Antiphospholipid Syndrome in Systemic Autoimmune Diseases [2nd Edition] 9780444636560, 9780444636553

Table of contents :
Content:
Antiphospholipid Syndrome in Systemic Autoimmune DiseasesPage i
Handbook of Systemic Autoimmune DiseasesPage ii
Antiphospholipid Syndrome in Systemic Autoimmune DiseasesPage iii
CopyrightPage iv
ContentsPages v-xii
List of ContributorsPages xiii-xv
ForewordPage xviiGraham Hughes
DedicationPage xix
Chapter 1 - History, Classification, and Subsets of the Antiphospholipid SyndromePages 1-16Roger A. Levy, Jose A. Gómez-Puerta, Ricard Cervera
Chapter 2 - Epidemiology of the Antiphospholipid SyndromePages 17-30Laura Durcan, Michelle Petri
Chapter 3 - Mechanisms of Action of the Antiphospholipid AntibodiesPages 31-46Cecilia B. Chighizola, Elena Raschi, Maria O. Borghi, Pier L. Meroni
Chapter 4 - Laboratory Markers With Clinical Significance in the Antiphospholipid SyndromePages 47-69Olga Amengual, Maria L. Bertolaccini, Tatsuya Atsumi
Chapter 5 - Genetic and Epigenetic Aspects of Antiphospholipid Syndrome: What we knew, what we knowPages 71-86Annamaria Iuliano, Gian D. Sebastiani, Mauro Galeazzi
Chapter 6 - Thrombotic Manifestations of the Antiphospholipid SyndromePages 87-106Ricard Cervera, Ignasi Rodríguez-Pintó, Gerard Espinosa, Joan C. Reverter
Chapter 7 - Obstetric Manifestations of the Antiphospholipid SyndromePages 107-120Angela Tincani, Cecilia Nalli, Rossella Reggia, Sonia Zatti, Andrea Lojacono
Chapter 8 - Thrombocytopenia in the Antiphospholipid SyndromePages 121-130Serena Fasano, David A. Isenberg
Chapter 9 - Nonclassification Criteria Manifestations of the Antiphospholipid SyndromePages 131-144Mohammad Hassan A. Noureldine, Imad Uthman
Chapter 10 - Paediatric Antiphospholipid SyndromePages 145-165Nataša Toplak, Tadej Avčin
Chapter 11 - Antiphospholipid Antibodies and Their Relationship With Infections, Vaccines, and DrugsPages 167-179Jiram Torres Ruiz, Miri Blank, Gisele Zandman-Goddard, Yaniv Sherer, Yehuda Shoenfeld
Chapter 12 - Antiphospholipid Syndrome Associated With MalignanciesPages 181-191Jose A. Gómez-Puerta, Wolfgang Miesbach
Chapter 13 - Antiphospholipid Antibodies and AtherosclerosisPages 193-214Joan T. Merrill
Chapter 14 - Global Antiphospholipid Syndrome ScorePages 215-219Savino Sciascia, Munther Khamashta, Dario Roccatello
Chapter 15 - Primary Prophylaxis in Patients With Positive Antiphospholipid AntibodiesPages 221-229María J. Cuadrado
Chapter 16 - Treatment of Thrombosis in Antiphospholipid SyndromePages 231-241Guillermo Ruiz-Irastorza, Munther Khamashta
Chapter 17 - Treatment of Catastrophic Antiphospholipid SyndromePages 243-255Ignasi Rodríguez-Pintó, Gerard Espinosa, Ricard Cervera
Chapter 18 - Treatment of Pregnancy Complications in Antiphospholipid SyndromePages 257-279Anwar Nassar, Imad Uthman, Joe Eid, Munther Khamashta
Chapter 19 - Prognosis in Antiphospholipid SyndromePages 281-294Rosa M. Serrano, Guillermo J. Pons-Estel, Gerard Espinosa, Ricard Cervera
IndexPages 295-306

Citation preview

Handbook of Systemic Autoimmune Diseases Volume 10

Antiphospholipid Syndrome in Systemic Autoimmune Diseases

Handbook of Systemic Autoimmune Diseases Series Editors: F. Atzeni and P. Sarzi-Puttini Volume 1

The Heart in Systemic Autoimmune Diseases, 2nd edition Edited by: Fabiola Atzeni, Andrea Doria, Mike Nurmohamed, and Paolo Pauletto

Volume 2 Pulmonary Involvement in Systemic Autoimmune Diseases Edited by: Athol U. Wells and Christopher P. Denton Volume 3 Neurologic Involvement in Systemic Autoimmune Diseases Edited by: Doruk Erkan and Steven R. Levine Volume 4 Reproductive and Hormonal Aspects of Systemic Autoimmune Diseases Edited by: Michael Lockshin and Ware Branch Volume 5

The Skin in Systemic Autoimmune Diseases Edited by: Piercarlo Sarzi-Puttini, Andrea Doria, Giampiero Girolomoni and Annegret Kuhn

Volume 6

Pediatrics in Systemic Autoimmune Diseases, 2nd edition Edited by: Rolando Cimaz and Thomas Lehman

Volume 7

The Kidney in Systemic Autoimmune Diseases Edited by: Justin C. Mason and Charles D. Pusey

Volume 8  Digestive Involvement in Systemic Autoimmune Diseases, 2nd edition Edited by: Fabiola Atzeni, Munter Khamashta, Manuel RamosCasals, Pilar Brito-Zerón, and Joan Rodés Teixidor Volume 9 Endocrine Manifestations of Systemic Autoimmune Diseases Edited by: Sara E. Walker and Luis J. Jara Volume 10 Antiphospholipid Syndrome in Systemic Autoimmune Diseases Edited by: Ricard Cervera, Gerard Espinosa and Munther Khamashta

Handbook of Systemic Autoimmune Diseases Volume 10

Antiphospholipid Syndrome in Systemic Autoimmune Diseases Second Edition Edited by:

Ricard Cervera, MD, PhD, FRCP Department of Autoimmune Diseases Hospital Clínic Barcelona, Catalonia, Spain

Gerard Espinosa, MD, PhD Department of Autoimmune Diseases Hospital Clínic Barcelona, Catalonia, Spain

Munther Khamashta, MD, FRCP, PhD Graham Hughes Lupus Research Laboratory Division of Women’s Health King’s College London The Rayne Institute St Thomas’ Hospital London, United Kingdom Series Editors:

F. Atzeni and P. Sarzi-Puttini

AMSTERDAM • BOSTON • HEIDELBERG • LONDON • NEW YORK • OXFORD PARIS • SAN DIEGO • SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, UK 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, USA Copyright © 2017, 2009 Elsevier B.V. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 978-0-444-63655-3 ISSN: 1571-5078 For Information on all Elsevier publications visit our website at http://www.elsevier.com/

Publisher: Sara Tenney Acquisition Editor: Linda Versteeg-Buschman Editorial Project Manager: Halima Williams Production Project Manager: Lucía Pérez Designer: Victoria Pearson Typeset by MPS Limited, Chennai, India

Contents List of Contributors xiii Foreword xvii Dedication xix

1. History, Classification, and Subsets of the Antiphospholipid Syndrome Roger A. Levy, Jose A. Gómez-Puerta and Ricard Cervera 1.1 Introduction 1.2 Historical Perspective 1.3 Classification of APS 1.4 Primary or Isolated APS 1.5 APS Associated With Other Diseases 1.5.1 APS Associated With Autoimmune Diseases 1.5.2 APS Associated With Infections 1.5.3 APS Associated With Drugs 1.5.4 APS Associated With Malignancies 1.6 Seronegative APS 1.7 Catastrophic APS 1.8 International aPL/APS Congresses 1.9 APS Action References

1 1 2 4 6 6 7 8 8 8 9 9 10 12

2. Epidemiology of the Antiphospholipid Syndrome Laura Durcan and Michelle Petri 2.1 Introduction 2.2 APS in the General Population 2.3 aPL and Venous Thrombosis 2.4 aPL and Arterial Thrombosis 2.5 aPL and Pregnancy Morbidity 2.6 aPL and SLE 2.7 Conclusions Acknowledgements References

17 18 22 23 23 24 25 25 25

v

vi  Contents

3. Mechanisms of Action of the Antiphospholipid Antibodies Cecilia B. Chighizola, Elena Raschi, Maria O. Borghi and Pier L. Meroni 3.1 Introduction 3.2 Antiphospholipid Antibodies 3.2.1 The β2 Glycoprotein I-Dependent Autoantibodies 3.2.2 Prothrombin-Dependent Antibodies 3.2.3 Antibodies Against Other PL Antigens 3.3 aPL-Mediated Mechanisms of Thrombosis 3.3.1 Endothelial Cells 3.3.2 Monocytes 3.3.3 Platelets 3.3.4 Neutrophils 3.3.5 Soluble Phase 3.3.6 Complement 3.4 aPL-Mediated Mechanism of Pregnancy Complications 3.5 Receptors for β2GPI/Anti-β2GPI Antibodies 3.6 Intracellular Pathways 3.7 Two-Hit Hypothesis 3.8 Genetics and Epigenetics 3.9 Conclusions References

31 31 32 33 34 34 35 35 35 36 36 37 37 39 40 40 41 42 43

4. Laboratory Markers With Clinical Significance in the Antiphospholipid Syndrome Olga Amengual, Maria L. Bertolaccini and Tatsuya Atsumi 4.1 Introduction 47 4.2 aPL Detected by Solid-Phase Immunoassays 49 4.2.1 Anticardiolipin Antibody Assay 50 4.2.2 Anti-β2GPI Antibody Assay 51 4.2.3 Antibodies Against Domain I of β2GPI 52 4.2.4 Antiprothrombin Antibodies 53 4.2.5 Antibodies to Negatively Charged Phospholipids Other Than Cardiolipin 54 4.2.6 Antibodies to Phosphatidylethanolamine 55 4.2.7 Other aPL Specificities 55 4.3 Lupus Anticoagulant 55 4.4 Annexin A5 Resistance Test: A Mechanistic Test for the Detection of Pathogenic aPL Antibodies 56 4.5 New Technologies for the Detection of aPL 57 4.6 Which aPL Should Be Tested in Patients With Suspicion of Having APS? 57 4.7 Conclusions 59 Acknowledgements 59 References 59

Contents  vii

5. Genetic and Epigenetic Aspects of Antiphospholipid Syndrome Annamaria Iuliano, Gian D. Sebastiani and Mauro Galeazzi 5.1 HLA, APS, and aPL 5.1.1 Family Studies 5.1.2 Population Studies on PAPS 5.1.3 Population Studies on aPL in Diseases Other Than PAPS 5.2 The Role of Non-MHC Genes in APS Susceptibility 5.3 Thrombophilic Hereditary Factors 5.4 Posttranscription Modifications of Anti-β2GPI Antibodies 5.5 Conclusions References

71 73 73 75 78 80 81 82 84

6. Thrombotic Manifestations of the Antiphospholipid Syndrome Ricard Cervera, Ignasi Rodríguez-Pintó, Gerard Espinosa and Joan C. Reverter 6.1 Introduction 6.2 Large Vessel Manifestations 6.2.1 Venous Thrombosis 6.2.2 Arterial Occlusions 6.3 Neurologic Manifestations 6.3.1 Cerebral Infarctions and Transient Ischaemic Attacks 6.3.2 Epilepsy 6.3.3 Dementia 6.3.4 Acute Ischaemic Encephalopathy 6.3.5 Migraine and Migranous Stroke 6.3.6 Pseudomultiple Sclerosis 6.3.7 Cerebral Venous Sinus Thrombosis 6.3.8 Psychosis 6.3.9 Movement Disorders 6.3.10 Spinal Syndromes 6.3.11 Guillain–Barré Syndrome 6.3.12 Ophthalmic Complications 6.4 Pulmonary Manifestations 6.4.1 Pulmonary Embolic Disease and Lung Infarct 6.4.2 Pulmonary Hypertension 6.4.3 Acute Respiratory Distress Syndrome 6.4.4 Bronchial Arterial Thrombosis 6.5 Cardiac Manifestations 6.5.1 Valve Disease 6.5.2 Myocardial Infarctions 6.5.3 Cardiomyopathy 6.5.4 Intracardiac Thrombus

87 87 87 90 90 90 90 90 91 91 91 91 92 92 92 93 93 93 93 93 94 94 94 94 95 95 95

viii  Contents 6.6 Renal Manifestations 6.6.1 Thrombotic Microangiopathy 6.6.2 Renal Vein Thrombosis 6.6.3 Renal Artery Thrombosis/Stenosis 6.6.4 Renal Infarction 6.7 Haematologic Manifestations 6.7.1 Thrombocytopenia 6.7.2 Thrombotic Microangiopathic Haemolytic Anaemia 6.7.3 Autoimmune Haemolytic Anaemia 6.8 Dermatologic Manifestations 6.8.1 Livedo Reticularis 6.8.2 Cutaneous Ulcerations/Necrosis 6.8.3 Pseudovasculitis Lesions 6.8.4 Digital Necrosis and Gangrene 6.8.5 Multiple Subungual Haemorrhages 6.8.6 Anetoderma 6.8.7 Other Dermatologic Manifestations 6.9 Hepatic and Digestive Manifestations 6.9.1 Thrombotic Liver Disease 6.9.2 Nonthrombotic Liver Disease 6.9.3 Mesenteric Manifestations 6.9.4 Splenic Infarction 6.9.5 Pancreatitis 6.9.6 Other Gastrointestinal Manifestations 6.9.7 Other Haematologic Manifestations 6.10 Adrenal Manifestations 6.11 Osteoarticular Manifestations 6.11.1 Osteonecrosis 6.12 Catastrophic APS References

96 96 96 96 96 97 97 97 97 97 97 98 98 98 98 98 98 99 99 99 99 99 99 100 100 100 100 100 100 101

7. Obstetric Manifestations of the Antiphospholipid Syndrome Angela Tincani, Cecilia Nalli, Rossella Reggia, Sonia Zatti and Andrea Lojacono 7.1 Introduction 7.2 Recurrent Early Pregnancy Loss 7.3 Foetal Death 7.4 Preeclampsia and PI 7.5 Risk Stratification 7.6 Conclusions References

107 109 112 113 114 117 117

8. Thrombocytopenia in the Antiphospholipid Syndrome Serena Fasano and David A. Isenberg 8.1 History and Definition 8.2 Prevalence in Primary and Secondary APS

121 122

Contents  ix

8.3 Pathogenesis 8.4 Treatment 8.5 Immune Thrombocytopenic Purpura 8.6 Heparin-Induced Thrombocytopenia 8.7 Thrombotic Microangiopathy 8.8 Pseudothrombocytopenia 8.9 Disseminated Intravascular Coagulation References

122 123 125 125 126 126 127 127

9. Nonclassification Criteria Manifestations of the Antiphospholipid Syndrome Mohammad Hassan A. Noureldine and Imad Uthman 9.1 Introduction 9.2 Nonclassification Criteria Manifestations 9.2.1 Obstetrical Morbidity 9.2.2 Haematologic Manifestations 9.2.3 Dermatologic Manifestations 9.2.4 Cardiac Valve Disease 9.2.5 Renal Thrombosis 9.2.6 Neurological Manifestations (Migraine, Seizures, Cognitive Dysfunction, Chorea, Transverse Myelitis) References

131 133 133 135 136 137 137 138 139

10. Paediatric Antiphospholipid Syndrome Nataša Toplak and Tadej Avčin 10.1 Introduction 10.2 Epidemiology 10.2.1 Primary APS 10.2.2 Systemic Lupus Erythematosus 10.2.3 Other Diseases 10.2.4 Environmental Triggers of aPL 10.2.5 Healthy Children 10.3 Clinical Manifestations 10.3.1 Thromboses 10.3.2 Nonthrombotic Manifestations 10.3.3 Catastrophic APS 10.3.4 Neonatal APS 10.4 Differential Diagnosis 10.5 Treatment and Outcome 10.5.1 Primary Thrombosis Prophylaxis 10.5.2 Secondary Thrombosis Prophylaxis 10.5.3 Treatment of CAPS 10.5.4 Treatment of Neonatal APS References

145 146 146 146 147 147 148 149 149 151 153 155 156 156 156 157 158 158 159

x  Contents

11. Antiphospholipid Antibodies and Their Relationship With Infections, Vaccines, and Drugs Jiram Torres Ruiz, Miri Blank, Gisele Zandman-Goddard, Yaniv Sherer and Yehuda Shoenfeld 11.1 Introduction 11.2 aPL Associated With Infections 11.2.1 Viral Infections 11.2.2 Bacterial, Mycobacterial, Yeast and Parasitic Infections, and aPL 11.3 The Infection Origin of APS 11.4 aPL and Vaccination 11.5 Drug-Induced aPL 11.6 Summary References

167 168 168 169 171 173 174 174 175

12. Antiphospholipid Syndrome Associated With Malignancies Jose A. Gómez-Puerta and Wolfgang Miesbach 12.1 Introduction 12.2 Solid and Haematological Malignancies and aPL 12.3 Catastrophic APS and Malignancies 12.4 Therapeutic Aspects 12.5 Conclusion References

181 182 187 189 189 190

13.
 Antiphospholipid Antibodies and Atherosclerosis Joan T. Merrill 13.1 Introduction 13.2 Clinical Evidence for or Against a Relationship Between aPL and Atherosclerosis 13.3 Classic aPL: Can They Explain All Autoimmune Mechanisms for Atherosclerosis or Are They the Tip of an Iceberg? 13.4 Intravascular Autoantibodies: Evidence for Effects on Traditional Cardiovascular Risk Factors 13.5 Natural Autoantibodies: Can They Protect Against Atherosclerosis, and Are They Related to aPL? References

193 195 198 200 204 206

14. Global Antiphospholipid Syndrome Score Savino Sciascia, Munther Khamashta and Dario Roccatello 14.1 Introduction 14.2 The Global APS Score 14.3 External Validations of GAPSS 14.4 Conclusion References

215 216 217 218 218

Contents  xi

15. Primary Prophylaxis in Patients With Positive Antiphospholipid Antibodies María J. Cuadrado 15.1 Introduction 15.1.1 Risk of Thrombotic Event in aPL-Positive Patients 15.1.2 Therapeutic Approach to Carriers of aPL Without Previous Thrombosis 15.1.3 Recommendations 15.2 Conclusion References

221 222 224 226 227 227

16.
 Treatment of Thrombosis in Antiphospholipid Syndrome Guillermo Ruiz-Irastorza and Munther Khamashta 16.1 Prevention of Recurrent Thrombosis 16.2 Primary Thromboprophylaxis 16.3 New Anticoagulant Drugs 16.4 Other Therapies 16.5 Final Remarks References

232 234 237 237 238 238

17. Treatment of Catastrophic Antiphospholipid Syndrome Ignasi Rodríguez-Pintó, Gerard Espinosa and Ricard Cervera 17.1 Introduction 17.2 Current Approach 17.2.1 Supportive General Measures 17.2.2 Trigger-Guided Therapy 17.2.3 Specific Therapies 17.3 New Approaches 17.3.1 Rituximab 17.3.2 Eculizumab 17.3.3 New Oral Anticoagulants 17.4 Conclusions References

243 245 245 246 246 249 250 250 251 252 253

18. Treatment of Pregnancy Complications in Antiphospholipid Syndrome Anwar Nassar, Imad Uthman, Joe Eid and Munther Khamashta 18.1 Introduction 18.2 Obstetric Complications Associated With APS 18.3 Mechanisms for Adverse Pregnancy Outcomes 18.4 Therapeutic Options for Women With APS 18.4.1 Aspirin 18.4.2 Heparin 18.4.3 LMWH Versus UFH 18.4.4 Corticosteroids

257 257 258 260 261 262 263 263

xii  Contents 18.4.5 Intravenous Immunoglobulin 18.4.6 Warfarin 18.4.7 Plasmapheresis 18.4.8 Hydroxychloroquine 18.5 Management Algorithm 18.5.1 Preconception and Antepartum 18.5.2 Intrapartum Management 18.5.3 Postpartum Management 18.6 Management of Specific Complications 18.6.1 Catastrophic APS 18.6.2 Refractory Cases 18.6.3 Patients With SLE 18.7 Conclusions References

264 264 265 265 266 266 267 268 268 268 268 269 269 270

19. Prognosis in Antiphospholipid Syndrome Rosa M. Serrano, Guillermo J. Pons-Estel, Gerard Espinosa and Ricard Cervera 19.1 Introduction 281 19.2 Mortality and Morbidity in Patients With Long-Term Follow-Up APS 281 19.2.1 Mortality 282 19.2.2 Morbidity 283 19.2.3 Thrombosis and Rethrombosis 285 19.2.4 Haemorrhagic Complications 287 19.3 Obstetric APS 287 19.4 Catastrophic APS 290 References 290 Index 295

List of Contributors Olga Amengual Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Tatsuya Atsumi Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo, Japan Tadej Avcˇin Department of Allergology, Rheumatology and Clinical Immunology, University Children’s Hospital Ljubljana, University Medical Center, Ljubljana, Slovenia Maria L. Bertolaccini Graham Hughes Lupus Research Laboratory, Division of Women’s Health, King’s College London, The Rayne Institute, St Thomas’ Hospital, London, United Kingdom Miri Blank The Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Maria O. Borghi Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy; Experiment Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano, Milan, Italy Ricard Cervera Department of Autoimmune Diseases, Hospital Clínic, Barcelona, Catalonia, Spain Cecilia B. Chighizola Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy; Experiment Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano, Milan, Italy María J. Cuadrado Louise Coote Lupus Unit, Guy’s and St Thomas’ NHS Foundation Trust, King’s College London, London, United Kingdom Laura Durcan Division of Rheumatology, Department of Medicine, University of Washington, Seattle, WA, United States Joe Eid Department of Obstetrics and Gynecology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon Gerard Espinosa Department of Autoimmune Diseases, Hospital Clínic, Barcelona, Catalonia, Spain Serena Fasano Rheumatology Unit, Second University of Naples, Naples, Italy Mauro Galeazzi Dip. Di Medicina Clinica e Scienze Immunologiche sez. di Reumatologia, Policlinico Le Scotte, Università di Siena, Siena, Italy

xiii

xiv  List of Contributors Jose A. Gómez-Puerta Grupo de Inmunología Celular e Inmunogenética, Sede de Investigación Universitaria, Universidad de Antioquia, Medellín, Colombia David A. Isenberg Centre for Rheumatology, Department of Medicine, University College London, London, United Kingdom Annamaria Iuliano U.O.C. Reumatologia, Ospedale San Camillo-Forlanini, Roma, Italy Munther Khamashta Graham Hughes Lupus Research Laboratory, Division of Women’s Health, King’s College London; The Rayne Institute, St Thomas’ Hospital, London, United Kingdom Roger A. Levy Department of Rheumatology, Hospital Universitário Pedro Ernesto, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil Andrea Lojacono Maternal-Foetal Medicine Unit, Department of Obstetrics and Gynecology, Spedali Civili and University of Brescia, Brescia, Italy Pier L. Meroni Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy; Experiment Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano, Milan, Italy; Division of Rheumatology, Istituto Gaetano Pini-CTO, Milan, Italy Joan T. Merrill Oklahoma Medical Research Foundation, Oklahoma City, OK, United States Wolfgang Miesbach Medical Clinic III, Institute of Transfusion Medicine, Goethe University, Frankfurt, Germany Cecilia Nalli Rheumatology and Clinical Immunology, Spedali Civili and University of Brescia, Brescia, Italy Anwar Nassar Department of Obstetrics and Gynecology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon Mohammad Hassan A. Noureldine Gilbert and Rose-Marie Chagoury School of Medicine, Lebanese American University Medical Center, Beirut, Lebanon Michelle Petri Hopkins Lupus Center, Hopkins University School of Medicine, Baltimore, MD, United States Guillermo J. Pons-Estel Department of Autoimmune Diseases, Hospital Clínic, Barcelona, Catalonia, Spain Elena Raschi Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy Rossella Reggia Rheumatology and Clinical Immunology, Spedali Civili and University of Brescia, Brescia, Italy Joan C. Reverter Department of Hemostasis and Hemotherapy, Hospital Clínic, Barcelona, Catalonia, Spain Dario Roccatello Center of Research of Immunopathology and Rare DiseasesCoordinating Center of the Network for Rare Diseases of Piedmont and Aosta Valley, Department of Rare, Immunologic, Hematologic, and Immunohematologic Diseases, S. Giovanni Bosco Hospital, Turin, Italy

List of Contributors  xv

Ignasi Rodríguez-Pintó Department of Autoimmune Diseases, Hospital Clínic, Barcelona, Catalonia, Spain Jiram Torres Ruiz Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran, Mexico City, Mexico; The Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, TelHashomer, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Guillermo Ruiz-Irastorza Autoimmune Diseases Research Unit, Department of Internal Medicine, Biocruces Health Research Institute, Hospital Universitario Cruces, University of the Basque Country, Bizkaia, Spain Savino Sciascia Center of Research of Immunopathology and Rare DiseasesCoordinating Center of the Network for Rare Diseases of Piedmont and Aosta Valley, Department of Rare, Immunologic, Hematologic, and Immunohematologic Diseases, S. Giovanni Bosco Hospital, Turin, Italy Gian D. Sebastiani U.O.C. Reumatologia, Ospedale San Camillo-Forlanini, Roma, Italy Rosa M. Serrano Department of Autoimmune Diseases, Hospital Clínic, Barcelona, Catalonia, Spain Yaniv Sherer The Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel; Barzilai Medical Center, Ashkelon, Faculty of Health Sciences, Ben-Gurion University of the Negev, Israel Yehuda Shoenfeld The Zabludowicz Center for Autoimmune Diseases, Sheba Medical Center, Tel-Hashomer, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Angela Tincani Rheumatology and Clinical Immunology, Spedali Civili and University of Brescia, Brescia, Italy Nataša Toplak Department of Allergology, Rheumatology and Clinical Immunology, University Children’s Hospital Ljubljana, University Medical Center, Ljubljana, Slovenia Imad Uthman Division of Rheumatology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon Gisele Zandman-Goddard Department of Medicine C, Wolfson Medical Center, Hospital Management, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv, Israel Sonia Zatti Maternal-Foetal Medicine Unit, Department of Obstetrics and Gynecology, Spedali Civili and University of Brescia, Brescia, Italy

Foreword Much has been learnt about the antiphospholipid syndrome (APS) since the publication of the first edition of this book. Advances have been made in the understanding of the mechanisms of thrombosis, including the relevance of the structural properties of β2-glycoprotein I (antibodies against its domain 1 may prove more prothrombotic) and of the involvement of complement, for example. Broader aspects of antiphospholipid antibody (aPL)-induced thrombosis have been explored – the role of the ‘two-hit’ phenomenon and genetic aspects, including the links with other autoimmune diseases such as Sjögren’s and Hashimoto’s. Critical advances reported in detail in this edition include the recognition of the importance of APS in the world of Neurology (including lesser known aspects such as autonomic neuropathy, temporal lobe epilepsy, and sleep disturbance). Likewise, in Cardiology the risk of cardiac ischaemia in APS is now more in focus. It seems a safe bet that aPL testing will become pivotal in the work-up of females aged 45 or younger with angina. Many clinical enigmas remain – the clinical differentiation of APS from multiple sclerosis and the extent of the link between aPL and bone fractures, or the unresolved issues of APS pregnancy (the links to stillbirth, the wisdom of waiting for three miscarriages before aPL testing, to mention two). In treatment the advent of new oral anticoagulants has widened our armamentarium, though these are early days as far as clinical experience is concerned. For me, one of the key issues is the patient with ‘seronegative APS’. The clinical response in these patients following anticoagulation is often as dramatic or successful as in aPL-positive patients. Such cases beg the question of how many ‘seronegative APS’ go unrecognized in migraine clinics, in obstetric practice, and, for example, in younger female cardiology clinics. I am positive that many of these questions will become answered by the atmosphere of collaboration that exists between those studying APS. Drs Cervera, Espinosa, and Khamashta have highlighted this collaboration with the stellar list of contributors to this volume. Graham Hughes London Lupus Centre ([email protected]) 2 Mar. 2016

xvii

Dedication Dr. Ricard Cervera wishes to dedicate this book to his wife Carme and his daughters Marta and Laura; Dr. Gerard Espinosa, to his wife Susanna and his daughters Júlia and Èlia; and Dr. Munther Khamashta, to his wife Francesca.

xix

Chapter 1

History, Classification, and Subsets of the Antiphospholipid Syndrome Roger A. Levya, Jose A. Gómez-Puertab and Ricard Cerverac a

Department of Rheumatology, Hospital Universitário Pedro Ernesto, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil bGrupo de Inmunología Celular e Inmunogenética, Sede de Investigación Universitaria, Universidad de Antioquia, Medellín, Colombia c Department of Autoimmune Diseases, Hospital Clínic, Barcelona, Catalonia, Spain

1.1 INTRODUCTION Antiphospholipid syndrome (APS) is defined by the occurrence of venous and arterial thromboses (often multiple) and pregnancy morbidity (abortions, foetal deaths, premature births), in the presence of antiphospholipid antibodies (aPL); namely, lupus anticoagulant (LA), anticardiolipin antibodies (aCL), or anti-β2 glycoprotein-I (anti-β2GPI) antibodies. APS can occur in patients having neither clinical nor laboratory evidence of another definable condition (primary or isolated APS), or it may be associated with other diseases, mainly systemic lupus erythematosus (SLE), and occasionally with other autoimmune conditions, infections, drugs, and malignancies. Rapid chronological occlusive events, occurring over days to weeks, have been termed as catastrophic APS (CAPS). Other postulated APS subsets include microangiopathic APS (MAPS) and seronegative APS.

1.2  HISTORICAL PERSPECTIVE Wassermann et al. [1] discovered reagin, an antibody against an antigen located in alcohol extracts of congenital syphilis foetal liver cells. Pangborn [2] showed that this antigen was a phospholipid named cardiolipin. The use of the mixture of cardiolipin with phosphatidylcholine and cholesterol led to the development of various precipitation complement fixation techniques to detect reagin. Antiphospholipid Syndrome in Systemic Autoimmune Diseases. DOI: http://dx.doi.org/10.1016/B978-0-444-63655-3.00001-6 © 2017 2016 Elsevier B.V. All rights reserved.

1

2  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

During World War II, individuals with positive serologic results for syphilis, with no clinical disease evidence, were identified. It became apparent that false-positive results might occur, usually as a result of an acute infection such as malaria or endocarditis. In 1955, it was shown that patients with endocarditis had a high incidence of SLE [3]. In 1952, an in vitro coagulation inhibitor was found in two SLE patients. This inhibitor was associated with false-positive serologic test results for syphilis and could be absorbed from plasma by phospholipids [4]. In 1972, this phenomenon was named LA, and although LA acts as an anticoagulant in vitro, it is associated with thrombotic events in vivo [5]. In the early 1980s, studies at the London Hammersmith Hospital by G.R.V. Hughes and colleagues led to the development of solid-phase immunoassays to detect aCL [6]. A high correlation between the IgG aCL and thrombosis was documented and a close relationship between aCL and LA was also demonstrated [7]. These findings led to the recognition of the so-called anticardiolipin syndrome, which was later named the APS [8]. In 1987, the group recognized that some individuals without lupus or antinuclear antibodies (ANA) developed APS. They were classified as having a primary APS (PAPS) [9], also known as isolated APS. The formal descriptions of PAPS were published simultaneously by two groups in 1989 [10,15]). At the 2007 International aPL Congress, held in Florence, Italy, it was established that PAPS will be called APS and the ‘secondary’ form will be called APS, associated with the other systemic autoimmune disorder. A major advance came in the early 1990s with the concomitant recognition by three groups that aPL required a plasma protein ‘cofactor’ (β2GPI) to bind to cardiolipin on enzyme-linked immunosorbent assay (ELISA) plates. Since then, other cofactors, including prothrombin (PT), have been described. In 1992, Asherson described a subset of patients with widespread coagulopathy affecting predominantly small vessels that led to rapid multiorgan failure, named CAPS [11].

1.3  CLASSIFICATION OF APS In 1998, a preliminary classification criteria was proposed by an expert workshop held in Sapporo, Japan [12] after the 8th International Congress. The need for consensus on an APS criteria was highlighted by the diversity of clinical and basic science disciplines that contribute to its diagnosis and treatment. Subsequently, in 2004, a criteria workshop was held in Sydney, Australia, during the 11th International Congress, and modifications were proposed, such as the inclusion of anti-β2GPI antibodies and the laboratory confirmation within 12 weeks. Although no new clinical criteria were added, some particular APS features were highlighted, such as cardiac valve involvement, livedo reticularis, thrombocytopenia, APS nephropathy and nonthrombotic central nervous system manifestations (Table 1.1) [13]. The international Task Force initiative that

History, Classification, and Subsets of the APS  Chapter | 1  3

TABLE 1.1 Revised Classification Criteria for the APS Clinical Criteria 1. Vascular Thrombosisa One or more clinical episodes of arterial, venous, or small-vessel thrombosis in any tissue or organ. Thrombosis must be confirmed by imaging or Doppler studies or histopathology, with the exception of superficial venous thrombosis. For histopathology, thrombosis should be present without significant evidence of inflammation in the vessel wall. 2. Pregnancy Morbidity a. One or more unexplained deaths of a morphologically normal foetus at or beyond the 10th week of gestation, with normal foetal morphology documented by ultrasound or by direct examination of the foetus, or; b. One or more premature births of a morphologically normal neonate before the 34th week of gestation because of (i) eclampsia or severe preeclampsia defined according to standard definitions, or (ii) recognized features of placental insufficiency,b or; c. Three or more unexplained consecutive spontaneous abortions before the 10th week of gestation, with maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded. In studies of populations of patients who have more than one type of pregnancy morbidity, investigators are strongly encouraged to stratify groups of subjects according to (a), (b), or (c). Laboratory Criteriac 1. Anticardiolipin antibody of IgG and/or IgM isotype in serum or plasma, present in medium or high titre (ie, >40 GPL or MPL, or >the 99th percentile, or >mean + 3SD of 40 healthy controls), on two or more occasions, at least 12 weeks apart, measured by a standardized enzyme-linked immunosorbent assay. 2. Lupus anticoagulant present in plasma, on two or more occasions at least 12 weeks apart, detected according to the guidelines of the International Society on Thrombosis and Hemostasis (Scientific Subcommittee on Lupus Anticoagulants/ Phospholipid-Dependent Antibodies). 3. Anti-β2 glycoprotein-I antibody of IgG and/or IgM isotype in serum or plasma, present on two or more occasions, at least 12 weeks apart, measured by a standardized ELISA, according to recommended procedures. 4. Definite APS is present if at least one clinical criteria and onec laboratory criteria are met, with the first measurement of the laboratory test performed at least 12 weeks from the clinical manifestation.d (Continued)

4  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

TABLE 1.1 Revised Classification Criteria for the APS (Continued) a

Coexisting inherited or acquired factors for thrombosis are not reasons for excluding patients from APS trials. However, two subgroups of APS patients should be recognized, according to (1) the presence and (2) the absence of additional risk factors for thrombosis. Indicative (but not exhaustive) such cases include age (>55 in men, and >65 in women), and the presence of any of the established risk factors for cardiovascular disease (hypertension, diabetes mellitus, elevated low-density lipoprotein (LDL) or low high-density lipoprotein (HDL) cholesterol, cigarette smoking, family history of premature cardiovascular disease, body mass index ≥30 kg/m2, microalbuminuria, estimated glomerular filtration rate (GFR) 1:320). Antimitochondrial antibodies (M5 type) were present in 11/40 patients, and 60 patients had LA and aCL, 5 had aCL alone, and 5 had only LA. Thrombocytopenia was present in 32 patients (46%) and Coombs test in 10 (14%), with autoimmune haemolytic anaemia in 3 cases (4%). Only 5 of 70 patients had relatives with SLE, rheumatoid arthritis (RA) or a clotting tendency. When compared to SLE-associated APS, PAPS patients had less valve lesions, livedo reticularis, chorea, fever, myalgia, and arthralgia. The presence of rheumatoid factor, cryoglobulinemia or low complement in a minority of patients also might be indicative of an immune-mediated mechanism in isolated APS. Piette et al. [16] proposed exclusion criteria to distinguish isolated APS from SLE-related APS. The presence of any of the following criteria excludes the diagnosis of PAPS: malar rash, discoid rash, oral or pharyngeal ulceration, frank arthritis, pleuritis in the absence of PE or left-sided heart failure, pericarditis in the absence of MI or uraemia, proteinuria greater than 0.5 g/day due to biopsyproven immune-complex-related glomerulonephritis, lymphopenia (1:320 and drug treatment. The authors proposed that a follow-up of more than 5 years after the first clinical manifestation is necessary to rule out the subsequent emergence of SLE. Vianna et al. [17] analysed the differences between PAPS and SLE-related APS. A total of 56 patients had APS plus SLE, and 58 had PAPS. There were no significant differences between the two groups with the exception of autoimmune haemolytic anaemia (p < 0.05), cardiac valve disease (p < 0.005), neutropenia (p < 0.01), and low C4 (p < 0.001); all were more frequent in SLErelated APS. Soltesz et al. [18] studied a large APS cohort; PAPS in 218 patients and SLE-associated APS in 288 subjects. There were significantly more men among the PAPS. Cerebrovascular thrombosis was significantly more frequent with SLE-related APS sufferers (128/288) than among PAPS patients (77/218). No differences were found between the two groups in the occurrence of DVT, coronary, carotid, and peripheral arterial thrombosis, as well as foetal losses. The frequency of LA, IgG, and IgM aCL was similar. PAPS is rare in children, and little information exists on its potential evolution into SLE. Gattorno et al. [19] reported one of the few series in children; they described 14 patients (9 boys) who presented with clinical APS between 3 and 13 years (median, 9 years) and were followed for 2–16 years (median, 6 years). A total of 6 patients presented with DVT, 5 with stroke, 2 with peripheral artery occlusion, 1 with Budd–Chiari’s syndrome and 1 with MI. During followup, 4 patients had one or more thrombosis recurrences. At the last observation,

6  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

10 patients could still be classified as PAPS, 2 had developed SLE (and both had anti-dsDNA), 1 had lupuslike syndrome and 1 had Hodgkin’s lymphoma 4 years after the PAPS onset. The authors suggested that some children who present with features of PAPS may progress to SLE or lupuslike syndrome. However, PAPS rarely progresses to SLE. Only 8% of 128 patients, followed up for about 9 years, developed lupus, a positive Coombs test was a clinically significant predictor of progression. Gómez-Puerta et al. [20] described 16 cases (11 with SLE and 5 with lupuslike syndrome) who developed clinical or serologic features of a ‘new’ autoimmune disease after long-term follow-up and 1 who developed myasthenia gravis. There are more than 30 reported cases whose PAPS evolved into SLE or lupuslike disease [19,21–27]. The percentage of progression to SLE or lupuslike disease (21.4%) observed in paediatric patients [19] followed for a median of 6 years is almost double that found in the adults with PAPS. Despite the clinical heterogeneity APS, some features can be grouped into different clusters. Krause et al. [28] analysed 246 patients with APS and, after stratification and statistical analysis (by factor analysis), found different clusters for APS. The first group is characterized by cardiac valve abnormalities, livedo reticularis, and neurologic manifestations (epilepsy and migraine). The second presents with arthritis, thrombocytopenia, and leukopenia. The third presents with recurrent foetal loss and intrauterine growth restriction; and the fourth illustrates the inverse correlation between arterial and venous thrombosis. The authors suggested that once any of these features or lesions is recognized in a patient, special attention should be paid to the future emergence of other manifestations of the cluster.

1.5  APS ASSOCIATED WITH OTHER DISEASES APS was first recognized in patients with SLE and later, in a lower frequency, in other autoimmune diseases. Additionally, aPL are occasionally found in individuals with a series of chronic infections or malignancies, or they can be induced by drugs. In a study of 552 randomly selected blood donors, IgG aCL was present in up to 9.4% in a first test and persisted in approximately 1.4% [29]. Increased levels of IgG or IgM aCL have been observed in 12–52% of the elderly. The prevalence of the LA has ranged from 1.7% of patients with suspected DVT who did not have the disease to 8% in blood donors [30]. In addition, aPL occur with increased frequency (10–15%) in women with more than three spontaneous recurrent abortions [31].

1.5.1  APS Associated With Autoimmune Diseases Both LA and aCL have been found in a variety of autoimmune and rheumatic diseases [32–34], including haemolytic anaemia; idiopathic thrombocytopenic purpura (up to 30%) [35]; juvenile arthritis (28–46%) [36]; RA (7–50%) [37];

History, Classification, and Subsets of the APS  Chapter | 1  7

psoriatic arthritis (28%) [38]; systemic sclerosis (25%), especially with severe disease [39]; Behçet’s syndrome (7–20%) [40]; Sjögren’s syndrome (25–42%) [41,42]; mixed connective tissue disease (22%) [43]; polymyositis/dermatomyositis [44]; polymyalgia rheumatica (20%) [44]; chronic discoid lupus [45]; eosinophilia myalgia [46]; vasculitis [47]; and autoimmune thyroid disease (43%) [48].

1.5.2  APS Associated With Infections Since 1983, many infections have been found to be associated with aPLpositivity, although the pathogenic role of these antibodies was not usually obvious. However, there have been reports that many infections may not only trigger aPL production, but also appear to be accompanied by APS clinical manifestations [49–51], particularly in CAPS [52]. Some authors have proposed that infections may trigger the induction of pathogenic aPL in predisposed subjects. The β2GPI induced by infections may bind to self/physiological aPL, thus forming an immunogenic complex. What constitutes this predisposition is unknown at this time, but clearly genetic factors might play a role. The antibodies produced by infectious triggers are, therefore, heterogeneous in their dependency on β2GPI, and a minority may behave as the autoimmune type [53]. Infectious agents may induce autoimmune disease by several mechanisms; for aPL production, molecular mimicry may play a role. A hexapeptide, TLRVYK, recognized specifically by a pathogenic anti-β2GPI monoclonal antibody, was identified [54]. The authors evaluated the pathogenic potential of this hexapeptide by infusing IgG specific to the peptide intravenously into naïve mice. High levels of anti-β2GPI were seen in mice immunized with Haemophilus influenzae, Neisseria gonorrhoeae, and tetanus toxoid. Significant thrombocytopenia, prolonged activated partial thromboplastin time and increased foetal loss followed. Thus, an experimental APS can be induced by immunization with certain microbial pathogens that share epitope homology with β2GPI [54]. Cervera et  al. [55] described the clinical and serological characteristics of 100 patients with APS related to infections; 59% were female. Their mean age was 32 ± 18 years (range 1–78). There were 24 young patients (under 18 years), who were affected mainly by skin and respiratory infections. A total of 68 patients had PAPS, 27 SLE, 2 lupuslike disease, 2 inflammatory bowel disease, and 1 RA. In 40/100 cases, the thrombotic events presented as CAPS. The main clinical manifestations of APS included pulmonary involvement (39%), skin (36%), and renal (35%; 9 with renal MAPS). The main associated infections and agents included skin infection (18%), human immunodeficiency virus (17%), pneumonia (14%), hepatitis C virus (13%), urinary tract infection (10%), upper respiratory infection (9%), sepsis (6%), and gastrointestinal infection (6%), among others. It is also well known that infections are common triggers of CAPS. The CAPS Registry (an international registry of patients with CAPS created in 2000

8  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

by the European Forum on aPL, http://ontocrf.costaisa.com/es/web/caps) shows that at least 60% of patients appear to have developed CAPS following an identifiable trigger factor, with infections dominating the list. These include nonspecific viral infections, pneumonia, infected leg ulcers, upper respiratory, urinary, gastrointestinal, and cutaneous infections, as well as specific infections such as typhoid fever, malaria, and dengue fever [52].

1.5.3  APS Associated With Drugs Several drugs have been implicated as potential inducers of aPL/APS, including phenothiazines (chlorpromazine), phenytoin, hydralazine, procainamide, quinidine, dilantin, ethosuximide, alfa interferon, amoxicillin, chlorothiazide, oral contraceptives, and propranolol [56,57]. Tumour necrosis factor (TNF) inhibitors may induce autoantibodies such as ANA, anti-dsDNA, and aCL. One possible explanation for the induction of aCL positivity in patients treated with anti-TNF is that downregulation of TNF leads to upregulation of IL-10, which in turn activates autoreactive B cells and thus induces autoantibody production [58]. Ferraccioli et al. [59] studied the induction of aCL in 8 RA patients treated with etanercept for 85 weeks. In the study, 5 patients presented with increased IgG aCL levels, while anti-dsDNA became positive in 3 of the 8 patients. The authors noted that the autoantibodies’ appearance correlated with bacterial urinary or upper respiratory tract infections and that antibiotic treatment restored aCL to normal levels. Bobbio-Pallavicini et al. [60] studied 39 RA patients on infliximab over 78 weeks and found a significant increase in aCL, starting at 30 weeks for IgM and at 78 weeks for IgG. However, in most cases, the levels did not exceed normal limits, even after 78 weeks, and no one developed APS.

1.5.4  APS Associated With Malignancies Since the discovery of aCL, there have been many case reports of its association with vascular events in patients with malignant conditions, including solid tumours, and lymphoproliferative and haematological malignancies [61,62]. Further data on aPL and malignancies is explained with more detail in Chapter 12.

1.6  SERONEGATIVE APS APS is defined by the occurrence of thrombotic events or obstetrical complications in patients with persistent aPL (either LA, aCL, anti-β2GPI, or a combination). However, some patients exhibit clinical features of APS like livedo reticularis, recurrent pregnancy losses, DVT and thrombocytopenia, with negative classical aPL on several occasions. The absence of aPL in symptomatic patients has led some to propose the term seronegative APS. This term was first

History, Classification, and Subsets of the APS  Chapter | 1  9

introduced in 2003 by Hughes and Khamashta [63] to describe patients with clinical manifestations highly suggestive of APS, but with persistently negative routine aPL. Some potential explanations for seronegative APS include (1) antibody consumption during an acute thrombotic episode, (2) transient negativity of previously positive aPL patients (unlikely), and (3) a more realistic one: antibodies to the heterogeneous aPL family against protein and protein-bound phospholipids which have not been identified to date. The most promising of this aPL family are antibodies to phospholipid-binding plasma proteins (prothrombin, protein C, protein S, annexin V, and domains of β2GPI) [64]; phospholipidprotein complexes (vimentin/cardiolipin complex); and anionic phospholipids other than cardiolipin (phosphatidylserine (PS), phosphatidylinositol (PI)) [65]; and antibodies to the complex PS/PT [66].

1.7  CATASTROPHIC APS The catastrophic variant of APS is an accelerated form of APS that results in multiorgan failure due to multiple small-vessel occlusions. Since the description by Asherson [11], more than 500 cases have been collected in the CAPS Registry. Patients with CAPS, also known as Asherson’s syndrome [67] have the following in common: (1) clinical evidence of multiple organ involvement developing over a very short period of time, (2) histopathological evidence of multiple small-vessel occlusions, and (3) laboratory confirmation of the presence of aPL. Most of these catastrophic episodes are preceded by a precipitating event (mainly infections), but also malignancies, trauma or surgical procedures; anticoagulation withdrawal; contraceptive use; or during pregnancy and the puerperium [68–72]. The heterogeneity of the different presentation forms led to consensus criteria for the definition and classification of these patients at a presymposium workshop held during the 10th International Congress in Taormina, Italy, in 2002 (Table 1.2) [73], which were later validated [74]. From the analysis of the initial 176 patients of the CAPS Registry, 89 (51%) were classified as having ‘definite’, and 70 (40%) as ‘probable’ CAPS. The sensitivity of the criteria was 90.3% and the specificity 99.4%. Positive and negative predictive values were 99.4% and 91.1%, respectively [74]. Patients may develop CAPS de novo, without any previous history of thrombosis associated with either PAPS or SLE. However, it has been shown that previous DVT, foetal loss, or thrombocytopenia are the most frequent preexisting aPL-associated manifestations.

1.8 INTERNATIONAL aPL/APS CONGRESSES Since the first meeting in 1984, the International aPL/APS Congresses have gathered increasing numbers of participants and presented the latest findings in

10  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

TABLE 1.2 Preliminary Criteria for the Classification of CAPS 1. Evidence of involvement of three or more organs, systems, or tissuesa 2. Development of manifestations simultaneously or in less than a week 3. Confirmation by histopathology of small-vessel occlusion in at least one organ or tissueb 4. Laboratory confirmation of the presence of aPL (LA, aCL, or both)c Definite CAPS: All four criteria Probable CAPS: All four criteria, except for only two organs, systems, or tissues involved. All four criteria, except for the absence of laboratory confirmation at least 6 weeks apart due to the early death of a patient never tested for aPL before the CAPS. Criteria 1, 2, and 4. Criteria 1, 3, and 4 and the development of a third event in more than a week but less than a month, despite anticoagulation. a Usually, clinical evidence of vessel occlusions, confirmed by imaging techniques when appropriate. Renal involvement is defined by a 50% rise in serum creatinine, severe systemic hypertension (>180 × 100 mmHg), or proteinuria (>500 mg/24 h). b For histopathological confirmation, significant evidence of thrombosis must be present, although vasculitis may coexist occasionally. c If the patient had not been previously diagnosed as having APS, the laboratory confirmation requires that the presence of aPL must be detected on two or more occasions at least 6 weeks apart (not necessarily at the time of the event), according to the proposed preliminary criteria for the classification of definite APS.

the field. It is intended to be a multidisciplinary meeting, involving basic and clinical sciences related to aPL and APS (Table 1.3). At the 13th International Congress in Galveston, Texas, in 2010, collaborative task forces were created, and their work is ongoing [75]. The task forces gathered again at the 14th International Congress in Rio de Janeiro, Brazil, and used the GRADE methodology to review the literature and the expert opinions on the five major areas of the field that led to articles in Autoimmunity Reviews on clinical characteristics [76], laboratory new tests for diagnostic [77], obstetrical complications [78], CAPS [79], and future treatment [80]. The ongoing international collaboration will lead to an update of the classification criteria and several new findings that we hope to see presented at the upcoming 15th International Congress in Istanbul, Turkey, in Sep. 2016.

1.9  APS ACTION In 2011, an international group, APS ACTION, gathered with the aim of planning several studies on aPL-related syndromes. Its primary mission involves the prevention, treatment, and cure of aPL-associated clinical manifestations through high-quality, multicentre, and multidisciplinary clinical research. It was initialized by the creation of a prospective registry, where 1000 aPL carriers and APS patients will be enroled and clinical and laboratory data are being collected. More than 500 patients are already included. Several centres are currently recruiting participants for a prospective open-label study on the effects

TABLE 1.3 International Antiphospholipid Congresses Event

Year

City, Country

Participants/Abstracts

Organizer(s)

I

1984

London, United Kingdom

120/60

G.R.V. Hughes, E.N. Harris, and A.E. Gharavi

II

1986

London, United Kingdom

100/80

G.R.V. Hughes, E.N. Harris, and A.E. Gharavi

III

1988

Kingston, Jamaica

120/120

E.N. Harris

IV

1990

Sirmione, Italy

200/150

A. Tincani, P.-L. Meroni, and G. Balestrieri

V

1992

San Antonio, TX, United States

–/–

R.L. Brey

VI

1994

Leuven, Belgium

–/–

J. Arnout

VII

1996

New Orleans, LA, United States

350/222

W. Wilson and A.E. Gharavi

VIII

1998

Sapporo, Japan

350/220

T. Koike

IX

2000

Tours, France

–/–

M.-C. Boffa and J.C. Piette

X

2002

Taormina, Italy

730/600

Y. Shoenfeld

XI

2004

Sydney, NSW, Australia

350/199

S. Krilis

XII

2007

Florence, Italy

500/250

A. Tincani and P.-L. Meroni

XIII

2010

Galveston, TX, United States

280/157

S.S. Pierangeli and R.L. Brey

XIV

2013

Rio de Janeiro, Brazil

780/330

R.A. Levy and Y. Shoenfeld

XV

2016

Istanbul, Turkey

Upcoming

D. Erkan

12  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

of hydroxychloroquine on primary thrombosis prophylaxis in persistently aPLpositive but thrombosis-free patients without systemic autoimmune diseases (ClinicalTrials.gov Identifier: NCT01784523).

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History, Classification, and Subsets of the APS  Chapter | 1  13 [19] Gattorno M, Falcini F, Ravelli A, Zulian F, Buoncompagni A, Martini G, et al. Outcome of primary antiphospholipid syndrome in childhood. Lupus 2003;12:449–53. [20] Gómez-Puerta JA, Martín H, Amigo MC, Aguirre MA, Camps MT, Cuadrado MJ, et al. Longterm follow-up in 128 patients with primary antiphospholipid syndrome: do they develop lupus? Medicine (Baltimore) 2005;84:225–30. [21] Blanco Y, Ramos-Casals M, Garcia-Carrasco M, Cervera R, Font J, Ingelmo M. Sindrome antifosfolipidico primario que evoluciona a lupus eritematoso sistemico: presentacion de tres nuevos casos y revision de la literatura. Rev Clin Esp 1999;199:586–8. [22] Carbone J, Orera M, Rodriguez-Mahou M, Rodriguez- Perez C, Sanchez-Ramon S, Seoane E, et al. Immunological abnormalities in primary APS evolving into SLE: 6 years follow-up in women with repeated pregnancy loss. Lupus 1999;8:274–8. [23] Derksen RHWM, Gmelig-Meijling FHJ, de Groot PG. Primary antiphospholipid syndrome evolving into systemic lupus erythematosus. Lupus 1996;5:77–80. [24] Asherson RA, Baguley E, Pal C, Hughes GRV. Antiphospholipid syndrome: five years follow up. Ann Rheum Dis 1991;50:805–10. [25] Mujic F, Cuadrado MJ, Lloyd M, Khamashta MA, Page G, Hughes GRV. Primary antiphospholipid syndrome evolving into systemic lupus eryhtematosus. J Rheumatol 1995;22:1589–92. [26] Queiro R, Weruaga A, Riestra JL. C4 deficiency state in antiphospholipid antibodyrelated recurrent preeclampsia evolving into systemic lupus erythematosus. Rheumatol Int 2002;22:126–8. [27] Seisedos L, Muñoz-Rodriguez FJ, Cervera R, Font J, Ingelmo M. Primary antiphospholipid syndrome evolving into systemic lupus erythematosus. Lupus 1997;6:285–6. [28] Krause I, Leibovici L, Brank M, Shoenfeld Y. Clusters of disease manifestations in patients with antiphospholipid syndrome demonstrated by factor analysis. Lupus 2007;16:176–80. [29] Vila P, Hernandez MC, Lopez-Fernandez MF, Battle J. Prevalence, follow-up and clinical significance of the anticardiolipin antibodies in normal subjects. Thromb Haemost 1994;72:209–13. [30] Shi W, Krilis SA, Chong BH, Gordon S, Chesterman CN. Prevalence of lupus anticoagulant in a healthy population: lack of correlation with anticardiolipin antibodies. Aust NZ J Med 1990;20:231–6. [31] Melk A, Mueller Eckhardt G, Polten B, Lattermann A, Heine O, Hoffmann O. Diagnostic and prognostic significance of anticardiolipin antibodies in patients with recurrent spontaneous abortions. Am J Reprod Immunol 1995;33:228–33. [32] Cervera R, Asherson RA. Clinical and epidemiological aspects in the antiphospholipid syndrome. Immunobiology 2003;207:5–11. [33] McNeil HP, Chesterman CN, Krilis SA. Immunology and clinical importance of antiphospholipid antibodies. Adv Immunol 1991;49:193–280. [34] Sebastiani GD, Galeazzi M, Tincani A, Piette JC, Font J, Allegri F, et  al. Anticardiolipin and anti-beta2GPI antibodies in a large series of European patients with systemic lupus erythematosus. Prevalence and clinical associations. European Concerted Action on the Immunogenetics of SLE. Scand J Rheumatol 1999;28:344–51. [35] Bidot CJ, Jy W, Horstman LL, Ahn ER, Yaniz M, Ahn YS. Antiphospholipid antibodies (APLA) in immune thrombocytopenic purpura (ITP) and antiphospholipid syndrome (APS). Am J Hematol 2006;81:391–6. [36] Avcin T, Ambrozic A, Bozic B, Acceto M, Kveder T, Rozman B. Estimation of anticardiolipin antibodies, anti-beta2 glycoprotein I antibodies and lupus anticoagulant in a prospective longitudinal study of children with juvenile idiopathic arthritis. Clin Exp Rheumatol 2002;20:101–8.

14  Antiphospholipid Syndrome in Systemic Autoimmune Diseases [37] Olech E, Merrill JT. The prevalence and clinical significance of antiphospholipid antibodies in rheumatoid arthritis. Curr Rheumatol Rep 2006;8:100–8. [38] Buchanan RR, Warlaw JR, Riglar AG, Littlejohn GO, Miller MH. Antiphospholipid antibodies in the connective tissue diseases: their relation to the antiphospholipid syndrome and forme fruste disease. J Rheumatol 1989;16:757–61. [39] Picillo U, Migliaresi S, Marcialis MR, Ferruzzi AM, Tirri G. Clinical significance of anticardiolipin antibodies in patients with systemic sclerosis. Autoimmunity 1995;20:1–8. [40] Tokay S, Direskeneli H, Yurdakul S, Akoglu T. Anticardiolipin antibodies in Behcet’s disease: a reassessment. Rheumatology (Oxford) 2001;40:192–5. [41] Fauchais AL, Lambert M, Launay D, Michon-Pasturel U, Queyrel V, Nguyen N, et  al. Antiphospholipid antibodies in primary Sjogren’s syndrome: prevalence and clinical signific� cance in a series of 74 patients. Lupus 2004;13:245–8. [42] Ramos-Casals M, Nardi N, Brito-Zeron P, Aguilo S, Gil V, Delgado G, et al. Atypical autoantibodies in patients with primary Sjogren syndrome: clinical characteristics and follow-up of 82 cases. Semin Arthritis Rheum 2006;35:312–21. [43] Sherer Y, Livneh A, Levy Y, Shoenfeld Y, Langevitz P. Dermatomyositis and polymyositis associated with the antiphospholipid syndrome – a novel overlap syndrome. Lupus 2000;9:42–6. [44] Chakravarty K, Pountain G, Merry P, Byron M, Hazleman B, Scott DG. A longitudinal study of anticardiolipin antibody in polymyalgia rheumatica and giant cell arteritis. J Rheumatol 1995;22:1694–7. [45] Ruffatti A, Veller-Fornasa C, Patrassi GM, Sartori E, Tonello M, Tonetto S, et  al. Anticardiolipin antibodies and antiphospholipid syndrome in chronic discoid lupus erythematosus. Clin Rheumatol 1995;14:402–4. [46] Carreira PE, Montalvo MG, Kaufman LD, Silver RM, Izquierdo M, Gomez-Reino JJ. Antiphospholipid antibodies in patients with eosinophilia myalgia and toxic oil syndrome. J Rheumatol 1997;24:69–72. [47] Rees JD, Lanca S, Marques PV, Gómez Puerta JA, Moco R, Oliveri C, et al. Prevalence of the antiphospholipid syndrome in primary systemic vasculitis. Ann Rheum Dis 2006;65:109–11. [48] Nabriski D, Ellis M, Ness-Abramof R, Shapiro M, Shenkman L. Autoimmune thyroid disease and antiphospholipid antibodies. Am J Hematol 2000;64:73–5. [49] Galrao L, Brites C, Atta ML, Atta A, Lima I, Gonzalez F, et al. Antiphospholipid antibodies in HIV-positive patients. Clin Rheumatol 2007;26:1825–30. [50] Ramos-Casals M, Cervera R, Lagrutta M, Medina F, Garcia-Carrasco M, de la Red G, et al. Clinical features related to antiphospholipid syndrome in patients with chronic viral infections (hepatitis C virus/HIV infection): description of 82 cases. Clin Infect Dis 2004;38:1009–116. [51] Uthman IW, Gharavi AE. Viral infections and antiphospholipid antibodies. Semin Arthritis Rheum 2002;31:256–63. [52] Rojas-Rodrıguez J, Garcıa-Carrasco M, Ramos-Casals M, Enriquez-Coronel G, Colchero C, Cervera R, et al. Catastrophic antiphospholipid syndrome: clinical description and triggering factors in 8 patients. J Rheumatol 2000;27:238–40. [53] Asherson RA, Cervera R. Antiphospholipid antibodies and infections. Ann Rheum Dis 2003;62:388–93. [54] Blank M, Krause I, Fridkin M, Keller N, Kopolovic J, Goldberg I, et al. Bacterial induction of autoantibodies to beta2-glycoprotein-1 accounts for the infectious etiology of antiphospholipid syndrome. J Clin Invest 2002;109:797–804. [55] Cervera R, Asherson RA, Acevedo ML, Gómez-Puerta JA, Espinosa G, De La Red G, et al. Antiphospholipid syndrome associated with infections: clinical and microbiological characteristics of 100 patients. Ann Rheum Dis 2004;63:1312–17.

History, Classification, and Subsets of the APS  Chapter | 1  15 [56] Merrill JT, Shen C, Gugnani M, Lahita RG, Mongey AB. High prevalence of antiphospholipid antibodies in patients taking procainamide. J Rheumatol 1997;24:1083–8. [57] Triplett DA. Many faces of lupus anticoagulants. Lupus 1998;7(Suppl. 2):S18–22. [58] Atzeni F, Turiel M, Capsoni F, Doria A, Meroni P, Sarzi-Puttini P. Autoimmunity and antiTNF-alpha agents. Ann NY Acad Sci 2005;1051:559–69. [59] Ferraccioli GF, Mecchia F, Di Poi E, Fabris M. Anticardiolipin antibodies in rheumatoid patients treated with etanercept or conventional combination therapy: direct and indirect evidence for a possible associations with infections. Ann Rheum Dis 2002;61:358–61. [60] Bobbio-Pallavicini F, Alpini C, Caporali R, Avalle S, Bugatti S, Montecucco C. Autoantibody profile in rheumatoid arthritis during long-term infliximab treatment. Arthritis Res Ther 2004;6:R264–72. [61] Gómez-Puerta JA, Cervera R, Espinosa G, Aguilo S, Bucciarelli S, Ramos-Casals M, et al. Antiphospholipid antibodies associated with malignancies: clinical and pathological characteristics of 120 patients. Semin Arthritis Rheum 2006;35:322–32. [62] Miesbach W, Scharrer I, Asherson R. Thrombotic manifestations of the antiphospholipid syndrome in patients with malignancies. Clin Rheumatol 2006;25:840–4. [63] Hughes GR, Khamashta MA. Seronegative antiphospholipid syndrome. Ann Rheum Dis 2003;62:112. [64] Cousins L, Pericleous C, Khamashta M, Bertolaccini ML, Ioannou Y, Giles I, et al. Antibodies to domain I of β-2-glycoprotein I and IgA antiphospholipid antibodies in patients with ‘seronegative’ antiphospholipid syndrome. Ann Rheum Dis 2015;74:317–19. [65] Nayfe R, Uthman I, Aoun J, Saad Aldin E, Merashli M, Khamashta MA. Seronegative antiphospholipid syndrome. Rheumatology (Oxford) 2013;52:1358–67. [66] Sciascia S, Khamashta MA, Bertolaccini ML. New tests to detect antiphospholipid antibodies: antiprothrombin (aPT) and anti-phosphatidylserine/prothrombin (aPS/PT) antibodies. Curr Rheumatol Rep 2014;16:415. [67] Piette JC, Cervera R, Levy R, Nasonov EL, Triplett DA, Shoenfeld Y. The catastrophic antiphospholipid syndrome – Asherson’s syndrome. Ann Med Intern (Paris) 2003;154:95–6. [68] Asherson RA, Espinosa G, Cervera R, Gómez-Puerta JA, Musuruana J, Bucciarelli S, et al. Disseminated intravascular coagulation in catastrophic antiphospholipid syndrome: clinical and haematological characteristics of 23 patients. Ann Rheum Dis 2005;64:943–6. [69] Bucciarelli S, Espinosa G, Cervera R, Erkan D, Gómez- Puerta JA, Ramos-Casals M, et al. Mortality in the catastrophic antiphospholipid syndrome. Causes of death and prognostic factors in a series of 250 patients. Arthritis Rheum 2006;54:2568–76. [70] Espinosa G, Bucciarelli S, Cervera R, Lozano M, Reverter JC, De la Red G, et al. Thrombotic microangiopathic haemolytic anaemia and antiphospholipid antibodies. Ann Rheum Dis 2004;63:730–6. [71] Asherson RA, Cervera R, Piette JC, Font J, Lie JT, Borcoglu A, et al. Catastrophic antibody syndrome. Clinical and laboratory features of 50 patients. Medicine (Baltimore) 1998;77:195–207. [72] Asherson RA, Cervera R, Piette JC, Shoenfeld Y, Espinosa G, Petri MA, et al. Catastrophic antiphospholipid syndrome: clues to the pathogenesis from a series of 80 patients. Medicine (Baltimore) 2001;80:355–76. [73] Asherson RA, Cervera R, de Groot PG, Erkan D, Boffa MC, Piette JC, et al. Catastrophic Antiphospholipid Syndrome Registry Project Group. Catastrophic antiphospholipid syndrome: international consensus statement on classification criteria and treatment guidelines. Lupus 2003;12:530–4. [74] Cervera R, Font J, Gómez-Puerta JA, Espinosa G, Cucho M, Bucciarelli S, et al. Validation of the preliminary criteria for the classification of catastrophic antiphospholipid syndrome. Ann Rheum Dis 2005;64:1205–9.

16  Antiphospholipid Syndrome in Systemic Autoimmune Diseases [75] Pierangeli SS, Brey RL. 13th International Congress on Antiphospholipid Antibodies (APLA 2010). Lupus 2011;20:152. [76] Abreu MM, Danowski A, Wahl DG, Amigo MC, Tektonidou M, Pacheco MS, et  al. The relevance of “non-criteria” clinical manifestations of antiphospholipid syndrome: 14th International Congress on Antiphospholipid Antibodies Technical Task Force Report on Antiphospholipid Syndrome Clinical Features. Autoimmun Rev 2015;14(5):401–14. [77] Bertolaccini ML, Amengual O, Andreoli L, Atsumi T, Chighizola CB, Forastiero R, et al. 14th International Congress on Antiphospholipid Antibodies Task Force. Report on antiphospholipid syndrome laboratory diagnostics and trends. Autoimmun Rev 2014;13(9):917–30. [78] de Jesús GR, Agmon-Levin N, Andrade CA, Andreoli L, Chighizola CB, Porter TF, et  al. 14th International Congress on Antiphospholipid Antibodies Task Force report on obstetric antiphospholipid syndrome. Autoimmun Rev 2014;13(8):795–813. [79] Cervera R, Rodríguez-Pintó I, Colafrancesco S, Conti F, Valesini G, Rosário C, et al. 14th International Congress on Antiphospholipid Antibodies Task Force Report on Catastrophic Antiphospholipid Syndrome. Autoimmun Rev 2014;13(7):699–707. [80] Erkan D, Aguiar CL, Andrade D, Cohen H, Cuadrado MJ, Danowski A, et al. 14th International Congress on Antiphospholipid Antibodies: task force report on antiphospholipid syndrome treatment trends. Autoimmun Rev 2014;13(6):685–96.

Chapter 2

Epidemiology of the Antiphospholipid Syndrome Laura Durcana and Michelle Petrib a

Division of Rheumatology, Department of Medicine, University of Washington, Seattle, WA, United States bHopkins Lupus Center, Hopkins University School of Medicine, Baltimore, MD, United States

2.1 INTRODUCTION The term antiphospholipid syndrome (APS) is used to describe an autoimmune disorder, characterized by thrombosis, either arterial or venous, and pregnancy morbidity in the setting of persistently positive antiphospholipid antibodies (aPL). These antibodies include the lupus anticoagulant (LA), anticardiolipin (aCL) and anti-β2-glycoprotein I antibodies. APS was first described in the early 1980s in association with systemic lupus erythematosus (SLE) [1], although abnormal clotting time in this population had been recognized decades earlier [2]. Since then, it has been recognized that this syndrome can occur both with (secondary APS) and without (primary APS) an underlying systemic autoimmune disorder. Catastrophic APS (CAPS) is the term used to describe the rare phenomenon of life-threatening, multiple-organ thrombosis associated with aPL. APS is classified according to the 2006 update of the Sapporo criteria [3], which are outlined in Box 2.1. The Sydney consensus conference also recommended that for the purposes of research, patients should be stratified according to their laboratory criteria (ie, more than one laboratory criterion, LA alone, anticardiolipin alone or anti-β2 glycoprotein I positivity in isolation). There are also a number of other possible, noncriteria manifestations associated with APS. These include, among others, thrombocytopenia, livedo reticularis, renal involvement, and valvular heart disease [4]. The classification criteria for CAPS have been outlined by Asherson et al. [5]. ‘Definite’ CAPS is defined as thromboses in three or more organs developing in less than a week, microthrombosis in at least one organ and persistent aPL positivity. If a patient has three out of these four criteria, then the patient is classified as having ‘probable’ CAPS. Antiphospholipid Syndrome in Systemic Autoimmune Diseases. DOI: http://dx.doi.org/10.1016/B978-0-444-63655-3.00002-8 © 2017 2016 Elsevier B.V. All rights reserved.

17

18  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

BOX 2.1  Classification Criteria for APS Classification criteria Clinical criteria

1. Vascular thrombosis: a. ≥1 clinical episodes of arterial, venous or small vessel thrombosis in any tissue or organ. 2. Pregnancy morbidity: a. ≥1 unexplained deaths of a morphologically normal foetus at or beyond the 10th week of gestation, b. ≥1 premature births of a morphologically normal neonate before the 34th week of gestation because of eclampsia, severe preeclampsia or recognized placental insufficiency, or c. ≥3 unexplained consecutive spontaneous abortions before the 10th week of gestation, with maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded. Laboratory criteria

1. Lupus anticoagulant present in plasma, on ≥2 occasions at least 12 weeks apart. 2. Anticardiolipin antibody of IgG and/or IgM isotype, in medium of high titre (>40GPL or MPL, or > the 99th percentile), on ≥2 occasions at least 12 weeks apart. 3. Anti-β2 glycoprotein 1 antibody of IgG and/or IgM isotype, in medium or high titre (>the 99th percentile), on ≥2 occasions, at least 12 weeks apart. Reproduced with permission from Miyakis S, Lockshin MD, Atsumi T, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4(2):295–306.

This chapter provides a summary of the available literature on the prevalence of APS and of aPL in the general population, in thrombotic diseases and in SLE.

2.2  APS IN THE GENERAL POPULATION The actual incidence of APS is unknown. Estimates have indicated an incidence of around 5 new cases per 100,000 persons per year, with a prevalence of around 40–50 cases per 100,000 persons [4]. A review by Andreoli et al. [6] examined the prevalence of aPL in different thrombotic conditions and used this estimate to calculate a crude estimation of incidence. They calculated that 6% of women with pregnancy morbidity have positive aPL, 13.5% with stroke, 11% with myocardial infarction, and 9.5% with deep venous thrombosis (DVT). Based on census data for the United States, they estimated 280,000 antiphospholipid-related events annually. CAPS is rare, accounting for only 1% of all APS, but the associated high mortality makes it an important entity and research agenda [7,8].

Epidemiology of the Antiphospholipid Syndrome  Chapter | 2  19

aPL can be found in healthy subjects with an increased prevalence in older populations, as outlined in Table 2.1. Generally, aCL and the LA have a prevalence of 1–5% in young healthy controls [9]. There is some heterogeneity in the earlier studies, which were limited by a lack of uniformity in assay methods. For the most part, in normal control populations, these antibodies were identified at low titres without an association with APS manifestation. In large populationbased studies involving older individuals, such as the Honolulu Heart study, aPL (anticardiolipin and anti-β2 glycoprotein I) were demonstrated in up to 12% of normal controls [10]. Viral, bacterial, and parasitic infections have been found to be associated with aPL positivity. These include hepatitis C, cytomegalovirus, Epstein– Barr, human immunodeficiency virus, adenovirus, and parvovirus B 19 [11]. Regarding bacterial infections in particular, aPL positivity is commonly associated with leprosy and syphilis infections [12]. A higher rate of aPL positivity has also been observed in patients with malignancies [13,14]. It is unknown whether aPL play a role in the increased rate of thrombosis observed in this population. There appears to be a higher prevalence of aPL positivity in children. The syndrome is diagnosed based on the adult classification criteria, with the exception of the obstetric components, which generally do not apply to this population. The estimated frequency of positive aPL in children without any underlying disorder ranges widely, from 3% to 28% for aCL and from 3% to 7% for anti-β2 glycoprotein I [11]. This is thought to be due to the high rate of infections in children [15]. In children, aPL from infection are often transient [16–18]. In children with paediatric APS, the rate of SLE is in the range of 9–14% [19,20]. A meta-analysis by Kenet et al. [21] evaluated 16 case-controlled studies [22–36] comparing aPL in children with and without a history of thrombosis. They demonstrated a strong association between aPL and both venous and arterial thrombosis with an odds ratio of 5.9. Without underlying autoimmune disease, those who have multiple positive aPL have been shown to have the highest rate of thrombosis. This was particularly true in those with ‘triple positivity’ (ie, a positive LA, anticardiolipin, and anti-β2 glycoprotein I). These individuals were shown to have a 9.8% risk of thrombosis after 2 years, which increased to 37% after a decade [37]. Mustonen et al. evaluated a large Finnish population with positive aPL who did not have a history of thrombosis at inception. They also demonstrated that double or triple positivity carried a higher risk of future thrombotic events. The rate of thrombotic events was lower in those who were taking aspirin as a prophylaxis [38]. In the Hopkins Lupus Cohort, LA was strongly related to lifetime thrombosis. After controlling for the LA, aCL were not predictive of thrombosis. This is in keeping with other SLE literature demonstrating that, of the aPL, the LA is the strongest predictor of thrombotic events [39]. The titre of aPL is also important in the risk stratification of those who have not had a thrombosis. Multiple studies have reported a higher rate of thrombosis

TABLE 2.1 Prevalence of Antiphospholipid Antibodies in Normal Controls Study and Year

Number

Source

LA

aCL

380

P + NP

64

Elderly

El-Roeiy et al. (1988) [66]

400

50% M

Briley et al. (1989) [67]

800

1.6%

Fields et al. (1989) [68]

543

2%

Lockwood et al. (1989) [69]

737

P

2.7%

2.2%

Shi et al. (1990) [70]

499

BD

3.6%

5.6%

Infante-Rivard et al. (1991) [71]

993

P

3.8%

1.5%

1200

P

Pattison et al. (1993) [73]

933

P

Phadke et al. (1993) [74]

504

Juby et al. (1998) [75]

250

H

1.2%

63

HE

0%

E (disease)

12.3%

Vaarala et al. (1986) [64] Manoussakis et al. (1987) [65]

Perez et al. (1991) [72]

301

IgG-aCL

IgM-aCL

1%

1%

1.8%

1%

1.8%

4.3%

IgA-aCL

50%

1.25% 1.2%

1% 4.2%

5%

2.2%

β2GP-IgM

β2GP-IgG

TABLE 2.1 Prevalence of Antiphospholipid Antibodies in Normal Controls Study and Year Avcin et al. (2001) [76]

Brey et al. (2001) [10]

Number 113

1360

Source

LA

aCL

IgG-aCL

61 Ch

11.4%

11.4%

52 BD

9.6%

5.7%

Controls

12.1% 0.8%

Pusterla et al. (2004) [13]

Meroni et al. (2004) [78]

268

200

77

IgA-aCL

β2GP-IgM

β2GP-IgG 3.2%

3.8%

a

Harrison et al. (2002) [77]

IgM-aCL

a

1.9%

a

4.4%

1.9%

a

9.0%

68 ET

23%

10.2%

2.9%

200 H

1.5%

0.5%

1.5%

8.6%

54%

100 L

7%

24%

100 H

1%

7%

Centenarians

20%

2.5%

P, pregnancy; NP, not pregnant; M, male; HE, healthy elderly; E, elderly; ET, essential thrombocytosis; H, healthy controls; Ch, children; BD, blood donors; L, lymphoma. a β2-Glycoprotein I dependent and independent results.

22  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

in those who have high-titre aPL and in those with and without underlying autoimmune disease [40,41]. The presence of the LA has been found to be a particularly strong predictor of future thrombosis in both normal populations and in those with SLE [39,41,42]. A total of 50% of SLE patients with a positive LA will develop a thrombosis over 20 years [41]. Other cardiovascular risk factors, such as smoking and hypertension, have also been found to contribute to the rate of thrombotic events in patients with positive aPL [38,39,41]. In particular, hypertriglyceridemia has been shown to be predictive of venous thrombotic events and hypertension is a strong predictor of arterial thrombosis [43]. These are particularly notable, as they are modifiable.

2.3  aPL AND VENOUS THROMBOSIS Venous thrombosis, usually DVT, is the most common APS clinical manifestation. The frequency of positive aPL in DVT has been reported in the range of 5.2–30% [77,80, 81–83,85]. In those with DVTs, the prevalence of the LA in the reported literature is 0.6–5.5%, with aCL in 4–24%. In venous sinus thrombosis, the rate of aPL positivity is 8–53% [78,79,84]. This is a rare condition; hence, the numbers in these studies were small (Table 2.2). A meta-analysis performed by Galli et  al. [42] evaluated studies involving 4184 patients and 3151 controls. They found that the LA was most

TABLE 2.2 Frequency of Antiphospholipid Antibodies in Venous Thrombosis Study and Year

Number

Any aPL Positive

LA

aCL

Mateo et al. (1997) [79]

2132

5.2%

0.6%

4%

Deschiens et al. (1996) [80]

40 (VST)

8%

Carhuapoma et al. (1997) [81]

15 (VST)

53%

Bick et al. (1999) [82]

100

4%

24%

Eschwège et al. (1998) [83]

122

Salomon et al. (1999) [84]

109

Zanon et al. (1999) [85]

227

30%

Saasatnia et al. (2004) [86]

30 (VST)

23.5%

Roldan et al. (2009) [87]

597 (first) 326 (recurrent)

24% 28%

15%

16% 5.5%

20%

VST, venous sinus thrombosis; aPL, antiphospholipid; LA, lupus anticoagulant; aCL, anticardiolipin antibody.

Epidemiology of the Antiphospholipid Syndrome  Chapter | 2  23

strongly predictive of thrombosis. This has also been the finding in the SLE literature [44]. aCL, particularly at high titres, have also been shown to be predictive of thrombotic events and recurrences with the cessation of anticoagulation [45].

2.4  aPL AND ARTERIAL THROMBOSIS In contrast with thromboses associated with many of the congenital thrombophilias, any vascular bed can be affected with APS. In the arterial system, the central nervous system is the most commonly affected site. Stroke is the most frequent arterial clinical manifestation. There is evidence that subclinical disease may also occur in the form of white mater changes on magnetic resonance imaging, the clinical significance of which is unknown [46]. The strongest association of aPL with stroke is in those who are less than 50 years old; 10% of the total stroke population are in that category. Sciascia et al. [47] performed a meta-analysis evaluating the prevalence of aPL in young people with stroke. A total of 43 studies demonstrated positive aPL in 17.4% of the 5217 participants. In older individuals, the relationship is less clear. A recent study by Arvanitakis et al. [48] included the postmortem evaluation of 607 brains for infarction, 23% of whom had at least one positive aPL. There was no relationship found between the prevalence of positive aPL and cerebral infarcts. In infants presenting with stroke, 12 of 62 (about 19%) were found to have positive aPL of unclear clinical significance [49]. An association has been found between myocardial infarction and aPL. This is thought to be due to both thrombotic and atherosclerotic mechanisms. aPL have been reported to play an important role in the development and progression of atherosclerosis, representing a nontraditional cardiovascular risk factor [50–52]. However, aPL have been associated with atherosclerosis mainly on the basis of animal models, whereas passive infusion of aPL or active induction of antibodies cross-reacting with murine anti-β2 glycoprotein I showed an enhanced formation of atherosclerotic plaque [53]. Belizna et al. [54] evaluated carotid intimal medial thickness and found that this increased, in both primary and secondary APS, regardless of the other disease features. Contrarily, comparing APS to normal controls, Ames et al. found that there were no differences in carotid intimal medial thickness between young patients with APS and controls. However, there were some differences found in older populations [55]. In SLE, LA positivity has been found to be associated with cardiovascular events, but not with the development of atherosclerosis; this points toward thrombotic, rather than predominantly atherosclerotic, mechanisms [56,57].

2.5  aPL AND PREGNANCY MORBIDITY There is some association between aPL and pregnancy morbidity. Recurrent miscarriage occurs in about 1% of the female population trying to have children

24  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

[58]. Approximately 10–15% of women with recurrent miscarriages are found to have positive aPL and are diagnosed with obstetric APS [59,60]. However, the association between pregnancy morbidity and aPL has not been consistently demonstrated [61]. Most studies, although not all were evaluated in a meta-analysis by the APS action group [61], point toward a higher incidence of pregnancy loss with positive aPL. When studies were pooled, there was no association found between aPL and preeclampsia or the HELLP syndrome, although there was a significant relationship demonstrated between those with severe preeclampsia and aPL. The PROMISSE study found that LA is the primary predictor of adverse pregnancy outcomes after 12 weeks of gestation and that aCL and anti-β2 glycoprotein I, in the absence of a positive LA, did not predict adverse pregnancy outcomes [62].

2.6  aPL AND SLE aPL are common in SLE, as outlined in Table 2.3. They are among the most frequent autoantibodies in SLE and were first described in this population [1]. These antibodies can fluctuate with time, which makes it difficult to firmly establish a point prevalence. In the Hopkins Lupus Cohort, a longitudinal study

TABLE 2.3 Antiphospholipid Antibodies in SLE Study and Year

Number

LA

aCL

Alarcón-Segovia et al. (1989) [88]

500

39%

Buchanan et al. (1989) [89]

117

30%

Worrall et al. (1990) [90]

100

38%

Mayumi et al. (1991) [91]

106

16%

Wong et al. (1991) [92]

91

11%

Jones et al. (1991) [93]

200

17%

Picillo et al. (1992) [94]

102

86%

Cervera et al. (1993) [8]

1000

Kutteh et al. (1993) [95]

125

25%

Axtens et al. (1994) [96]

127

24%

Somers et al. (2002) [41]

678

Petri (2010) [63] Woo et al. (2010) [97]

88

15%

ANTI β2GP

24%

27%

48%

26%

47%

32.5%

34%

31.8%

11.4%

LA, lupus anticoagulant; aCL, anticardiolipin antibody; ANTI β2GP, anti-β2 glycoprotein I.

Epidemiology of the Antiphospholipid Syndrome  Chapter | 2  25

in which aPL are measured quarterly, the prevalence of aCL positivity was 46% and that of the LA was 26% [63]. In the setting of LA positivity in SLE, the 20-year risk of thrombosis is 50% [11]. Neither aCL nor LA are associated with carotid intimal medial thickness, carotid plaque or coronary calcium score by helical computed tomography in SLE [57].

2.7 CONCLUSIONS aPL occur at a low frequency in the general population. Longitudinal studies have shown that they are a risk factor for thrombosis. In the general population, the risk of thrombosis increases with the number of positive antibodies. In SLE, only the LA is an independent risk factor for thrombosis. The relationship between aPL and stroke is strongest in those who are young. The association with stroke weakens with advancing age (although the incidence of antibodies is highest in those who are elderly). The exact role played by these antibodies in the pathogenesis of thrombi and mechanisms of possibly enhanced atherosclerosis in this population is presently unknown.

ACKNOWLEDGEMENTS Supported by NIH RO-1 AR 43727. Dr Durcan was supported by the Bresnihan Molloy Scholarship, awarded by the Royal College of Physicians of Ireland.

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30  Antiphospholipid Syndrome in Systemic Autoimmune Diseases [84] Salomon O, Steinberg DM, Zivelin A, et  al. Single and combined prothrombotic factors in patients with idiopathic venous thromboembolism: prevalence and risk assessment. Arterioscler Thromb Vasc Biol 1999;19(3):511–8. [85] Zanon E, Prandoni P, Vianello F, et al. Anti-beta2-glycoprotein I antibodies in patients with acute venous thromboembolism: prevalence and association with recurrent thromboembolism. Thromb Res 1999;96(4):269–74. [86] Saadatnia M, Mousavi SA, Haghighi S, Aminorroaya A. Cerebral vein and sinus thrombosis in Isfahan-Iran: a changing profile. Can J Neurol Sci 2004;31(4):474–7. [87] Roldan V, Lecumberri R, Muñoz-Torrero JF, et  al. Thrombophilia testing in patients with venous thromboembolism. Findings from the RIETE registry. Thromb Res 2009;124(2):174–7. [88] Alarcón-Segovia D, Delezé M, Oria CV, et al. Antiphospholipid antibodies and the antiphospholipid syndrome in systemic lupus erythematosus. A prospective analysis of 500 consecutive patients. Medicine (Baltimore) 1989;68(6):353–65. [89] Buchanan RR, Wardlaw JR, Riglar AG, Littlejohn GO, Miller MH. Antiphospholipid antibodies in the connective tissue diseases: their relation to the antiphospholipid syndrome and forme fruste disease. J Rheumatol 1989;16(6):757–61. [90] Worrall JG, Snaith ML, Batchelor JR, Isenberg DA. SLE: a rheumatological view. Analysis of the clinical features, serology and immunogenetics of 100 SLE patients during long-term follow-up. Q J Med 1990;74(275):319–30. [91] Mayumi T, Nagasawa K, Inoguchi T, et  al. Haemostatic factors associated with vascular thrombosis in patients with systemic lupus erythematosus and the lupus anticoagulant. Ann Rheum Dis 1991;50(8):543–7. [92] Wong KL, Liu HW, Ho K, Chan K, Wong R. Anticardiolipin antibodies and lupus anticoagulant in Chinese patients with systemic lupus erythematosus. J Rheumatol 1991;18(8):1187–92. [93] Jones HW, Ireland R, Senaldi G, et al. Anticardiolipin antibodies in patients from Malaysia with systemic lupus erythematosus. Ann Rheum Dis 1991;50(3):173–5. [94] Picillo U, Migliaresi S, Marcialis MR, Longobardo A, La Palombara F, Tirri G. Longitudinal survey of anticardiolipin antibodies in systemic lupus erythematosus. Relationships with clinical manifestations and disease activity in an Italian series. Scand J Rheumatol 1992;21(6):271–6. [95] Kutteh WH, Lyda EC, Abraham SM, Wacholtz MC. Association of anticardiolipin antibodies and pregnancy loss in women with systemic lupus erythematosus. Fertil Steril 1993;60(3):449–55. [96] Axtens RS, Miller MH, Littlejohn GO, Topliss DJ, Morand EF. Single anticardiolipin measurement in the routine management of patients with systemic lupus erythematosus. J Rheumatol 1994;21(1):91–3. [97] Woo KS, Kim KE, Kim JM, Han JY, Chung WT, Kim KH. Prevalence and clinical associations of lupus anticoagulant, anticardiolipin antibodies, and anti-beta2-glycoprotein I antibodies in patients with systemic lupus erythematosus. Korean J Lab Med 2010;30(1):38–44.

Chapter 3

Mechanisms of Action of the Antiphospholipid Antibodies Cecilia B. Chighizolaa,b, Elena Raschia, Maria O. Borghia,b and Pier L. Meronia,b,c a

Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy Experiment Laboratory of Immunological and Rheumatologic Researches, IRCCS Istituto Auxologico Italiano, Milan, Italy cDivision of Rheumatology, Istituto Gaetano Pini-CTO, Milan, Italy b

3.1 INTRODUCTION Antiphospholipid syndrome (APS) is a chronic autoimmune condition clinically characterized by vascular thrombosis and/or pregnancy complications. The laboratory diagnosis of APS relies on the identification of circulating antiphospholipid antibodies (aPL) using three tests: two solid-phase assays detecting anticardiolipin (aCL) and anti-β2 glycoprotein I antibodies (anti-β2GPI), plus the functional assay lupus anticoagulant (LA) [1]. Medium/high-titre aPL positivity, confirmed 12 weeks apart, of at least one test is required to diagnose APS [1]. In addition, aPL not only provide diagnostic biomarkers of APS, but also exert a pathogenic role (Fig. 3.1). aPL with the same autoantigen specificity and titres can be related to different clinical pictures and pathogenic mechanisms in experimental models [2]; each aPL profile might associate with merely vascular events or pregnancy complications [1]. These observations explain why aPL are regarded as a risk-factor for APS, with additional hits required to trigger clinical manifestations [2]. Such cofactors might account for the clinical and biological disparity between vascular and obstetric variants of the syndrome.

3.2  ANTIPHOSPHOLIPID ANTIBODIES aPL are a heterogeneous family of autoantibodies, but there is evidence that only antibodies reacting with phospholipid (PL)-binding proteins, particularly β2 glycoprotein I (β2GPI) and prothrombin (PT), display a pathogenic potential. Antiphospholipid Syndrome in Systemic Autoimmune Diseases. DOI: http://dx.doi.org/10.1016/B978-0-444-63655-3.00003-X © 2017 2016 Elsevier B.V. All rights reserved.

31

32  Antiphospholipid Syndrome in Systemic Autoimmune Diseases

FIGURE 3.1  Schematic representation of the pathogenic effects mediated by anti-β2GPI antibodies.

3.2.1 The β2 Glycoprotein I-Dependent Autoantibodies β2GPI is a single-chain 43-kDa glycoprotein synthesized by endothelial cells (EC), hepatocytes, and trophoblast cells. A member of complement control protein (CCP) family, β2GPI consists of 326 amino acids arranged in five CCP-repeat domains (D). DI-IV comprise 60 amino acids and contain two disulphide bridges each; DV is aberrant, consisting of 82 amino acids crosslinked by an additional disulphide bond. DV is responsible for binding to PL and cell membranes. Three β2GPI configurations have been described: (1) a circular form, adopted by circulating plasma β2GPI, (2) a J-shaped configuration, assumed upon binding to anionic surfaces, such as cardiolipin (CL) and other PL or lipopolysaccharide (LPS), and (3) an intermediate S-shape. β2GPI interacts specifically with LPS through the C-terminal, potentially acting as an LPS carrier or scavenger [3].

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aPL reacting with β2GPI are currently regarded as the main antibody subset. Affinity-purified anti-β2GPI IgG trigger a pathogenic effect in all in vivo models, while specific absorption of anti-β2GPI activity inhibits the thrombotic effect [4]. Antibodies against β2GPI are the main mediators of LA, an in vitro elongation of PL-dependent clotting time. High-titre antibodies, often of IgG isotype, mediate this functional phenomenon. Anti-β2GPI antibodies partially overlap with autoantibodies identified with aCL enzyme-linked immonosorbent assay (ELISA) test, which can employ CL-coated matrix and bovine or human serum, thus detecting antibodies against β2GPI-bound CL (β2GPI-dependent aCL) or CL alone (β2GPI-independent aCL). A positively charged discontinuous structure in β2GPI-DI is the main epitope involved in β2GPI/anti-β2GPI antibody binding. This structure is cryptic and conformation-dependent, available for antibody binding only when β2GPI opens to a J-configuration. In the circular conformation, DI interacts with DV, thus hiding the critical epitope [5]. The immunogenicity of β2GPI also depends on its conformation, as supported by in vivo evidence: mice develop antibodies against DI only when injected with misfolded β2GPI or β2GPI-CL [6]. Anti-DI antibodies can be detected in most APS patients, and are significantly associated with LA. The pathogenicity of such autoantibody subset has been progressively characterized: infusion of a synthetic DI peptide partially protects naive mice from the thrombogenic effects of polyclonal aPL IgG [7]. A direct demonstration of their pathogenic effect was obtained using MBB2, a human monoclonal IgG antibody targeting β2GPI-DI. Its infusion induces foetal losses in pregnant mice and blood clots in rats after LPS priming [8]. Further support comes from tolerogenic dendritic cells (tDCs) pulsed with the whole molecule or β2GPI-DI: infusion of tDCs to β2GPI-immunized BALB/c mice results in reduced foetal loss rate, decreased anti-β2GPI antibody titres, and raised expression of anti-inflammatory cytokines. A greater effect is obtained with DC pulsed with DI than the whole molecule [9]. It could be concluded that anti-DI are the pathogenic antibodies, and antiDIV/V are innocent players because IgG reacting with β2GPI from asymptomatic carriers preferentially recognize DIV/V epitopes. However, the real scenario is much more complicated: approximately one third of APS patients carrying anti-β2GPI antibodies are negative for anti-DI IgG [10,11]. A multicentre study exploiting several peptides spanning the different domains confirms that a consistent rate of APS patients display autoantibodies reacting with β2GPI epitopes other than DI [12].

3.2.2  Prothrombin-Dependent Antibodies PT (factor (F) II) is a 72-kDa vitamin K-dependent glycoprotein synthesized in the liver. PT physiological activation is mediated by the prothrombinase complex, which enlists activated FX, FV, calcium, and PL. Prothrombinase complex converts PT into thrombin only when negatively charged PL bind to

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PT. To be antigenically recognized, human PT has to be coated on activated plates or exposed to immobilized phosphatidylserine (PS) via calcium ions. ELISA-detecting antibodies against PS/PT complex (anti-PS/PT) identify a partially different autoantibody population from the assay using PT as the only antigen [13]. In vitro experimental findings suggest antibodies against PT exert thrombogenic effects interfering with fluid-phase coagulation components and activating EC. Together with anti-β2GPI antibodies, antibodies against PT (anti-PT) constitute the major contributor to LA phenomenon: approximately two-thirds of IgG anti-PT display in vitro anticoagulant activity. Such elongation of clotting time could be explained by PT/anti-PT/PL trimolecular complexes inhibiting the activation of prothrombinase and tenase complexes and competing with clotting factors for PL surfaces [14]. Due to the lack of cross-reactivity of human antibodies with animal PT, evidence from animal models is weak. Anti-PS/PT are more closely related with clinical events than anti-PT, suggesting that pathogenic antibodies may recognize a conformational epitope(s) expressed when PT complexes with anionic PL in the presence of calcium ions [15]. Human monoclonal or affinity-purified polyclonal anti-β2GPI antibodies from a serum reacting with both β2GPI and PS/PT react toward β2GPI only, clearing the contentious issue of the potential cross-reactivity between anti-PS/ PT and anti-β2GPI antibodies [16].

3.2.3  Antibodies Against Other PL Antigens The pathogenic roles of several autoantibodies that target negatively charged PL other than CL have been evaluated in APS. PS, phosphatidylinositol, and phosphatidic acid are among the best-characterized antigens. aCL are well-known to broadly cross-react with antibodies targeting both PS and phosphatidylinositol, due to the recognition of β2GPI/PL complex. Therefore, cross-reactivity is mainly mediated by autoantibodies reacting with β2GPI [17]. Antibodies targeting phosphatidylethanolamine (PE) deserve more attention. PE, a zwitterionic PL, promotes thrombosis by activating FX and PT, and works as anticoagulant potentiating activated protein C (APC) activity. Antibodies against PE (anti-PE) bind to kininogen, leading to antibody/PE/ kininogen trimolecular complexes that enhance thrombin-induced platelet aggregation. An in vivo demonstration of anti-PE pathogenicity in vascular events is lacking; anti-PE infusion to pregnant mice triggers placental thrombosis and haemorrhage [18].

3.3  aPL-MEDIATED MECHANISMS OF THROMBOSIS The association between aPL and thrombosis is supported by several epidemiological studies, with clot formation as the key-event [2]. Most evidence about aPL-mediated thrombus formation has been gained from in vitro models, with

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further support coming from three different in vivo models of thrombosis [2]. aPL can increase the size of thrombi triggered by mechanical or chemical stimuli; in a third model, infusion of human aPL IgG with a small LPS amount is sufficient to induce clotting [19]. aPL procoagulant mechanisms are mainly mediated by antibody reactivity against PL-binding proteins on membranes of different cells. It is still unclear whether aPL significantly react with PL-binding proteins in fluidphase. Complex formation in fluid-phase requires stoichiometric antigen– antibody ratios uncommon in patients because aPL are low-avidity antibodies [20]. Furthermore, circulating β2GPI adopts the circular form and opens upon binding to PL on the cell membrane, where the high antigenic density allows for easier autoantibodies engagement.

3.3.1  Endothelial Cells Endothelium acts as the main player in APS pathogenesis: indeed, aPL induce a proinflammatory and procoagulant endothelial phenotype leading to clotting events. aPL drive a significant upregulation of cellular adhesion molecules (CAMs) in in vitro EC. CAM expression on vascular surface favours leucocyte adhesion to the endothelium, contributing to APS prothrombotic diathesis. Accordingly, in vivo aPL infusion increases endothelial adhesion of leucocytes. Ex vivo studies, although inconsistently, report raised levels of soluble CAM in APS subjects. Moreover, aPL upregulate proinflammatory cytokines, such as interleukin (IL)-1β, IL-6, and IL-8 in in vitro EC, modulate vascular tone inhibiting endothelial nitric oxide synthase, and altering prostaglandin metabolism [21]. APS patients consistently display endothelial perturbation, particularly impaired brachial artery flow-mediated vasodilation response, increased circulating EC, tissue plasminogen activator (tPA), and von Willebrand factor (vWF) compared with controls [2,22].

3.3.2 Monocytes Monocytes contribute to APS pathogenesis providing the main source of tissue factor (TF), which is the major initiator of clotting cascade. aPL significantly increase TF expression in both monocytes and EC. Vascular endothelial growth factor and its receptor Flt-1, two mediators upstream of TF, are upregulated in monocytes from APS patients [2,21].

3.3.3 Platelets Even though aPL are well-known to induce aggregation and activation of platelets, prestimulation by agonists such as thrombin or collagen is a required step. Since PS is a negatively charged PL, it favours β2GPI adhesion on platelet

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membrane with the antigen in the optimal conformation for interaction with aPL. Furthermore, aPL affect β2GPI inhibition of vWF: aPL – upon binding to β2GPI – neutralize such interaction, thus interfering with vWF-dependent platelet adhesion. This observation might explain the mild thrombocytopenia frequently observed in aPL carriers. Additional evidence of platelet activation by aPL comes from rat models: (1) in animals pretreated with low concentration of adenosine diphosphate, aPL infusion produces a platelet-rich thrombus, and (2) platelets contribute to thrombus formation induced by photochemical trauma. Ex vivo studies provide concordant findings: elevated levels of platelet-derived thromboxane metabolic products are found in urine of APS patients [2,21].

3.3.4 Neutrophils Neutrophils act as additional players in coagulation: upon cell death, they release extracellular traps (NET), consisting in decondensed chromatin with nuclear proteins. NET actively participate in coagulation processes: NET-derived proteases activate the coagulation cascade and their structure serves as scaffolding for clot assembly. NET might also damage the endothelium, providing potential mediators of atherosclerosis and arterial thrombosis. NET have been recently evaluated specifically in APS: compared with healthy volunteers, sera and plasma from patients display elevated levels of both cell-free DNA and NET, APS neutrophils spontaneously release higher levels of NET. Sera and IgG from APS patients and human aPL monoclonals, especially those targeting β2GPI, stimulate NET release from control neutrophils, a mechanism abrogated by inhibitors of reactive oxygen species formation and toll-like receptor (TLR) 4 signalling [23].

3.3.5  Soluble Phase The evidence of aPL interference with fluid-phase components of coagulation has been gained mostly from in vitro models and a few ex vivo experiments [2,24,25]. It has been found that aPL react against several members of serine protease (SP) family, which enlists procoagulant factors as thrombin, PT, FVIIa, FIXa, and FXa, anticoagulants such as protein C (pC) and agents involved in fibrinolysis such as plasmin and tPA. aPL interaction with these proteins is mediated by conformational epitopes shared by β2GPI and SP enzymatic domain. Importantly, aPL interaction with thrombin and FXa interferes with formation of thrombin–antithrombin (AT) and FXa-AT complexes, thus hindering AT-inactivation of thrombin and FXa. Moreover, aPL disrupt pC and protein S (pS) pathways: aPL reacting against pS or pC have been found in APS subjects, and are associated with decreased levels of pC or pS. Positivity rates of anti-pC and anti-pS antibodies in APS populations vary widely across reports, as well as their association with clinical events [26,27]. In addition, aPL decrease APC activity by competing for PL binding, an increased APC resistance has been demonstrated in APS patients.

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Some aPL inhibit plasmin-mediated fibrinolysis, particularly impairing fibrin dissolution by plasmin or inhibiting tPA-mediated conversion of plasminogen to plasmin. Antibodies against tPA have been described in APS patients, inversely correlating with plasma tPA activity. Other than the inhibitory effects on anticoagulants, aPL may increase the enzymatic activity of procoagulants: some aPL subsets induce a gain-of-function of PT, which leads to increased fibrin production. Furthermore, aPL disrupt the crystallization on EC of AnnexinA5, a potent anticoagulant that prevents PL bioavailability for coagulation enzymes [2,24,25].

3.3.6 Complement Complement activation provides a necessary step in aPL-mediated thrombosis. Most APS sera fix complement in vitro; however, the strongest evidence of complement role in aPL thrombotic events pertains to in vivo models. Animals deficient in complement components or complement receptors or treated with inhibitors of complement activation are protected from aPL thrombogenic effects. The involvement of complement cascade is further confirmed by the efficacy of the monoclonal anti-DI MBB2 and the failure of the parent monoclonal antibody MBB2ΔCH2 to induce vascular thrombosis in rats [8]. MBB2ΔCH2, which displays the same antigen specificity of MBB2 but does not activate complement because it lacks the CH2 domain, prevents aPL procoagulant effects in vivo by competing with autoantibodies for binding to β2GPI [8]. Indirect evidence also comes from the in vivo effectiveness of complement C5-inhibitor rEV576 coversin, which inhibits aPL-mediated venous thrombosis and TF production in a mouse model [28]. However, a clear decrease of complement levels has not been described in patients, only two studies report mild hypocomplementemia in primary APS [2].

3.4  aPL-MEDIATED MECHANISM OF PREGNANCY COMPLICATIONS aPL provide the most frequently acquired risk-factor for pregnancy complications [1]. This association is clearly supported by experimental models: passive transfer of aPL IgG induces foetal loss and growth retardation in pregnant naive mice [2]. The placental tropism of aPL could be explained by the high β2GPI amount found on trophoblast: β2GPI binds to PS on external membranes of trophoblast undergoing syncytium formation. Since the earliest histopathological report of APS placentas, aPL have been thought to induce spiral artery thrombosis leading to placental infarction, and the resulting impairment of maternal–foetal blood exchange was believed to interfere with pregnancy physiology [29]. Surely aPL can induce a procoagulant state disrupting the anticoagulant AnnexinA5 shield on trophoblast. A reduced amount of AnnexinA5 consistently covers the intervillous surfaces in placentas of aPL-positive women.

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However, histopathological studies report a similar prevalence of intervillous thrombosis in aPL-positive and aPL-negative women; most miscarriage samples and placentas from APS patients do not display any histopathological finding that suggest thrombosis [30]. Placental thrombosis and infarction are unlikely causes of early loss since a significant maternal blood flow does not occur in intervillous spaces until the end of the first trimester. It is increasingly acknowledged that nonthrombotic mechanisms might be implicated in APS-associated pregnancy complications [29]. This is the case of placental inflammation. In humans, aPL can induce first-trimester trophoblasts, a potent proinflammatory cytokine, IL-1β, via the inflammasome. Injections of large amounts of human aPL to pregnant naive mice after embryo implantation elicit strong placental inflammatory damage that results in foetal resorption and growth retardation. Immunohistochemical and histological examinations of decidua show neutrophil infiltration and local tumour necrosis factor (TNF)−α secretion, with transient increase in blood TNFα. Mice deficient in D6, a placental receptor that targets to degradation inflammatory chemokines, are more susceptible to foetal loss when infused with small amount of human aPL IgG than pregnant wildtype mice [31]. On the other hand, when small amounts of human aPL IgG are administered to mice before implantation, placental histological analysis fails to show clear signs of inflammation [32]. Accordingly, abortive material or term placentae from APS women do not show any sign of acute local inflammatory events [2]. In vivo experimental models are also strongly suggestive for complement role in mediating aPL-induced pregnancy complications. Indeed, pregnant mice deficient in complement C3, C5, C5a receptor, or treated with an inhibitor of C3 convertase, do not experience aPL-induced foetal loss. Furthermore, the monoclonal anti-DI MBB2 induces foetal loss while the noncomplement fixing CH2deleted variant does not [8]. In humans, a retrospective study finds complement deposition in placentae from aPL-positive women; a case study reported no complement deposition in foetuses miscarried by APS women, while a more recent prospective study on full-term placentas and abortive specimens shows only mild complement deposition without any relationship to pregnancy outcome or therapy [2]. Lastly, there is sound evidence for a direct effect of aPL on placentation. Polyclonal IgG from APS patients and human anti-β2GPI IgM monoclonals can react in vitro with β2GPI both at the foetal (trophoblast cells) and maternal (stromal decidual cells and human endometrial EC (HEEC)) sides of human placenta. The direct interaction of aPL with the foetal side results in (1) inhibition of trophoblast differentiation, as shown by the reduced secretion of human chorionic gonadotropin, (2) impairment of the invasiveness of extravillous trophoblast cells, with a significant downregulation of matrix metalloproteinases, integrins, cadherins, and heparin-binding epidermal growth factor, (3) trophoblast injury and apoptosis, and (4) extrusion of necrotic trophoblast debris able

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to activate the maternal endothelium. Upon interaction with the maternal side of the placenta, aPL are able to (1) induce a proinflammatory phenotype in stromal decidual cells and (2) block endometrial angiogenesis, by inhibiting HEEC angiogenic differentiation and production of proangiogenic factors [33].

3.5  RECEPTORS FOR β2GPI/ANTI-β2GPI ANTIBODIES aPL-induced effects are mainly mediated by the reactivity of autoantibodies with β2GPI expressed on cell membrane. β2GPI adhesion to EC is mediated by several different receptors. AnnexinA2, a tPA and plasminogen receptor, directly binds β2GPI on EC and monocytes/macrophages. A coreceptor is required to trigger the signalling cascade because AnnexinA2 lacks an intracytoplasmatic tail. TLR2 and TLR4, heparan-sulphate and Apolipoprotein E Receptor 2′ (ApoER2′) have been shown to bind β2GPI on the endothelial surface [2,21]. In particular, TLR4 is the key player in driving endothelial perturbation: tlr4- but not AnnexinA2-silencing prevents the upregulation of adhesion molecules [34]. TLR4 is part of a multiprotein complex, which also includes AnnexinA2, calreticulin, and nucleolin, involved in aPL-induced EC activation [35]. On monocytes, aPL interact with β2GPI, AnnexinA2, and TLR4 within lipid rafts; however, β2GPI interaction with LPS accounts for apparent TLR4 activation by aPL. LPS/β2GPI complexes mediates β2GPI binding to cell membrane; both LPS and TLR4 are required for β2GPI to bind and activate macrophages, and treatment with LPS-inactivator polymyxin abolishes β2GPI binding to macrophages [36]. LPS/β2GPI complexes do not display the same effect on the endothelium. Indeed, low LPS contamination does not affect β2GPI/TLR4 interaction and EC activation; at high EC-activating concentrations, LPS increases β2GPI binding, likely through TLR4 upregulation. Cosilencing for AnnexinA2 and TLR4 does not completely inhibit anti-β2GPI antibody binding, a finding consistent with potential additional surface β2GPI receptors [34]. TLR1, TLR2, and TLR6 have indeed been appointed as candidate β2GPI coreceptors. TLR2 contributes to mediating intracellular aPL signalling in EC; to note, TLR2 is expressed by EC only upon cell activation while TLR4 is constitutively expressed. Other authors provide indirect evidence of TLR2 involvement in mediating aPL-induced monocyte activation [2,37]. TLR1, TLR2, and TLR6 colocalize with aPL IgG; antibodies blocking TLR1, TLR2, and TLR6 decrease aPL-mediated upregulation of TNF and TF in human monocytes [38]. AnnexinA2, TLR4, and ApoER2′ have also been investigated in vivo. Animals deficient in any of these molecules are only partially protected against aPL thrombogenic effects, suggesting redundancy in the signalling cascade [2]. The role of TLR2 and TLR4 was confirmed in a recent ex vivo study: peripheral blood mononuclear cells from APS patients display an increased mRNA expression of these innate immunity receptors and a markedly raised phosphorylation level of IRAK-1, a major mediator in the TLR transduction pathway [38].

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To date, two cell membrane receptors appear to mediate aPL interaction with platelets. ApoER2′, a member of the low-density lipoprotein (LDL) receptor family, recognizes a positively charged patch of lysine residues in β2GPI-DV via its LDL-binding DI. An inhibitor of LDL receptors also blocks the platelet activation and thromboxane synthesis induced by aPL. Lastly, β2GPI binds directly to glycoprotein (GP) Iba, a subunit of GPIb-IX-V platelet receptor. The role of GP in APS pathogenesis is supported by in vivo findings: thrombus formation is not affected by aPL infusion in GPIIb/IIIa-deficient mice and pretreatment with a monoclonal anti-GPIIb/IIIa antibody inhibits aPL-mediated reduced thrombus formation [21].

3.6  INTRACELLULAR PATHWAYS Upon the engagement of β2GPI receptors on target cells, aPL lead to the recruitment of nuclear factor κB (NFκB) and p38 mitogen-activated protein kinase (MAPK) in both EC and monocytes [2]. aPL engagement of NFκB occurs via a clathrin-dependent endocytic pathway, a mechanism requiring CD14 (TLR4 coreceptor) and AnnexinA2 [39]. The phosphatidylinositol 3-kinase (PI3K)-AKT pathway is an additional signalling cascade used by aPL. It culminates in the recruitment of mammalian target of rapamycin (mTOR), a kinase-modulating cellular growth, proliferation, and apoptosis. In human microvascular EC, stimulation with aPL IgG results in PI3K-mediated activation of two components of the mTOR pathway, S6 ribosomal protein (S6RP), and AKT [40]. Lastly, human monoclonal aPL and IgG fractions of APS patients induce transcription of NLRP3 and caspase-1 via the activation of endosomal NADPHoxidase-2 (NOX2), resulting in inflammasome activation. Accordingly, mononuclear cells from APS patients show an increased expression of caspase-1 and NLRP3, with a threefold increased serum concentration of IL-1β [41]. The heterogeneity in the clinical spectrum of APS suggests that IgG from thrombotic versus obstetric patients might elicit different biological effects. In vitro support for this intriguing theory was first raised using monocytes: IgG from thrombotic APS patients, but not IgG from patients with obstetric APS, asymptomatic aPL carriers or healthy controls, cause NFκB and p38MAPK phosphorylation and TF upregulation [42]. The differential effects of IgG have been recently described using first-trimester trophoblast cells: aPL from women with pure obstetric APS, differently from those purified from thrombotic patients, which significantly reduce trophoblast invasion via TLR4 [43].

3.7  TWO-HIT HYPOTHESIS Thrombotic events occur occasionally in patients with aPL, despite the persistent presence of autoantibodies. To tentatively explain this apparent paradox, a two-hit hypothesis has been formulated. In this, aPL, which act as the first

aPL Pathogenic Mechanisms  Chapter | 3  41

hit, might induce a thrombophilic condition that is not sufficient to trigger a clinically evident thrombosis. A second hit (namely, an additional thrombophilic condition) is required for clotting to take place [2]. Support for this hypothesis comes from animal models. aPL exert their pathogenic prothrombotic potential exclusively in animals already primed with LPS or mechanical, chemical, or photochemical trauma [2,19]. As infections frequently precede aPL-associated events, it has been suggested that infectious processes could be the second hit in humans [44]. This hypothesis fits well with the potential involvement of TLR2 and TLR4 in EC and monocyte activation by β2GPIdependent aPL: β2GPI amount on resting endothelium is not enough to allow a sufficient aPL binding in order to trigger clotting. LPS may increase β2GPI vascular distribution by upregulating TLR2 and TLR4, thus overcoming the threshold for thrombosis [34]. In line with this hypothesis, LPS priming leads to the upregulation of β2GPI expression in murine tissues [45]. It is conceivable that the gut’s resident microbiota could affect LPS uptake, since it is the main source of LPS in healthy individuals. Gut microbiota is indeed increasingly recognized as a major player in the development of autoimmunity; commensal bacteria might contribute to APS pathogenesis inducing autoreactive CD4+ T-cells and anti-β2GPI antibody production via molecular mimicry mechanisms or favouring conformational changes in β2GPI. Consistent evidence comes from in vivo models: depletion of gut microbiome with broadspectrum antibiotics in APS-prone animals markedly prevents thrombotic events, increases survival, and reduces anti-β2GPI IgG titres [46]. The two-hit hypothesis does not apply to obstetric APS: aPL IgG induces foetal loss in naive pregnant mice without requiring a second hit. β2GPI is largely expressed in placental tissues even in physiological conditions and binding of labelled exogenous β2GPI infused into naive pregnant mice to trophoblast and EC has been documented in vivo [45]. The high expression of β2GPI at the placental level together with pregnancy hormonal and blood-flow modifications might be sufficient to favour autoantibody pathogenic activity [4].

3.8  GENETICS AND EPIGENETICS The relevance of a genetic background for APS was first postulated in 1966, when Harvey described a family whose members, some with previous thrombosis, tested falsely positive for syphilis. When aPL positivity rates are evaluated in family members of APS patients, relatives of probands are more likely to carry aPL. A segregation study on seven families with at least two members with APS (diagnosed according to a semi-quantitative scoring index different from international criteria) rejects an environmental and autosomal recessive hypothesis, suggesting an autosomal dominant model of disease inheritance. Since then, many genetic studies have focused on major histocompatability complex genes that identify a strong association of APS with human leucocyte antigen (HLA)

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genes. DRB1*04, DR7, DQB1*0301/4, DQB1*0604/5/6/7/8/9, DQA1*0301/2 are described with increased frequency in patients with primary APS; antiβ2GPI antibody positivity is strongly associated with DRB1*1302 and DQB1*0604/0605 haplotypes in African-American and white British patients with primary APS, while in Caucasians and Mexican-Americans, DQB1*0302 strongly correlates with anti-β2GPI antibodies. Associations with non-HLA genes have also been investigated, with particular attention paid to singlenucleotide polymorphisms (SNP) in β2GPI. However, studies on Cys/Gly, Trp/ Ser, and Val/Leu SNP provide conflicting results. A recent meta-analysis, specifically focusing on Val247Leu polymorphism, concludes that APS patients have a significantly higher prevalence of Val/Val genotype compared with controls; in particular, APS patients carrying anti-β2GPI antibodies have a higher prevalence, while no significant association emerges for arterial or venous thrombosis [47]. Weak associations have been observed with several SNP of immunoglobulin receptor Fcγ RIIA; a proinflammatory genotype, defined by SNP in the genes for IL1β, TNFα, TGFβ, IL6, and TLR4, that has been identified in APS patients within a single family. STAT4 and BLK, both associated with an increased susceptibility to lupus, exhibit a strong genetic association with APS, while a weak association for IRF5 and no association with BANK1 are observed. Many studies report increased positivity rates of aPL in family members of lupus patients. This finding is also confirmed by a genome-wide linkage analyses on 1506 individuals: IgM, but not IgG aCL, exhibit a strong familial aggregation in these lupus pedigrees. Inherited prothrombotic factors may also modulate the thrombotic risk of aPL-positive subjects. FV Leiden is associated with increased thrombotic risk, while gain-of-function mutations in PT and loss-of-function mutations in AT, pC, and pS are linked to venous thrombotic events [48]. Epigenetics may also contribute to APS aetiopathogenesis, lowering the threshold for coagulation-cascade activation. An epigenetic mechanism that potentially plays a role in APS involves miR-19b and miR-20a, both downregulating TF on monocytes; in particular, miR-20a might exert a direct regulating effect as it binds TF mRNA. In monocytes from APS patients, miR-19b and miR-20a are decreased, inversely correlating with TF expression on the cell membrane. However, the comparable expression of miR-19b and miR-20a in monocytes from aPL-negative lupus patients suggests the phenomenon is not APS-specific [49].

3.9 CONCLUSIONS Research in APS has recently focused on the identification of novel mediators involved in the pathogenesis of the syndrome, also aiming at characterizing the diagnostic and prognostic value of each autoantibody subset. Several tools have been proposed to better discriminate between pathogenic versus nonpathogenic antibodies, such as the domain specificity of anti-β2GPI antibodies. Several

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other antibody features are under evaluation; for example, the Fc glycosylation rate of anti-β2GPI antibodies might limit autoantibody pathogenicity [50]. Additional risk factors, such as ABO blood types, have been proposed as determinant of the hazard of each patient to develop clinical events [51]. Hopefully the next few years will yield the progressive unravelling of aPL-mediated pathogenic mechanisms and translate into a more accurate estimate of the thrombotic and obstetric risk, possibly leading to a treatment strategy tailored upon the characteristic profile of each patient.

REFERENCES [1] Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4:295–306. [2] Meroni PL, Borghi MO, Raschi E, Tedesco F. Pathogenesis of antiphospholipid syndrome: understanding the antibodies. Nat Rev Rheumatol 2011;7:330–9. [3] Chighizola CB, Gerosa M, Meroni PL. New tests to detect antiphospholipid antibodies: antidomain I beta-2-glycoprotein-I antibodies. Curr Rheumatol Rep 2014;16:402–9. [4] Meroni PL, Chighizola C. Pathophysiology of the antiphospholipid syndrome (APS). Rev Med Interne 2012;33:A2–A4. [5] de Laat B, Derksen RHWM, van Lummel M, Pennings MTT, de Groot PG. Pathogenic anti-beta2-glycoprotein I antibodies recognize domain I of beta2-glycoprotein I only after a conformational change. Blood 2006;107:1916–24. [6] de Laat B, van Berkel M, Urbanus RT, Siregar B, de Groot PG, Gebbink MF, et al. Immune responses against domain I of β(2)-glycoprotein I are driven by conformational changes: domain I of β(2)-glycoprotein I harbors a cryptic immunogenic epitope. Arthritis Rheum 2011;63:3960–8. [7] Ioannou Y, Romay-Penabad Z, Pericleous C, Giles I, Papalardo E, Vargas G, et al. In vivo inhibition of antiphospholipid antibody-induced pathogenicity utilizing the antigenic target peptide domain I of beta2-glycoprotein I: proof of concept. J Thromb Haemost 2009;7:833–42. [8] Agostinis C, Durigutto P, Sblattero D, Borghi MO, Grossi C, Guida F, et  al. A noncomplement-fixing antibody to β2 glycoprotein I as a novel therapy for antiphospholipid syndrome. Blood 2014;123:3478–87. [9] Zandman-Goddard G, Pierangeli SS, Gertel S, Blank M. Tolerogenic dendritic cells specific for β2-glycoprotein-I domain-I, attenuate experimental antiphospholipid syndrome. J Autoimmun 2014;54:72–80. [10] Andreoli L, Chighizola CB, Nalli C, Gerosa M, Borghi MO, Pregnolato F, et  al. Clinical characterization of antiphospholipid syndrome by detection of IgG antibodies against β2-glycoprotein I domain 1 and domain 4/5: ratio of anti-domain 1 to anti-domain 4/5 as a useful new biomarker for antiphospholipid syndrome. Arthritis Rheumatol 2015;67:2196–204. [11] Pengo V, Ruffatti A, Tonello M, Cuffaro S, Banzato A, Bison E, et  al. Antiphospholipid syndrome: antibodies to domain 1 of β2-glycoprotein 1 correctly classify patients at risk. J Thromb Haemost 2015;136:161–3. [12] Artenjak A, Locatelli I, Brelih H, Simonič DM, Ulcova-Gallova Z, Swadzba J, et  al. Immunoreactivity and avidity of IgG anti-β2-glycoprotein I antibodies from patients with autoimmune diseases to different peptide clusters of β2-glycoprotein I. Immunol Res 2014;61:35–44.

44  Antiphospholipid Syndrome in Systemic Autoimmune Diseases [13] Pregnolato F, Chighizola CB. Phospholipid autoantibodies (nonanticardiolipin)antiprothrombin antibodies. In: Shoenfeld Y, Meroni PL, Gershwin E, editors. Autoantibodies. Oxford: Elsevier; 2014. p. 741–9. [14] Bevers EM, Zwaal RFA, Willems GM. The effect of phospholipids on the formation of immune complexes between autoantibodies and beta2-glycoprotein I or prothrombin. Clin Immunol 2004;112:150–60. [15] Bertolaccini M. Antibodies to prothrombin. Lupus 2012;21:729–31. [16] Pregnolato F, Chighizola CB, Encabo S, Shums Z, Norman GL, Tripodi A, et  al. Antiphosphatidylserine/prothrombin antibodies: an additional diagnostic marker for APS? Immunol Res 2013;56:432–8. [17] Bertolaccini M, Amengual O, Atsumi T, Binder WL, de Laat B, Forastiero R, et al. “Noncriteria” aPL tests: report of a task force and preconference workshop at the 13th International Congress on Antiphospholipid Antibodies, Galveston, TX, USA, April 2010. Lupus 2011;20:191–205. [18] Velayuthaprabhu S, Matsubayashi H, Sugi T, Nakamura M, Ohnishi Y, Ogura T, et  al. A unique preliminary study on placental apoptosis in mice with passive immunization of antiphosphatidylethanolamine antibodies and anti-factor XII antibodies. Am J Reprod Immunol 2011;66:373–84. [19] Fischetti F, Durigutto P, Pellis V, Debeus A, Macor P, Bulla R, et al. Thrombus formation induced by antibodies to beta2-glycoprotein I is complement dependent and requires a priming factor. Blood 2005;106:2340–6. [20] Tincani A, Spatola L, Prati E, Allegri F, Ferremi P, Cattaneo R, et al. The anti-beta2-glycoprotein I activity in human anti-phospholipid syndrome sera is due to monoreactive low-affinity autoantibodies directed to epitopes located on native beta2-glycoprotein I and preserved during species’ evolution. J Immunol 1996;157:5732–8. [21] Meroni PL, Chighizola CB. Anti-phospholipid antibody mechanisms of thrombosis. In: Mackay I, Rose N, Diamond B, Davidson A, editors. Encyclopedia of medical immunology. New York, NY: Springer; 2014. p. 63–70. [22] Erkan D, Willis R, Murthy VL, Basra G, Vega J, Ruiz-Limon P, et al. A prospective open-label pilot study of fluvastatin on proinflammatory and prothrombotic biomarkers in antiphospholipid antibody positive patients. Ann Rheum Dis 2014;73:1176–80. [23] Yalavarthi S, Gould TJ, Rao AN, Mazza LF, Morris AE, Nunez-Alvarez C, et  al. Antiphospholipid antibodies promote the release of neutrophil extracellular traps: a new mechanism of thrombosis in the antiphospholipid syndrome. Arthritis Rheumatol 2015;67(11). http://dx.doi.org/10.1002/art.39247. [24] Pierangeli SS, Chen PP, Raschi E, Scurati S, Grossi C, Borghi MO, et al. Antiphospholipid antibodies and the antiphospholipid syndrome: pathogenic mechanisms. Semin Thromb Hemost 2008;34:236–50. [25] Giannakopoulos B, Passam F, Rahgozar S, Krilis SA. Current concepts on the pathogenesis of the antiphospholipid syndrome. Blood 2007;109:422–30. [26] Wahl D, Membre A, Perret-Guillaume C, Regnault V, Lecompte T. Mechanisms of antiphospholipid-induced thrombosis: effects on the protein C system. Curr Rheumatol Rep 2009;11:77–81. [27] Arachchillage DRJ, Efthymiou M, Mackie IJ, Lawrie AS, Machin SJ, Cohen H. Anti-protein C antibodies are associated with resistance to endogenous protein C activation and a severe thrombotic phenotype in antiphospholipid syndrome. J Thromb Haemost 2014;12:1801–9. [28] Romay-Penabad Z, Carrera Marin A, Willis R, Weston-Davies W, Machin S, Cohen H, et al. Complement C5-inhibitor rEV576 (coversin) ameliorates in-vivo effects of antiphospholipid antibodies. Lupus 2014;23:1324–6.

aPL Pathogenic Mechanisms  Chapter | 3  45 [29] Viall CA, Chamley LW. Histopathology in the placentae of women with antiphospholipid antibodies: a systematic review of the literature. Autoimmun Rev 2015;14:446–71. [30] Meroni PL, Tedesco F, Locati M, Vecchi A, Di Simone N, Acaia B, et al. Anti-phospholipid antibody mediated fetal loss: still an open question from a pathogenic point of view. Lupus 2010;19:453–6. [31] de la Torre YM, Buracchi C, Borroni EM, Dupor J, Bonecchi R, Nebuloni M, et al. Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6. Proc Natl Acad Sci USA 2007;104:2319–24. [32] La Torre de YM, Pregnolato F, D’Amelio F, Grossi C, Di Simone N, Pasqualini F, et al. Antiphospholipid induced murine fetal loss: novel protective effect of a peptide targeting the β2 glycoprotein I phospholipid-binding site. Implications for human fetal loss. J Autoimmun 2012;38:J209–15. [33] Di Simone N, D’Ippolito S. The pathogenic mechanisms for antiphospholipid antibodies (aPL)-mediated pregnancy loss. In: Meroni PL, editor. Antiphospholipid antibody syndrome. From bench to bedside. Cham: Springer; 2014. p. 37–46. [34] Raschi E, Chighizola CB, Grossi C, Ronda N, Gatti R, Meroni PL, et al. β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: three players in the two hit theory for anti-phospholipid-mediated thrombosis. J Autoimmun 2014;55:42–50. [35] Allen KL, Fonseca FV, Betapudi V, Willard B, Zhang J, McCrae KR. A novel pathway for human endothelial cell activation by antiphospholipid/anti-β2 glycoprotein I antibodies. Blood 2012;119:884–93. [36] Laplante P, Amireault P, Subang R, Dieudé M, Levine JS, Rauch J. Interaction of β2-glycoprotein I with lipopolysaccharide leads to Toll-like receptor 4 (TLR4)-dependent activation of macrophages. J Biol Chem 2011;286:42494–503. [37] Boles J, Mackman N. Role of tissue factor in thrombosis in antiphospholipid antibody syndrome. Lupus 2010;19:370–8. [38] Benhamou Y, Bellien J, Armengol G, Brakenhielm E, Adriouch S, Iacob M, et al. Role of Toll-like receptors 2 and 4 in mediating endothelial dysfunction and arterial remodeling in primary arterial antiphospholipid syndrome. Arthritis Rheumatol 2014;66:3210–20. [39] Brandt KJ, Fickentscher C, Boehlen F, Kruithof EKO, de Moerloose P. NF-κB is activated from endosomal compartments in antiphospholipid antibodies-treated human monocytes. J Thromb Haemost 2014;12:779–91. [40] Canaud G, Kamar N, Anglicheau D, Esposito L, Rabant L, Noel LH, et  al. Eculizumab improves posttransplant thrombotic microangiopathy due to antiphospholipid syndrome recurrence but fails to prevent chronic vascular changes. Am J Transplant 2013;13:2179–85. [41] Müller-Calleja N, Köhler A, Siebald B, Canisius A, Orning C, Radsak M, et al. Cofactorindependent antiphospholipid antibodies activate the NLRP3-inflammasome via endosomal NADPH-oxidase: implications for the antiphospholipid syndrome. Thromb Haemost 2015;113:1071–83. [42] Lambrianides A, Carroll CJ, Pierangeli SS, Pericleous C, Branch W, Rice J, et al. Effects of Polyclonal IgG derived from patients with different clinical types of the antiphospholipid syndrome on monocyte signaling pathways. J Immunol 2010;184:6622–8. [43] Poulton K, Ripoll VM, Pericleous C, et al. Purified IgG from patients with obstetric but not IgG from non-obstetric antiphospholipid syndrome inhibit trophoblast invasion. Am J Reprod Immunol 2015;73:390–401. [44] Shoenfeld Y, Blank M, Cervera R, Font J, Raschi E, Meroni PL. Infectious origin of the antiphospholipid syndrome. Ann Rheum Dis 2006;65:2–6. [45] Agostinis C, Biffi S, Garrovo C, Durigutto P, Lorenzon A, Bek A, et al. In vivo distribution of β2 glycoprotein I under various pathophysiologic conditions. Blood 2011;118:4231–8.

46  Antiphospholipid Syndrome in Systemic Autoimmune Diseases [46] Ruff WE, Vieira SM, Kriegel MA. The role of the gut microbiota in the pathogenesis of antiphospholipid syndrome. Curr Rheumatol Rep 2014;17:472. [47] Chamorro A-J, Marcos M, Mirón-Canelo J-A, Cervera R, Espinosa G. Val247Leu β2-glycoprotein-I allelic variant is associated with antiphospholipid syndrome: systematic review and meta-analysis. Autoimmun Rev 2012;11:705–12. [48] Soriano A, Blank M, Shoenfeld Y. Genetics and origin of antiphospholipid syndrome. In: Meroni PL, editor. Antiphospholipid antibody syndrome. From bench to bedside. Cham: Springer I; 2014. p. 1–12. [49] Meroni PL, Penatti AE. Epigenetics and systemic lupus erythematosus: unmet needs. Clin Rev Allergy Immunol 2015. http://dx.doi.org/10.1007/s12016-015-8497-4. [50] Fickentscher C, Magorivska I, Janko C, Biermann M, Bilyy R, Nalli C, et  al. The pathogenicity of anti-β2GP1-IgG autoantibodies depends on Fc glycosylation. J Immunol Res 2015;7:1–12. [51] Nascimento NM, Bydlowski SP, Soares RP, de Andrade DC, Bonfá E, Seguro LP, et al. ABO blood group in primary antiphospholipid syndrome: influence in the site of thrombosis? J Thromb Thrombolysis 2015;40:374–8.

Chapter 4

Laboratory Markers With Clinical Significance in the Antiphospholipid Syndrome Olga Amenguala, Maria L. Bertolaccinib and Tatsuya Atsumia a

Division of Rheumatology, Endocrinology and Nephrology, Hokkaido University Graduate School of Medicine, Sapporo, Japan bGraham Hughes Lupus Research Laboratory, Division of Women’s Health, King’s College London, The Rayne Institute, St Thomas’ Hospital, London, United Kingdom

4.1 INTRODUCTION Antiphospholipid syndrome (APS) is an autoimmune disorder clinically characterized by recurrent arterial and/or venous thrombosis and/or pregnancy complications. The demonstration of circulating antiphospholipid antibodies (aPL) is mandatory for the diagnosis of APS. The latest classification criteria for definite APS (Sydney-revised Sapporo criteria [1]) includes the persistent presence of lupus anticoagulant (LA), anticardiolipin antibodies (aCL), and/or anti-β2glycoprotein I (β2GPI) antibodies (anti-β2GPI) of IgG or IgM isotype at medium to high titres in the patient’s plasma [1] (Table 4.1). The aPL family is an heterogeneous group of autoantibodies, which despite their name, are not directed against negatively charged phospholipids, but recognize several plasma proteins with affinity for anionic phospholipids. Among them, β2GPI and prothrombin are regarded as the main antigenic targets for the majority of aPL and subgroups of anti-β2GPI and antiprothrombin antibodies display LA in vitro [5–8]. The aPL were first discovered in 1906, when Wassermann et al. [9] noticed that sera from syphilitic patients could agglutinate a lipoid tissue extract. In the early 1940s, Pangborn [10] found that the relevant antigenic component of the tissue extracts used in these tests was a phospholipid called cardiolipin. The antibodies reactive to cardiolipin were termed reagins. Reagins in serum samples can be detected with the antigen mixture of cardiolipin, cholesterol, and phosphatidylcholine (Venereal Disease Research Laboratory, or VDRL antigen), resulting in a visible flocculation in in vitro assays. Although reagins are found Antiphospholipid Syndrome in Systemic Autoimmune Diseases. DOI: http://dx.doi.org/10.1016/B978-0-444-63655-3.00004-1 © 2017 2016 Elsevier B.V. All rights reserved.

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TABLE 4.1 Revised Classification Criteria for the Antiphospholipid Syndrome Clinical Criteria 1. Vascular thrombosis ≥1 clinical episodes of arterial, venous, or small-vessel thrombosis in any tissue or organ confirmed by objective validated criteria by imaging or histopathology in the absence of significant evidence of inflammation in the vessel wall. 2. Pregnancy morbidity a. ≥1 unexplained deaths of a morphologically normal foetus at or beyond the 10th week of gestation, or b. ≥1 premature births of a morphologically normal neonate before the 34th week of gestation due to eclampsia, severe preeclampsia, or placental insufficiency, or ≥3 unexplained consecutive spontaneous abortions before the 10th week of gestation (maternal anatomic or hormonal abnormalities and paternal and maternal chromosomal causes excluded). Laboratory Criteria 1. Lupus anticoagulant present in plasma, on ≥2 occasions at least 12 weeks apart, detected according to the guidelines of the International Society on Thrombosis and Haemostasis [2,3]. 2. IgG and/or IgM anticardiolipin antibodies present in medium or high titre in serum or plasma, on ≥2 occasions at least 12 weeks apart, measured by a standardized ELISA [4]. 3. IgG and/or IgM anti-β2glycoprotein I antibodies present in titre >99th percentile, in serum or plasma, on ≥2 occasions at least 12 weeks apart, measured by a standardized ELISA [4]. Antiphospholipid syndrome is present if at least one of the clinical criteria and one of the laboratory criteria are met. ELISA, enzyme-linked immunosorbent assay. From Miyakis S, Lockshin MD, Atsumi T, Branch DW, Brey RL, Cervera R, et al. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS). J Thromb Haemost 2006;4(2):295–306.

predominantly in association with syphilitic infection, they are not specific to antigens of Treponema pallidum. The development of tests for treponemaspecific antigens in the 1950s, revealed that individuals with persistently biological false-positive serological test for syphilis (BFP-STS), when monitored for many years, often developed systemic lupus erythematosus (SLE). Some patients with SLE and persistent BFP-STS had recurrent, spontaneous foetal loss, thrombocytopenia, and thromboembolic events [11]. In 1952, Conley and Hartmann [12] described the lupus inhibitor in two SLE patients. These patients had prolonged coagulation tests and BFP-STS results. Feinstein and Rapaport coined the term lupus anticoagulant (LA) for this inhibitor [13], the first of many major misnomers in this field. These antibodies, termed LA because they were originally detected in the plasma of patients with SLE, are not confined to patients with SLE. These anticoagulants prolong coagulation reactions in vitro, but do not inhibit individual coagulation factors and are not associated with a

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bleeding diathesis unless a second coagulation defect is also present. The paradoxical association between LA and thrombosis in SLE patients was suspected in the early 1960s [14], but it was not until 1980 that this association was widely recognized [15]. The laboratory diagnosis of LA became clinically relevant and LA phenomenon caused by antibodies reacting to negatively charged phospholipids was reported [16]. In the early 1980s, a radioimmunoassay and an enzyme-linked immunosorbent assay (ELISA) using solid-phase phospholipids were established [17– 19]. Cardiolipin was selected in these assays to directly detect circulating aCL because it is the main antigen in the serological test for syphilis. Investigators noticed that aCL cross-reacted with negatively charged phospholipids, such as phosphatidylserine and phosphatidylglycerol [20]. Thus, the name of aCL was expanded to aPL. Further work demonstrated that LA and aPL defined two distinct but related patient populations, each associated with an increased risk of thrombosis, and the aCL ELISA was established as a routine laboratory test in addition to those for LA [21]. In 1990, the requirement of β2GPI for the binding of autoimmune aCL to the phospholipids in the solid-phase assays was described [5,22,23]. When β2GPI is adsorbed on polyoxygenated polystyrene plates or interacts with negatively charged surfaces, the antigenic sites are exposed [24]. Prothrombin is the second major antigenic target of autoimmune aPL. Many other proteins with high affinity for phospholipids were recognized in the serum of patients with APS, including annexin V, protein S, protein C, high and lowmolecular-weight kininogens, and factor XII [7,25–27]. The expression of epitopes by at least some of these phospholipid-binding proteins aPL does not depend on the presence of phospholipids. In the laboratory, there is not one unique or gold-standard test for the diagnosis of APS. Individual patients often have a mixture of antibodies with different specificities. However, diagnostic aPL can be broadly categorized into two groups. Antibodies that are detected by solid-phase assays such as aCL, anti-β2GPI, antiprothrombin antibodies, or phosphatidylserine-dependent antiprothrombin antibodies (aPS/PT), and those antibodies detected by their ability to prolong phospholipid-dependent coagulation tests, known as LA. In this chapter, we detail the currently available laboratory assays for the detection of aPL, and discuss the value of each aPL as laboratory markers for APS.

4.2  aPL DETECTED BY SOLID-PHASE IMMUNOASSAYS This group comprises autoimmune antibodies assessed using a solid (ie, immobilized) phase methodology – mainly ELISA procedures. Chemiluminescencebased automated technologies are recently emerging. This technology seems to perform well for available aPL testing and results are comparable with the classical ELISA. Moreover, they are easy to apply and have the advantage of being fully automated [28].

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4.2.1  Anticardiolipin Antibody Assay The first aCL test was a radioimmunoassay, using cardiolipin as antigen, a mixture of gelatin/phosphate-buffered saline (PBS) to dilute patient’s serum and radiolabelled antihuman IgG or IgM to detect bound aCL. The test was subsequently modified. Foetal calf or adult bovine serum replaced gelatin/PBS as sample diluents and enzyme-labelled antihuman IgG or IgM antibodies were used [29]. The use of foetal calf or adult bovine serum increased the optical density reading of bound aCL [30]. Although unknown at the time, this increase of the signal was probably due to the presence of antibodies specific to β2GPI [5,22,23]. Therefore, the aCL assay detects not only antibodies to cardiolipin, but also those directed against β2GPI bound to cardiolipin, present in the serum or plasma samples and/or present in the sample diluent or blocking buffers. Positive IgG and/or IgM aCL at medium or high titres in at least two occasions, at least 2 weeks apart, is one of the laboratory criteria included in the current classification criteria for definite APS [1]. According to these criteria, detected aCL must be dependent on the presence of β2GPI. The assay should include wells without β2GPI to distinguish APS-unrelated aCL from the β2GPIdependent ones. Numerous in-house and commercial methods for the quantification of aCL have been developed [31], and international workshops have been conducted to standardize and evaluate the various modifications of the tests [32]. International standards consisting of affinity-purified aCL immunoglobulin for the calculation of IgG or IgM aCL units were first developed in 1987 [33]. Calibrator standards prepared by chimeric-monoclonal antibodies directed to β2GPI were introduced to assist laboratories worldwide in establishing the aCL assay [34–36]. The aCL ELISA is a sensitive, simple, and rapid method to aid the diagnosis of APS in patients with relevant symptoms. However, aCL are not specific for APS, and false-positive results can be found in patients with infections or other autoimmune diseases [37]. The IgM isotype of aCL is less often associated with the APS manifestations than the IgG isotype [38]. There is evidence that high titres of IgG aCL are associated with increased risk of arterial thrombosis and venous thrombosis [38–40] and represent a risk factor for obstetric complications [41]. In addition, the value of low aCL titres is still debated [42]. The value of IgA aCL test in diagnosing APS is uncertain. Numerous studies have investigated the possible association between raised levels of IgA, showing substantial differences in the performance of IgA assays [42–44]. Some reports found an association between IgA aCL and some clinical features related to APS, specifically thrombosis, pregnancy loss, and thrombocytopenia [45–49]. Other studies failed to find any relationship between the presence of IgA aCL and clinical signs of APS [50,51]. The ethnic group composition of patients can influence the isotypic distribution of aCL. IgA aCL appeared to be the most prevalent isotype in African-American [52], Afro-Caribbean [53], and Japanese patients [54]. Isolated IgA aCL are uncommon. In most cases where major APS

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manifestations occur, aCL IgA are found in association with IgG and/or IgM. Based on the current evidence, testing for IgA aCL can be restricted to patients with a strong suspicion of APS and negative aPL tests [42,55].

4.2.2 Anti-β2GPI Antibody Assay Since β2GPI has been shown to be the relevant antigen in the aCL test, specific ELISAs for detecting anti-β2GPI have been developed [56,57]. There is vast evidence confirming that anti-β2GPI are more specific for thrombosis and pregnancy complications [58–60], and are less likely to be detected in infections or be drug-induced [61]. For this reason, IgG and IgM anti-β2GPI were included in the revised criteria for APS [1]. Human β2GPI is a single-chain glycoprotein composed of 326 amino acid residues with five oligosaccharide attachment sites [62,63]. β2GPI belongs to a superfamily of proteins characterized by repeating stretches of approximately 60 amino acid residues, each with a set of 16 conserved residues and 2 fully conserved disulphide bonds. These repeating units are designated as short consensus repeat (SCR), complement control protein repeats, or sushi domains. β2GPI consists of five SCR domains. The first four are regular SCR domains with respect to their amino acid sequences and are characterized by a framework of four conserved halfcysteine residues related to the formation of two internal disulphide bridges [64]. The fifth C-terminal domain deviates significantly from the common SCR folds. Domain V is aberrant, including 82 amino acid residues, 6 half cysteins, and is stabilized by 3 internal disulphide bonds. Domain V is responsible for β2GPI binding to anionic phospholipids via a cluster of positively charges amino acids [65–67]. Antibodies against β2GPI recognize cryptic epitope(s) on the β2GPI molecule that are exposed when β2GPI interacts with lipid membranes composed of negatively charged phospholipids, or when β2GPI is adsorbed on a polyoxygenated polystyrene plate treated with γ-irradiation or electrons [68,69]. The antiβ2GPI ELISA is preferred over the aCL ELISA because the microtitre plates used are coated with a single and well-defined antigen. Despite the higher specificity of the solid-phase anti-β2GPI assay when compared with the aCL ELISA, the anti-β2GPI assay has some diagnostic weaknesses. Anti-β2GPI are a heterogeneous group of antibodies containing subclasses directed at different epitopes located in all five domains of β2GPI [69–72]. The anti-β2GPI antibody ELISA detects all antibodies reacting with β2GPI, including nonpathogenic antibodies and low-affinity anti-β2GPI, which makes them less suitable as a diagnostic test because it was reported that antiβ2GPI with high avidity are the most specific antibodies for APS detection [73]. Several attempts have been made to standardize and harmonize the antiβ2GPI ELISA, but a considerable degree of inconsistency still exists. A high interassay and interlaboratory variation in the anti-β2GPI assay was reported by the European forum on aPL [74]. Recommended protocols for the measurement

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of anti-β2GPI have been published [44,75,76] and, recently, a set of guidelines was made available from the International Society of Thrombosis and Haemostasis [77]. In addition, efforts have been made to identify the optimal candidate material for standardization of anti-β2GPI assays and the preparation of adequate quality controls for those materials [42,78]. Anti-β2GPI results are reported in a range of different and noncomparable arbitrary units (ie, IU/mL, U/mL, SGU, SMU, SAU, ng/mL, or μg/mL) depending on the assay manufacturer or the laboratory performing the test [4]. The establishment of international units for measurement of anti-β2GPI will greatly improve the comparability of results among different assays. In addition to IgA aCL, IgA anti-β2GPI have not been included in the criteria for APS classification. At the present, the diagnostic value of the IgA antiβ2GPI is limited due to the lack of standardized assays, limited availability of appropriate standards tests, and interlaboratory variety of the results using different commercial assays. IgA anti-β2GPI are prevalently found in SLE patients and associated with an increase in thromboembolic events. In most cases, the simultaneous presence of IgA with IgG and/or IgM anti-β2GPI makes interpreting this finding difficult [55]. However, several studies reported clinical manifestation of APS in patients with isolated IgA anti-β2GPI [48,79,80], suggesting that testing for IgA anti-β2GPI could contribute to the assessment of risk of thrombosis and/or pregnancy morbidity for SLE patients [42]. A subgroup of IgA anti-β2GPI that binds to domain IV/-V of β2GPI might have clinical relevance [81], however, the available data is not sufficient for a definite conclusion [82–84].

4.2.3  Antibodies Against Domain I of β2GPI β2GPI is recognized as the major antigen in APS, but not all patients carrying anti-β2GPI develop aPL-related clinical manifestations. As discussed previously, anti-β2GPI comprise a heterogeneous group of autoantibodies reacting toward different epitopes on β2GPI [6,85–89]. The N-terminal domain of β2GPI-designed domain I (DI) has been identified as the most relevant antigenic target involved in β2GPI/anti-β2GPI antibody binding [87,90]. Numerous studies suggested that antibodies against DI (anti-DI antibodies) are important in the pathogenesis of APS. Anti-DI antibodies induce the prolongation of clotting time in vitro [91]. The administration of a synthetic-DI peptide in a naive mouse inhibits the thrombus enhancement mediated by polyclonal human aPL IgG fractions [92]. In addition, when infused together with lipopolysaccharide, human monoclonal anti-DI IgG induced clotting and foetal loss in naive mice, providing a direct demonstration of the pathogenic effects of anti-DI antibodies [93]. The presence of anti-DI antibodies associates with thrombosis [6]. In an international multicentre study, a strong association between anti-DI antibodies and a history of thrombosis was reported. To a lesser extent, anti-DI antibodies

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were found to be related to pregnancy complications [94]. It is increasingly recognized that patients with multiple aPL are at higher risk for developing clinical complications [95], and these patients tend to have higher prevalence and higher titres of anti-DI antibodies [96]. A number of different methods for the detection of anti-DI antibodies have been reported. The majority of studies used two-step ELISA tests with a different source of antigen coated to hydrophilic and hydrophobic microtitre plates. Anti-DI antibodies react toward their target epitope only when DI is coated onto hydrophobic, but not hydrophilic, plates. In addition, a capture ELISA using N-terminally biotinylated DI on streptavidin plates has been described. In contrast with nonbiotinylated DI ELISA, the capture assays are able to discriminate between APS patients and controls. Two additional tests have also been established, a liquid phase inhibition assay using whole β2GPI immobilized on the solid-phase and a synthetic β2GPI-DI as inhibitor and the novel chemiluminescence immunoassay based on the BIOFLASH system (Inova Diagnostics, United States), using recombinant DI coupled to paramagnetic beads [97]. Anti-DI antibodies have proven high specificity for the diagnosis of APS and, at high titres, enabled the identification of APS patients with a more aggressive clinical presentation [96,98]. On the other hand, tests for anti-DI have shown a lower sensitivity compared with anti-β2GPI tests. In fact, some anti-β2GPI found in patients with APS react with β2GPI epitopes other than DI [99] and therefore, not all anti-β2GPI antibodies are detected by the anti-DI test. The number of studies is still limited and further research is needed to clearly define the clinical utility of testing for anti-DI antibodies.

4.2.4  Antiprothrombin Antibodies Prothrombin (factor II) is another important antigenic target for aPL in APS. Prothrombin is a vitamin K-dependent single-chain glycoprotein of 579 amino acid residues with a molecular weight of 72-kDa, and is present at a concentration of approximately 100 μg/mL in normal plasma [100]. Antibodies targeting human prothrombin are detected by ELISA and strongly associated with the APS. The antiprothrombin antibody ELISA identifies two antibody populations, those binding only to prothrombin (antiprothrombin antibodies or aPT-A), and those that bind to phosphatidylserine-prothrombin complexes (aPS/PT) [101,102]. Here, aPT-A and aPS/PT belong to different populations of autoantibodies, even though they can both be present in the same patient at once [102,103]. Several studies investigated the relationship between APS-related clinical features and the presence of aPT-A with conflicting conclusions. In two prospective studies, the presence of aPT-A appeared as a predictor of thromboembolic events in patients with aPL, mainly in those patients positive for LA [59,104]. A recent systematic review of the literature suggested that antibodies

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to prothrombin, both aPT-A and aPS/PT, increase the risk of thrombosis. aPS/ PT seemed to represent a stronger risk factor for arterial and/or venous thrombosis when compared with aPT-A [105], concluding that aPS/PT might be useful in establishing the thrombotic risk of patients with previous thrombosis or SLE [105]. The clinical utility of the aPS/PT in the diagnosis of APS has been reported. Several studies found an association between aPS/PT and thrombosis and pregnancy complications [102,103]. aPS/PT have also been significantly correlated with LA [102], suggesting that these antibodies may be a surrogate test for LA. Whilst aPS/PT appears to confer a higher risk of venous thrombosis and pregnancy loss in LA-positive patients when compared with the LA-negative group, the risk is present in both groups, with titres of aPS/PT significantly higher in the LA-positive group [106]. Furthermore, LA and aPS/PT were found to be independently associated to thrombosis and pregnancy loss after multivariate analysis [106]. In addition, a retrospective evaluation of 23 combinations of aPL in patients with SLE showed that the combination LA, anti-β2GPI, and aPS/PT has the best diagnostic accuracy for APS, even better than that when applying the Sydney laboratory criteria tests [107]. Preliminary unpublished data from an in-progress, multicentre study evaluating the diagnostic value of aPS/PT indicated that aPS/PT represent a population of autoantibodies associated with APS and that testing for IgG aPS/ PT could be a useful tool for identification of patients at-risk of developing APS [108]. Based on the available data, routine testing for aPT-A is not recommended for the evaluation of APS. On the other hand, the presence of aPS/PT represents a risk for thrombosis; therefore testing for aPS/PT can contribute to assess the risk of thrombosis and to better identify patients with APS [42].

4.2.5  Antibodies to Negatively Charged Phospholipids Other Than Cardiolipin Antibodies directed against negatively charged phospholipids, such as phosphatidic acid (anti-PA), phosphatidylinositol (anti-PI), and phosphatidylserine (anti-PS), have been reported in patients with APS. Assays using this phospholipids, particularly PS, haven been suggested to be more specific for APS when compared with the aCL assay. The properties of these antibodies have been reviewed extensively [43]. Some reports have suggested that assays using phospholipids other than cardiolipin may help identify women with recurrent pregnancy loss [109], whilst other studies did not observe improvement in the diagnosis performance when these were measured simultaneously with aCL and LA [43]. Based on the current evidence, testing for anti-PA, anti-PI, and anti-PS antibodies in the initial diagnostic work-up for APS is not clinically useful because these antibodies may have overlapping properties with the markers considered diagnostic for this disease [42,110,111].

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4.2.6  Antibodies to Phosphatidylethanolamine Antibodies directed against phosphatidylethanolamine (anti-PE), a zwitterionic phospholipid with an important role in phospholipid-dependent reactions of the protein-C system, have been described as the sole aPL in plasma of some patients suspected to have APS. Although anti-PE have been associated with thrombosis and pregnancy morbidity [112,113], other reports failed to find any association. The current available data do not provide evidence to support the association between anti-PE and thrombosis or pregnancy morbidity [42].

4.2.7  Other aPL Specificities A number of other autoantibodies have been reported in patients with APS, including antibodies to annexin V [114,115], high- and low-molecular-weight kininogens, prekallikrein and factor XI [26,116], vascular heparan sulphate proteoglycan [117], heparin [118], factor XII [27,119–121], and thrombin [122]. Some data suggest that autoantibodies could be directed against components of protein-C pathway [25], which includes protein C [123], protein S [124,125], and thrombomodulin [126].

4.3  LUPUS ANTICOAGULANT The LA test measures the ability of aPL to prolong phospholipid-dependent clotting reactions. The heterogeneous nature of these aPL makes it necessary to perform more than one coagulation test to reach the diagnosis according to the classification criteria [2,3]. A number of features need to be demonstrated: (1) prolongation of a phospholipid-dependent clotting time; (2) evidence of inhibition, shown by mixing studies; (3) evidence of phospholipid dependence; and (4) exclusion of specific inhibition of any one coagulation factor. In principle, the laboratory tests to detect LA should use a sensitive screening test followed by a specific confirmation test [127]. There are several tests available for the detection of LA, all with variable sensitivity. The most commonly used is the activated partial thromboplastin time, followed by the dilute Russell’s viper venom time. The presence of LA should always be confirmed by performing the assays in the presence of excess of phospholipids, with a correction of the prolongation of the times as a result [128]. It is important to mention that in some subjects receiving oral anticoagulation, accurate detection of the LA might be challenging. Guidelines from the International Society on Haemostasis and Thrombosis indicates that LA can be tested on undiluted plasma if the international normalized ratio (INR) is 10 weeks of gestation), suggesting that antibody-mediated damage can similarly affect different pregnancy periods. After this systematic review, a more recent, larger multicentre and multiethnic prospective population-based study shed light on the association between aPL and stillbirth [30] showing that elevated levels of aCL and anti-β2GPI antibodies confer a three- to fivefold increased risk. Strengths of this study were the inclusion of 582 cases and the centralization of aPL testing for homogeneous

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assessment of positive results. However, the lack of LA testing and the absence of any longitudinal confirmation of aPL persistence are limitations that do not allow the precise identification of the true positive patients. In order to minimize the interlaboratory variability, the APLA ObTF [8] limited its analysis only to studies in which patients have been investigated for all three criteria tests. A total of 10 papers tested a complete aPL panel in women with foetal death and a stratification of the risk according to the aPL profile was performed for a majority of them. In conclusion, a positive association between foetal death and aPL seems likely according to a discrete number of studies. Nevertheless, future studies will be crucial to clarify the picture.

7.4  PREECLAMPSIA AND PI Uteroplacental insufficiency inducing IUGR, premature delivery [31], and early PE are other possible pregnancy complications for women with aPL positivity. As already detailed previously in chapter ‘Mechanisms of Action of Antiphospholipid Antibodies’, poor placentation has been demonstrated to impair the physiological changes of spiral arteries into low-resistance vessels during the first half of pregnancy. The absence of this remodelling impairs uteroplacental blood flow, and consequently the physiological foetal growth. PE (defined in Table 7.2) is a major obstetric problem leading to substantial maternal and perinatal morbidity and mortality worldwide. Maternal and perinatal outcomes after the onset of PE depend on gestational age at time of onset, severity, presence or absence of preexisting medical disorders, and quality of management. It has been estimated that PE affects about 2–8% of the pregnancies in the general obstetric population [32], while for pregnant women with APS (with or without SLE), it has been reported in 20–50% of cases [33]. In particular, there is a higher rate of early (ie,