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