- Left: calcifying cells treated with protein S Gla peptide; right: control. tion of these ticking time bombs. ‘We see fewer microcalcifications in the treated cell cultures than in the negative control cultu- res’, says Gentier. ‘The exact mechanism of action is still unknown, but we think the A peptide against peptide binds to the phosphatidylserine resi- dues of the cell membrane of smooth mus- cle cells that are exposed to a calcifying en- vironment. Through this binding the cells atherosclerosis seem less prone to calcification.’ These findings may lead to a promising new treatment against atherosclerosis, hopes One of the leading causes of death worldwide is Gentier. She and her colleagues have ideas on how this could take shape. ‘We have yet atherosclerosis. PhD student Anouk Gentier to finalise this, but we are planning on ma- developed a peptide that can both trace and block king extracellular vesicles in which we intro- duce both protein S Gla and annexin A2.
    [Show full text]
  • Bioinformatic Characterization of Genes and Proteins Involved in Blood Clotting in Lampreys
    UC San Diego UC San Diego Previously Published Works Title Bioinformatic Characterization of Genes and Proteins Involved in Blood Clotting in Lampreys. Permalink https://escholarship.org/uc/item/0d21p5zb Journal Journal of molecular evolution, 81(3-4) ISSN 0022-2844 Author Doolittle, Russell F Publication Date 2015-10-05 DOI 10.1007/s00239-015-9701-0 Peer reviewed eScholarship.org Powered by the California Digital Library University of California Bioinformatic Characterization of Genes and Proteins Involved in Blood Clotting in Lampreys Russell F. Doolittle Journal of Molecular Evolution ISSN 0022-2844 Volume 81 Combined 3-4 J Mol Evol (2015) 81:121-130 DOI 10.1007/s00239-015-9701-0 1 23 Your article is protected by copyright and all rights are held exclusively by Springer Science +Business Media New York. This e-offprint is for personal use only and shall not be self- archived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”. 1 23 Author's personal copy J Mol Evol (2015) 81:121–130 DOI 10.1007/s00239-015-9701-0 ORIGINAL ARTICLE Bioinformatic Characterization of Genes and Proteins Involved in Blood Clotting in Lampreys Russell F.
    [Show full text]
  • The Linker Connecting the Two Kringles Plays a Key Role in Prothrombin Activation
    The linker connecting the two kringles plays a key role in prothrombin activation Nicola Pozzi, Zhiwei Chen, Leslie A. Pelc, Daniel B. Shropshire, and Enrico Di Cera1 Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104 Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved April 22, 2014 (received for review February 28, 2014) The zymogen prothrombin is proteolytically converted by factor between the autolysis loop (9) and exosite I (10). Factor Xa Xa to the active protease thrombin in a reaction that is accelerated interacts with kringle-2 (11) and residues near exosite II of the >3,000-fold by cofactor Va. This physiologically important effect is catalytic B chain (12), and with the Gla domain (13). These studies paradigmatic of analogous cofactor-dependent reactions in the have targeted regions of the zymogen also present in other vitamin coagulation and complement cascades, but its structural determi- K-dependent clotting factors and proteolytic enzymes (14). The nants remain poorly understood. Prothrombin has three linkers recent crystal structure of Gla-domainless prothrombin (GD-ProT; connecting the N-terminal Gla domain to kringle-1 (Lnk1), the residues 45–579) has revealed the architecture of the kringles two kringles (Lnk2), and kringle-2 to the C-terminal protease do- and protease domain arranged into an overall bent conformation main (Lnk3). Recent developments indicate that the linkers, and with the domains not vertically stacked (15). Importantly, none particularly Lnk2, confer on the zymogen significant flexibility in of the three linkers could be resolved in the electron density solution and enable prothrombin to sample alternative conforma- map.
    [Show full text]
  • The Structure, Function and Evolution of the Extracellular Matrix: a Systems-Level Analysis
    The Structure, Function and Evolution of the Extracellular Matrix: A Systems-Level Analysis by Graham L. Cromar A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Department of Molecular Genetics University of Toronto © Copyright by Graham L. Cromar 2014 ii The Structure, Function and Evolution of the Extracellular Matrix: A Systems-Level Analysis Graham L. Cromar Doctor of Philosophy Department of Molecular Genetics University of Toronto 2014 Abstract The extracellular matrix (ECM) is a three-dimensional meshwork of proteins, proteoglycans and polysaccharides imparting structure and mechanical stability to tissues. ECM dysfunction has been implicated in a number of debilitating conditions including cancer, atherosclerosis, asthma, fibrosis and arthritis. Identifying the components that comprise the ECM and understanding how they are organised within the matrix is key to uncovering its role in health and disease. This study defines a rigorous protocol for the rapid categorization of proteins comprising a biological system. Beginning with over 2000 candidate extracellular proteins, 357 core ECM genes and 524 functionally related (non-ECM) genes are identified. A network of high quality protein-protein interactions constructed from these core genes reveals the ECM is organised into biologically relevant functional modules whose components exhibit a mosaic of expression and conservation patterns. This suggests module innovations were widespread and evolved in parallel to convey tissue specific functionality on otherwise broadly expressed modules. Phylogenetic profiles of ECM proteins highlight components restricted and/or expanded in metazoans, vertebrates and mammals, indicating taxon-specific tissue innovations. Modules enriched for medical subject headings illustrate the potential for systems based analyses to predict new functional and disease associations on the basis of network topology.
    [Show full text]
  • From the Gla Domain to a Novel Small-Molecule Detector of Apoptosis
    Cell Research (2009) 19:625-637. © 2009 IBCB, SIBS, CAS All rights reserved 1001-0602/09 $ 30.00 npg ORIGINAL ARTICLE www.nature.com/cr From the Gla domain to a novel small-molecule detector of apoptosis Avi Cohen1, *, Anat Shirvan1, *, Galit Levin1, Hagit Grimberg1, Ayelet Reshef1, Ilan Ziv1 1Aposense Ltd, 5-7 Ha’Odem St, Kiryat Matalon, PO Box 7119, Petach Tikva, Israel Apoptosis plays a pivotal role in the etiology or pathogenesis of numerous medical disorders, and thus, target- ing of apoptotic cells may substantially advance patient care. In our quest for novel low-molecular-weight probes for apoptosis, we focused on the uncommon amino acid γ-carboxyglutamic acid (Gla), which plays a vital role in the binding of clotting factors to negatively charged phospholipid surfaces. Based on the alkyl-malonic acid motif of Gla, we have developed and now present ML-10 (2-(5-fluoro-pentyl)-2-methyl-malonic acid, MW=206 Da), the pro- totypical member of a novel family of small-molecule detectors of apoptosis. ML-10 was found to perform selective uptake and accumulation in apoptotic cells, while being excluded from either viable or necrotic cells. ML-10 uptake correlates with the apoptotic hallmarks of caspase activation, Annexin-V binding and disruption of mitochondrial membrane potential. The malonate moiety was found to be crucial for ML-10 function in apoptosis detection. ML- 10 responds to a unique complex of features of the cell in early apoptosis, comprising irreversible loss of membrane potential, permanent acidification of cell membrane and cytoplasm, and preservation of membrane integrity.
    [Show full text]
  • Substrate Softness Promotes Terminal Differentiation of Human
    Substrate softness promotes terminal differentiation of human keratinocytes without altering their ability to proliferate back into a rigid environment Choua Ya, Mariana Carrancá, Dominique Sigaudo-Roussel, Philippe Faure, Berengere Fromy, Romain Debret To cite this version: Choua Ya, Mariana Carrancá, Dominique Sigaudo-Roussel, Philippe Faure, Berengere Fromy, et al.. Substrate softness promotes terminal differentiation of human keratinocytes without altering their ability to proliferate back into a rigid environment. Archives of Dermatological Research, Springer Verlag, 2019, 311 (10), pp.741-751. 10.1007/s00403-019-01962-5. hal-02322166 HAL Id: hal-02322166 https://hal.archives-ouvertes.fr/hal-02322166 Submitted on 7 Dec 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Substrate softness promotes terminal differentiation of human keratinocytes without altering their ability to proliferate back into a rigid environment Choua Ya1,2, Mariana Carrancá1, Dominique Sigaudo-Roussel1, Philippe Faure3, Bérengère Fromy1†, Romain Debret1†* 1 CNRS, University Lyon 1, UMR 5305, Laboratory of Tissue Biology and Therapeutic Engineering, IBCP, 7 Passage du Vercors, 69367 Lyon Cedex 07, France. 2 Isispharma, 29 Rue Maurice Flandin, 69003 Lyon, France. 3 Alpol Cosmétique, 140 Rue Pasteur, 01500 Château-Gaillard, France.
    [Show full text]
  • Integrated Bioinformatics Analysis Reveals Novel Key Biomarkers and Potential Candidate Small Molecule Drugs in Gestational Diabetes Mellitus
    bioRxiv preprint doi: https://doi.org/10.1101/2021.03.09.434569; this version posted March 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Integrated bioinformatics analysis reveals novel key biomarkers and potential candidate small molecule drugs in gestational diabetes mellitus Basavaraj Vastrad1, Chanabasayya Vastrad*2, Anandkumar Tengli3 1. Department of Biochemistry, Basaveshwar College of Pharmacy, Gadag, Karnataka 582103, India. 2. Biostatistics and Bioinformatics, Chanabasava Nilaya, Bharthinagar, Dharwad 580001, Karnataka, India. 3. Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Mysuru and JSS Academy of Higher Education & Research, Mysuru, Karnataka, 570015, India * Chanabasayya Vastrad [email protected] Ph: +919480073398 Chanabasava Nilaya, Bharthinagar, Dharwad 580001 , Karanataka, India bioRxiv preprint doi: https://doi.org/10.1101/2021.03.09.434569; this version posted March 10, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Gestational diabetes mellitus (GDM) is one of the metabolic diseases during pregnancy. The identification of the central molecular mechanisms liable for the disease pathogenesis might lead to the advancement of new therapeutic options. The current investigation aimed to identify central differentially expressed genes (DEGs) in GDM. The transcription profiling by array data (E-MTAB-6418) was obtained from the ArrayExpress database. The DEGs between GDM samples and non GDM samples were analyzed with limma package. Gene ontology (GO) and REACTOME enrichment analysis were performed using ToppGene. Then we constructed the protein-protein interaction (PPI) network of DEGs by the Search Tool for the Retrieval of Interacting Genes database (STRING) and module analysis was performed.
    [Show full text]
  • Fine Tuning HCN Channel Activity Tural Basis of This Coupling Has Only Been Well Characterized in Kcsa
    Wednesday, February 15, 2017 465a Symposium: Protein Dynamics and Allostery example, in-silico models of human cardiac electrophysiology are being considered by the FDA for prediction of proarrhythmic cardiotoxicity as a 2285-Symp core component of the preclinical assessment phase of all new drugs. How- Coupled Residue-Residue Dynamics in Protein Allosteric Mechanisms ever, one issue that exists with current models is that they each respond Donald Hamelberg. differently to insults such as drug block of ion channels or mutation of cardiac Department of Chemistry, Georgia State University, Atlanta, GA, USA. ion channel genes. Clearly this poses a problem in relation to the utility of Although the relationship between structure and function in biomolecules is these models in making quantitative predictions that are physiologically or well established, it is not always adequate to provide a complete understanding clinically meaningful. To examine this in detail we tested the ability of three of biomolecular function. The dynamical fluctuations of biomolecules can also models of the human ventricular action potential, the O’hara-Rudy the Grandi- play an essential role in function. Detailed understanding of how conforma- Bers and the Ten Tusscher models, to reproduce the clinical phenotype of tional dynamics orchestrates function in allosteric regulation of recognition different subtypes of the long QT syndrome. All models, in their original and catalysis at atomic resolution remains ambiguous. The overarching goal form, produce markedly different and unrealistic predictions of QT prolonga- is to understand how biomolecular dynamics are coupled to function by using tion. To address this, we used a global optimization approach to constrain ex- atomistic molecular simulations to complement experiments.
    [Show full text]
  • Structure of Human Factor Viia and Its Implications for the Triggering of Blood Coagulation
    Proc. Natl. Acad. Sci. USA Vol. 96, pp. 8925–8930, August 1999 Biochemistry Structure of human factor VIIa and its implications for the triggering of blood coagulation ASHLEY C. W. PIKE*, ANDRZEJ M. BRZOZOWSKI*, SHIRLEY M. ROBERTS*, OLE H. OLSEN†, AND EGON PERSSON‡§ *Structural Biology Laboratory, Chemistry Department, University of York, York YO10 5DD, United Kingdom; and †Medicinal Chemistry Research IV and ‡Tissue Factor/Factor VII Research, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark Communicated by Earl W. Davie, University of Washington, Seattle, WA, May 26, 1999 (received for review February 17, 1999) ABSTRACT Factor VIIa (EC 3.4.21.21) is a trypsin-like of fVIIa at the proper distance above the surface of the serine protease that plays a key role in the blood coagulation membrane for alignment with the scissile bond (4). cascade. On injury, factor VIIa forms a complex with its The crystal structure of fVIIa in complex with the extracel- allosteric regulator, tissue factor, and initiates blood clotting. lular part of TF (sTF) reveals an extensive interface between Although the structure of the binary complex has already been the two proteins encompassing all four domains in fVIIa, with determined [Banner, D. W., D’Arcy, A., Che`ne, C., Winkler, the light chain contributing the majority of the binding energy F. K., Guha, A., Konigsberg, W. H., Nemerson, Y. & Kirch- (5), and both extracellular domains of TF (6, 7). The intrado- hofer, D. (1996) Nature (London) 380, 41–46], the conforma- main structural changes that accompany the allosteric en- tional effects of cofactor binding to factor VIIa are not known hancement of fVIIa’s activity appear to be confined to the in detail because of a lack of structural information on free protease domain (8), but the precise molecular switch by which factor VIIa.
    [Show full text]