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 Platelets
Fourth Edition
Platelets

FOURTH EDITION

Editor
ALAN D. MICHELSON, MD
Professor of Pediatrics, Harvard Medical School/Boston Children’s Hospital
Professor of Medicine, Harvard Medical School/Brigham and Women’s Hospital
Director, Center for Platelet Research Studies
Director, Thrombosis and Anticoagulation Program
Dana-Farber/Boston Children’s Cancer and Blood Disorders Center
Boston, Massachusetts, USA

Associate Editors

MARCO CATTANEO, MD
Professor of Internal Medicine
Director, Unit of Internal Medicine
Ospedale San Paolo
Università degli Studi di Milano
Milan, Italy

ANDREW L. FRELINGER III, PhD

Assistant Professor of Pediatrics, Harvard Medical School
Associate Director, Center for Platelet Research Studies
Staff Scientist, Dana-Farber/Boston Children’s Cancer and Blood Disorders Center
Boston, Massachusetts, USA

PETER J. NEWMAN, PhD

Vice President for Research, BloodCenter of Wisconsin
Jacquelyn Fredrick Endowed Chair for Foundational Research and Associate
Director, Blood Research Institute
Milwaukee, Wisconsin, USA
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Cover image: Platelets Adherent to a Surgical Suture. Colorized scanning electron micrograph of activated, aggregated and spread
platelets (blue) adherent to a synthetic-polymer surgical suture (straw-colored). Original magnification x800. Sample provided by
Alan D. Michelson, Emma E. Forde and Andrew L. Frelinger, Center for Platelet Research Studies, Dana-Farber/Boston Children's
Cancer and Blood Disorders Center, Harvard Medical School, Boston, Massachusetts, U.S.A. Image provided by Steve
Gschmeissner (https://theworldcloseup.com).
 Contributors
Joseph Alsousou Chapel Hill, NC Carol Briggs
Institute of Translational Medicine United States Department of Haematology
University of Liverpool University College London
Gerald Bertrand
Liverpool Hospitals
EFS
United Kingdom London
Rennes
United Kingdom
Dominick J. Angiolillo France
Division of Cardiology Tomasz Brzoska
Deepak L. Bhatt
University of Florida College of Pittsburgh Heart, Lung and Blood
Department of Cardiovascular
Medicine-Jacksonville Vascular Medicine Institute;
Medicine
Jacksonville, FL Sickle Cell Center of Excellence
Brigham and Women’s Hospital
United States University of Pittsburgh-School of
Harvard Medical School
Amal Arachiche Boston, MA Medicine
Department of Pharmacology United States Pittsburgh, PA
Case Western Reserve University United States
Thomas A. Blair
Cleveland, OH James B. Bussel
Center for Platelet Research Studies
United States Department of Pediatrics
Dana-Farber/Boston Children’s Cancer
Richard H. Aster and Blood Disorders Center Weill Medical College of Cornell
Blood Research Institute Harvard Medical School University
BloodCenter of Wisconsin Boston, MA New York, NY
Department of Medicine United States United States
Medical College of Wisconsin
Kamila Bledzka Marco Cattaneo
Milwaukee, WI
Joseph J. Jacobs Center for Thrombosis Unità di Medicina 2
United States
and Vascular Biology ASST Santi Paolo e Carlo
Tiziano Barbui Department of Molecular Cardiology Dipartimento di Scienze della Salute
FROM Research Foundation Cleveland Clinic Università degli Studi di Milano
Papa Giovanni XXIII Hospital Cleveland, OH Milan
Bergamo United States Italy
Italy
Oliver Borst Subarna Chakravorty
Stefania Basili University Hospital King’s College NHS Foundation
I Clinica Medica Department of Cardiology and Trust
Department of Internal Medicine Cardiovascular Medicine London
and Medical Specialties Eberhard Karls University Tuebingen United Kingdom
SAPIENZA-University of Rome Tuebingen
Rome Germany Noel C. Chan
Italy Thrombosis and Atherosclerosis
Emma G. Bouck
Research Institute
Elisabeth M. Battinelli Department of Pharmacology
Department of Medicine
Division of Hematology Case Western Reserve University
McMaster University
Brigham and Women’s Hospital Cleveland, OH
Hamilton, ON
Harvard Medical School United States
Canada
Boston, MA
Lawrence F. Brass
United States Shruti Chaturvedi
Division of Hematology-Oncology
Anthony A. Bavry Department of Medicine Division of Hematology
Department of Medicine Perelman School of Medicine Johns Hopkins University
University of Florida University of Pennsylvania Baltimore, MD
North Florida/South Georgia Veterans Philadelphia, PA United States
Health System United States Beng H. Chong
Gainesville, FL Department of Medicine
Paul F. Bray
United States St. George Clinical School
Molecular Medicine Program
Wolfgang Bergmeier Division of Hematology and University of New South Wales;
McAllister Heart Institute Hematologic Malignancies in the Department of Haematology
Department of Biochemistry and Department of Internal Medicine NSW Health Pathology
Biophysics University of Utah St George Hospital
University of North Carolina at Salt Lake City, UT Sydney
Chapel Hill United States Australia
vii
viii Contributors

Kenneth J. Clemetson Centro de Hemodonacion Department of Medicine

Department of Haematology IMIB-Murcia Harvard Medical School
University of Berne Inselspital CIBERER (CB15/00055) Boston, MA
Theodor Kocher Institute UCAM United States
University of Berne Murcia
Berne Spain Andreas Greinacher
Switzerland Department of Immunology and
Guido Finazzi Transfusion Medicine
Jeannine M. Clemetson Hematology Unit Universit€atsmedizin
Theodor Kocher Institute Papa Giovanni XXIII Hospital Greifswald
University of Berne Bergamo Germany
Berne Italy
Switzerland Thomas Gremmel
Barry S. Coller Robert Flaumenhaft Department of Internal Medicine II
Allen and Frances Adler Laboratory of Division of Hemostasis and Medical University of Vienna
Blood and Vascular Biology Thrombosis Vienna;
Rockefeller University Hospital Department of Medicine Department of Internal Medicine
Rockefeller University Beth Israel Deaconess Medical Center Cardiology and Nephrology
New York, NY Harvard Medical School Landesklinikum Wiener Neustadt
United States Boston, MA Wiener Neustadt
United States Austria
Gregory J. del Zoppo
Division of Hematology Jane E. Freedman Paul A. Gurbel
Department of Medicine Department of Medicine Inova Center for Thrombosis Research
University of Washington School of University of Massachusetts Medical and Drug Development
Medicine School Inova Heart and Vascular Institute
Harborview Medical Center Worcester, MA Falls Church, VA
Seattle, WA United States United States
United States
Andrew L. Frelinger, III Elizabeth J. Haining
Jenny M. Despotovic Center for Platelet Research Studies
Department of Pediatrics Institute of Cardiovascular Sciences
Dana-Farber/Boston Children’s College of Medical and Dental
Baylor College of Medicine Cancer and Blood Disorders
Houston, TX Sciences
Center University of Birmingham
United States Harvard Medical School Birmingham
Scott L. Diamond Boston, MA United Kingdom
Department of Chemical and United States
Biomolecular Engineering Xu Han
Kathleen Freson
University of Pennsylvania Department of Pharmacology
Department of Cardiovascular
Philadelphia, PA Case Western Reserve University
Sciences
United States Cleveland, OH
Center for Molecular and Vascular
United States
J. Donald Easton Biology
Department of Neurology KU Leuven Paul Harrison
University of California, San Francisco Leuven Institute of Inflammation and
Sandler Neuroscience Center Belgium Ageing
San Francisco, CA College of Medical and Dental
United States Aleksandra Gasecka
First Chair and Department of Sciences
Koji Eto Cardiology University of Birmingham
Department of Clinical Application Medical University of Warsaw Birmingham
Center for iPS Cell Research and Warsaw United Kingdom
Application Poland;
Kyoto University Catherine P.M. Hayward
Laboratory of Experimental Clinical
Kyoto; Departments of Pathology
Chemistry
Department of Innovative Medicine and Molecular Medicine and
Vesicle Observation Centre
Chiba University Graduate School Medicine
Academic Medical Centre of the
of Medicine McMaster University
University of Amsterdam
Chiba Hamilton, ON
Amsterdam
Japan Canada
The Netherlands

Falet
Herve Karin Hoffmeister
Blood Research Institute Meinrad Gawaz
University Hospital Division of Hematology
BloodCenter of Wisconsin Brigham and Women’s Hospital;
Department of Cell Biology, Department of Cardiology and
Cardiovascular Medicine Department of Medicine
Neurobiology, and Anatomy Harvard Medical School
Medical College of Wisconsin Eberhard Karls University
Tuebingen Boston, MA;
Milwaukee, WI Blood Research Institute
United States Tuebingen
Germany BloodCenter of Wisconsin;
Francisca Ferrer-Marin Department of Biochemistry
Unidad de Hematología y Oncología Silvia Giannini Medical College of Wisconsin
M
edica Division of Hematology Milwaukee, WI
Hospital Morales-Meseguer Brigham and Women’s Hospital; United States
 Contributors ix

Anne-Mette Hvas Kumaran Kolandaivelu Charleston, SC

Department of Clinical Biochemistry Department of Cardiovascular United States
Aarhus University Hospital; Medicine
Anqi Li
Department of Clinical Medicine Brigham and Women’s Hospital
Department of Microbiology and
Faculty of Health Harvard Medical School
Immunology
Aarhus University Boston, MA;
Hollings Cancer Center
Aarhus Institute for Medical Science and
Medical University of South Carolina
Denmark Engineering
Charleston, SC
Massachusetts Institute of
Sara J. Israels United States
Technology
Department of Pediatrics and Child
Cambridge, MA Rossella Liani
Health
United States Internal Medicine
University of Manitoba
Department of Medicine and Aging
Winnipeg Milka Koupenova
and Haemostasis and Thrombosis
Canada Department of Medicine
Unit
University of Massachusetts Medical
Joseph E. Italiano, Jr Center of Aging Science and
School
Hematology Division Translational Medicine
Worcester, MA
Brigham and Women’s Hospital; (CESI-Met)
United States
Vascular Biology Program University of Chieti
Boston Children’s Hospital; David J. Kuter Chieti, Italy
Harvard Medical School Department of Hematology


Marie Lordkipanidze
Boston, MA Massachusetts General Hospital
Faculty of Pharmacy
United States Harvard Medical School
University of Montreal;
Boston, MA
Young-Hoon Jeong Research Center
United States
Department of Internal Medicine Montreal Heart Institute
Gyeongsang National University Michele P. Lambert Montr
eal, QC
School of Medicine and Department of Pediatrics Canada
Cardiovascular Center Children’s Hospital of Philadelphia
Viola Lorenz
Gyeongsang National University Perelman School of Medicine at the
Division of Newborn Medicine
Changwon Hospital University of Pennsylvania
Boston Children’s Hospital
Changwon Philadelphia, PA
Harvard Medical School
South Korea United States
Boston, MA
Andrew D. Johnson Robert H. Lee United States
National Heart, Lung and Blood McAllister Heart Institute
Institute University of North Carolina at Chapel Kellie R. Machlus
The Framingham Heart Study Hill Hematology Division
Framingham, MA Chapel Hill, NC Brigham and Women’s Hospital
United States United States Harvard Medical School
Boston, MA
Cecile Kaplan Jack Levin United States
Retired, formerly Platelet Immunology Departments of Laboratory Medicine
Department and Medicine Dhruv Mahtta
I.N.T.S. University of California School of Department of Medicine
Paris Medicine University of Florida
France San Francisco, CA Gainesville, FL
United States United States
Peter Karagiannis
Department of Clinical Application Renhao Li Pier Mannuccio Mannucci
Center for iPS Cell Research and Aflac Cancer and Blood Disorders Angelo Bianchi Bonomi Hemophilia
Application Center and Thrombosis Center
Kyoto University Children’s Healthcare of Atlanta Fondazione IRCCS Ca’ Granda-
Kyoto Department of Pediatrics Ospedale Maggiore Policlinico
Japan Emory University School of Medicine Università degli Studi di Milano
Atlanta, GA Milan
Gregory J. Kato Italy
United States
Pittsburgh Heart, Lung and Blood
Vascular Medicine Institute; Zhenyu Li Keith R. McCrae
Division of Hematology and Oncology; Division of Cardiovascular Medicine Department of Hematology-Oncology
Sickle Cell Center of Excellence The Gill Heart and Vascular Cleveland Clinic
University of Pittsburgh-School of Institute Cleveland, OH
Medicine University of Kentucky United States
Pittsburgh, PA Lexington, KY
Alessandra Metelli
United States United States
Department of Microbiology and
Samuel Kemble Zihai Li Immunology
Institute of Inflammation and Ageing Department of Microbiology and Hollings Cancer Center
College of Medical and Dental Sciences Immunology Medical University of South
University of Birmingham Hollings Cancer Center Carolina
Birmingham Medical University of South Charleston, SC
United Kingdom Carolina United States
x Contributors

Alan D. Michelson Marie-Blanche Onselaer Julie Rayes

Center for Platelet Research Studies Institute of Cardiovascular Sciences Institute of Cardiovascular Sciences
Dana-Farber/Boston Children’s Cancer College of Medical and Dental Sciences College of Medical and Dental Sciences
and Blood Disorders Center University of Birmingham University of Birmingham
Harvard Medical School Birmingham Birmingham
Boston, MA United Kingdom United Kingdom
United States
Carlo Patrono Alexander P. Reiner
Karen A. Moffat Department of Pharmacology Department of Epidemiology
Department of Medicine Catholic University School of Medicine School of Public Health
McMaster University Rome Fred Hutchinson Cancer Research Center
Hamilton, ON Italy University of Washington
Canada Seattle, WA
Edward F. Plow United States
Jae Youn Moon Joseph J. Jacobs Center for Thrombosis
Division of Cardiology and Vascular Biology Brian Riesenberg
University of Florida College of Department of Molecular Cardiology Department of Microbiology and
Medicine-Jacksonville Cleveland Clinic Immunology
Jacksonville, FL Cleveland, OH Hollings Cancer Center
United States United States Medical University of South Carolina
Charleston, SC
James H. Morrissey Mortimer Poncz United States
Department of Biological Chemistry Department of Pediatrics
University of Michigan Medical School Irene A.G. Roberts
Children’s Hospital of Philadelphia Department of Paediatrics
Ann Arbor, MI Perelman School of Medicine at the
United States University of Oxford
University of Pennsylvania John Radcliffe Hospital
Nicola J. Mutch Philadelphia, PA Oxford
Institute of Medical Sciences United States United Kingdom
University of Aberdeen Man-Chiu Poon
Aberdeen Matthew T. Rondina
Division of Hematology and Department of Internal Medicine
United Kingdom Hematologic Malignancies University of Utah
Zoltan Nagy Department of Medicine Eccles Institute of Human Genetics;
Institute of Cardiovascular Sciences University of Calgary–Foothills Medical Molecular Medicine Program
College of Medical and Dental Sciences Centre University of Utah School of Medicine;
University of Birmingham Calgary, AB George E. Wahlen VAMC GRECC
Birmingham Canada Salt Lake City, UT
United Kingdom United States
Natalie S. Poulter
Heyu Ni Institute of Cardiovascular Sciences Jesse W. Rowley
St. Michael’s Hospital College of Medical and Dental Sciences Molecular Medicine Program
University of Toronto; University of Birmingham Division of Pulmonary Medicine in the
Canadian Blood Services Centre for Birmingham Department of Internal Medicine
Innovation United Kingdom University of Utah
Toronto, ON Salt Lake City, UT
Canada Izmarie Poventud-Fuentes United States
Division of Hematology-Oncology
Phillip L.R. Nicolson Department of Medicine Francesca Santilli
Institute of Cardiovascular Sciences Perelman School of Medicine Internal Medicine
College of Medical and Dental University of Pennsylvania Department of Medicine and Aging and
Sciences Philadelphia, PA Haemostasis and Thrombosis Unit
University of Birmingham United States Center of Aging Science and
Birmingham Translational Medicine
United Kingdom Patrick Provost (CESI-Met)
CHUQ Research Center/CHUL Pavilion; University of Chieti
Marvin T. Nieman Department of Microbiology Chieti
Department of Pharmacology Infectious Diseases and Immunology Italy
Case Western Reserve University Faculty of Medicine
Cleveland, OH Universit
e Laval € diger E. Scharf
Ru
United States Quebec City, QC Institute of Transplantation Diagnostics
Canada and Cell Therapeutics
Rienk Nieuwland Division of Clinical and Experimental
Laboratory of Experimental Clinical Jun Qin Hemostasis, Hemotherapy and
Chemistry; Joseph J. Jacobs Center for Thrombosis Transfusion Medicine
Vesicle Observation Centre and Vascular Biology University Blood Center and
Academic Medical Centre of the Department of Molecular Cardiology Hemophilia Comprehensive Care
University of Amsterdam Cleveland Clinic Center
Amsterdam Cleveland, OH Heinrich Heine University Medical
The Netherlands United States Center
 Contributors xi

and Biological Medical Research Center Lexington, KY Centre of Membrane Proteins and
Heinrich Heine University United States Receptors (COMPARE)
D€usseldorf University of Birmingham and
Germany Edward L. Snyder University of Nottingham
Department of Laboratory Medicine Midlands
Yotis A. Senis Yale University School of Medicine United Kingdom
Institute of Cardiovascular Sciences New Haven, CT
College of Medical and Dental Sciences United States Mark R. Thomas
University of Birmingham Institute of Cardiovascular Sciences
Birmingham Martha Sola-Visner College of Medical and Dental Sciences
United Kingdom Division of Newborn Medicine University of Birmingham
Boston Children’s Hospital Birmingham
Anish Sharda Harvard Medical School United Kingdom
Division of Hemostasis and Thrombosis Boston, MA
Department of Medicine United States Maurizio Tomaiuolo
Beth Israel Deaconess Medical Center Division of Hematology-Oncology
Harvard Medical School Timothy J. Stalker Department of Medicine
Boston, MA Division of Hematology-Oncology Perelman School of Medicine
United States Department of Medicine University of Pennsylvania
Perelman School of Medicine Philadelphia, PA
Alexa J. Siddon University of Pennsylvania United States
Department of Laboratory Medicine Philadelphia, PA
Christopher A. Tormey
Department of Pathology United States
Department of Laboratory Medicine
Yale University School of Medicine Yale University School of Medicine
Lucia Stefanini
New Haven, CT; New Haven, CT;
Department of Internal Medicine and
Pathology & Laboratory Medicine Pathology & Laboratory Medicine
Medical Specialties
VA Connecticut Healthcare VA Connecticut Healthcare
Sapienza University of Rome
West Haven, CT West Haven, CT
Rome
United States United States
Italy
Pia R.-M. Siljander Naoshi Sugimoto Han-Mou Tsai
EV Group Department of Clinical Application State University of New York Downstate
Molecular and Integrative Biosciences Center for iPS Cell Research and Medical Center
Research Programme Application Brooklyn, NY
Faculty of Biological and Environmental Kyoto University United States
Sciences; Kyoto;
EV Core Facility Francesco Violi
Department of Hematology I Clinica Medica
University of Helsinki Kagawa University Hospital
Helsinki Department of Internal Medicine and
Kagawa Medical Specialties
Finland Japan SAPIENZA-University of Rome
Pierluigi Tricoci Prithu Sundd Rome
Division of Cardiology Pittsburgh Heart, Lung and Blood Italy
Duke Clinical Research Institute Vascular Medicine Institute; Theodore E. Warkentin
Duke University Division of Pulmonary, Allergy and Department of Pathology and Molecular
Durham, NC Critical Care Medicine; Medicine
United States Sickle Cell Center of Excellence Department of Medicine
Paola Simeone University of Pittsburgh-School of Michael G. DeGroote School of
Internal Medicine Medicine Medicine
Department of Medicine and Aging Pittsburgh, PA McMaster University;
and Haemostasis and Thrombosis United States Hamilton Regional Laboratory
Unit Udaya S. Tantry Medicine Program
Center of Aging Science and Inova Center for Thrombosis Research Hamilton, ON
Translational Medicine (CESI-Met) and Drug Development Canada
University of Chieti Inova Heart and Vascular Institute Steve P. Watson
Chieti Falls Church, VA Institute of Cardiovascular
Italy United States Sciences
Stephanie A. Smith College of Medical and Dental Sciences
Ayalew Tefferi
Department of Biological Chemistry University of Birmingham
Division of Hematology
University of Michigan Medical School Birmingham
Department of Internal Medicine
Ann Arbor, MI United Kingdom
Mayo Clinic
United States Rochester, MN Jeffrey I. Weitz
United States Thrombosis and Atherosclerosis
Susan S. Smyth Research Institute;
Division of Cardiovascular Medicine Steven G. Thomas Department of Medicine
The Gill Heart and Vascular Institute Institute of Cardiovascular Sciences McMaster University
University of Kentucky; University of Birmingham Hamilton, ON
Lexington VA Medical Center Birmingham; Canada
xii Contributors

John Welsh The BloodCenter of Wisconsin Li Zhu

Division of Hematology-Oncology Milwaukee, WI Cyrus Tang Hematology Center
Department of Medicine United States Soochow University
Perelman School of Medicine Suzhou
Bill X. Wu
University of Pennsylvania China
Department of Microbiology and
Philadelphia, PA
Immunology
United States Guy A. Zimmerman
Hollings Cancer Center
Department of Internal
Andrew S. Weyrich Medical University of South
Medicine
Molecular Medicine Program Carolina
University of Utah
Division of Pulmonary Medicine Charleston, SC
Eccles Institute of Human Genetics;
in the Department of Internal United States
Molecular Medicine Program
Medicine
Michael R. Yeaman University of Utah School of
University of Utah
Department of Medicine Medicine
Salt Lake City, UT
David Geffen School of Medicine at Salt Lake City, UT
United States
University of California, Los Angeles United States
David A. Wilcox (UCLA)
Department of Pediatrics Torrance and Los Angeles Biomedical Elizabeth R. Zunica
Medical College of Wisconsin; Research Institute at Department of Pharmacology
Children’s Research Institute Harbor UCLA Medical Center Case Western Reserve University
The Children’s Hospital of Wisconsin Torrance, CA Cleveland, OH
Blood Research Institute United States United States
 Preface

Platelets are small cells of great importance in many patho- Marco Cattaneo, A.L. “Larry” Frelinger, and Peter J. Newman.
physiological processes including thrombosis, hemorrhage, There are 142 contributing authors from 18 countries.
inflammation, antimicrobial host defense, wound healing, The 67 chapters in this book are organized into six parts:
angiogenesis, and tumor growth and metastasis. In addition
Part I: Platelet Biology
to primary disorders of platelet number and function, platelets
Part II: The Role of Platelets in Disease
have a critical role in many other very common diseases,
Part III: Clinical Tests of Platelet Function
including coronary artery disease, stroke, peripheral vascular
Part IV: Disorders of Platelet Number and/or Function
disease, and diabetes mellitus.
Part V: Antiplatelet Therapy
The goal of this book is to be a comprehensive and definitive
Part VI: Therapy to Increase Platelet Numbers and/or Function
source of knowledge about platelets. Platelets integrates the
entire field of platelet biology, pathophysiology, and clinical Each chapter has been completely revised and updated, and
medicine. there are 11 new chapters on topics such as platelet glycobiol-
The intended audience for Platelets includes hematologists, ogy, the platelet transcriptome, platelet inhibitory receptors,
cardiologists, stroke physicians, blood bankers, pathologists, platelet function testing in clinical research trials, therapeutic
and researchers in thrombosis and hemostasis, as well as stu- platelet-rich plasma in wound healing, and new antiplatelet
dents and fellows in these fields. drugs.
The first edition of this book was the winner of the Best Book
in Medical Science Award from the Association of American Pub- Alan D. Michelson
lishers, and the third edition won the Highly Commended
Marco Cattaneo
Prize in the category of Internal Medicine from the British Med-
ical Association (BMA) Medical Book Awards. These awards A.L. “Larry” Frelinger
reflect the outstanding international group of contributing
Peter J. Newman
authors, all of whom are world leaders in their field.
In this fourth edition of Platelets, Alan Michelson, the editor
of the first three editions, is joined by three associate editors:

xiii
 Foreword: A Brief History
of Ideas About Platelets
in Health and Disease
Barry S. Coller
Allen and Frances Adler Laboratory of Blood and Vascular Biology, Rockefeller University Hospital, Rockefeller University, New York, NY, United States
Credible claims have been made by, or on behalf of, a number
of different scientists to be recognized as the first to describe the
blood platelet.1,2 For example, Osler undoubtedly described
platelets in his 1873 and 1874 papers (Fig. 1),1,3 recognizing
their disk-like structure, and that while they circulated singly
in the blood, they rapidly formed aggregates when removed
from the blood vessel. Osler was not certain, however, whether
the platelets were normal blood elements or exogenous “organ-
isms.”1,3 Seven years later, the elegant and compelling studies
of Bizzozero in 1881 and 1882 not only identified the platelet
anatomically, but also assigned it roles in both hemostasis and
experimental thrombosis.4–9 Thus, using intravital microscopy
of mesenteric venules in guinea pigs, Bizzozero also observed
that platelets are disk-shaped and circulate in isolation. He
went on to demonstrate that they adhere to the blood vessel
wall at sites of injury and then form aggregates. He additionally
noted that leukocytes were recruited into the platelet aggre-
gates, thus also offering the first description of platelet-
leukocyte interactions (Fig. 2). Bizzozero also developed the
first “flow chamber,” with the flow achieved by capillary action,
by an ingenious construction of a microscope slide, a piece of
thread, and blotting paper (Fig. 3).7,8 Using human biopsy
materials, Osler later built on Bizzozero’s observations and,
in studies conducted from 1881 to 1886, clearly established
the role of blood platelets (which he termed blood “plaques”)
in human thrombotic disease, identifying them in white
thrombi that formed on “atheromatous ulcers,” in vegetations
on heart valves, and in aortic aneurysms (Fig. 4).1,13
Ironically, 12 years before his publications on platelets, Biz-
zozero was also the first to identify bone marrow megakaryo-
cytes (Fig. 5),10 but he never recognized them as the
precursors of platelets. That discovery was made by Wright in
1906, who noted the similarities in shape and color of the
red to violet granules in platelets and megakaryocytes using
his new polychrome staining solution (Wright’s stain).14
The intervening 135 years since Bizzozero’s pioneering stud-
ies have seen a remarkable increase in our understanding of
hemostasis and thrombosis, and the important roles that plate-
lets play in both of these phenomena. Thus, it is timely that
Dr. Alan Michelson, who himself has made many important
discoveries regarding platelet function and pioneered the inno-
vative application of flow cytometry to the study of platelets,
has once again brought together contributions from the leaders
in this discipline to provide a comprehensive review of the
topic in this fourth edition of Platelets. In this Foreword, I offer
a brief and highly selective historical analysis of topics in plate-
let history, with the hope of providing both a sense of the sweep
of intellectual development in the field and a context for under-
standing our current concepts and likely future directions.
Those interested in a more systematic history of the discovery
of the platelet and its functions can consult a number of excel-
lent references.1, 9, 15–29 In addition, the Historical Sketch sec-
tion of the Journal of Thrombosis and Haemostasis has provided
extremely valuable first-hand accounts of discoveries related
to platelets by prominent members of the platelet research
community.30–42
THE ROLE OF PLATELETS IN HUMAN HEMOSTASIS,
PLATELET TRANSFUSION, AND BLEEDING TIME
Among the earliest evidence that platelets are crucial for human
hemostasis and that platelet transfusions can restore hemo-
static competence to individuals with low platelet counts, are
the compelling data from one of the three patients reported
by Duke, a student of Wright, in 1910 (Fig. 6).12,20,27,43 A
20-year-old Armenian man was admitted to the Massachusetts
General Hospital on May 8, 1909 with a platelet count of
6  109/L and profound mucocutaneous bleeding. On May
11, he developed uncontrollable epistaxis that left him nearly
moribund. In desperation, a donor was selected from among
his young Armenian friends and a direct blood transfusion
was arranged. A “large” amount of blood was transfused, as
judged by the increase in pulse of the donor as well as the rise
in the platelet count in the recipient to 123  109/L! All overt
signs of bleeding improved immediately after the transfusion.
To monitor the response, Duke used the bleeding time assay he
had just developed, analyzing the time for bleeding to stop
from a standardized wound in the ear lobe. The improvement
in the patient’s bleeding time correlated with his higher platelet
count and his improvement in clinical bleeding (Fig. 7). Since
the patient’s coagulation time was normal throughout, Duke’s
data provided strong evidence that the low platelet count itself
was responsible for both the clinical hemorrhage and the pro-
longed bleeding time, and that normal coagulation tests do not
insure a normal bleeding time or adequate hemostasis in the
face of thrombocytopenia.
Despite this remarkable success, many obstacles to routine
platelet transfusion remained. These began to be systematically
addressed 35 years later when U.S. government support of
research in this area was spurred by the observation that throm-
bocytopenic purpura was a major cause of death after radiation
exposure from atomic weapons (Fig. 8).44,45 Further impetus
for improving platelet transfusion support came soon thereaf-
ter when it became apparent that thrombocytopenia was a
xv
xvi Foreword

Fig. 1 Drawings by Osler (1874)1, 3 of “corpuscles” from rat

blood (5–7) and within a vein in the loose connective tissue of a
rat (8 and 9). (From Osler,3 with permission.)

Fig. 2 Drawing by Bizzozero (1882) of intravital microscopic image

of platelet and leukocyte deposition on damaged blood vessel in Fig. 4 Figs. 5 and 6 from Osler (1886)13 demonstrating “white
guinea pig mesentery.5 (From Bizzozero,5 with permission.) thrombi” in the aorta of a patient who died with cancer of the
stomach, and the “plaques” (platelets) that could be retrieved from
the thrombi. (From Osler,13 with permission.)

Fig. 3 Bizzozero’s “flow chamber” in which he watched platelets

deposit on a piece of thread, which was purposely frayed at both
ends (z). The chamber was formed by placing a coverslip (x) over two
strips of regular paper (a) and a strip of blotting paper (b) connected
to a wad of blotting paper. To encourage blood flow, the blood entry side
of the slide (o’) was slightly raised. (From Brewer,8 with permission.)

major cause of death from the then new chemotherapeutic

agents used to treat malignancies. In fact, successful platelet
transfusion therapy was absolutely crucial for the development
of modern chemotherapy.45,46
Many dramatic improvements in platelet transfusion ther-
apy were made over the next decades, including: improved anti-
coagulants; development of a differential centrifugation Fig. 5 Drawings by Bizzozero (1869)10 of giant bone marrow cells with
technique to produce platelet concentrates; recognition of the multilobular nuclei, now recognizable as megakaryocytes. (From
benefits of room temperature storage; improved plastics and Baserga A,11 with permission.)

Fig. 6 Bleeding time results from a patient described by Duke in
1910.12 The series from rows A–D are blots from the ear wound made
when the patient’s platelet count was 3000/μL. The blots in series A
were taken immediately after the ear was pricked; series B began at
20 minutes; series C began at 40 minutes; series D began at 60 minutes;
and series E began at 80 minutes. The bleeding time was 90minutes. In
row F are the blots obtained after transfusion (platelet count 110,000/μL),
demonstrating a bleeding time of 3 minutes. (From Duke,12 with
permission.)
conditions of bag storage to facilitate gas exchange and the
maintenance of pH; cryopreservation; HLA matching or partial
matching to improve platelet responses in patients refractory to
random donor platelets; platelet “cross-matching;” single
donor platelet collection via continuous flow centrifugation;
blood screening assays to prevent viral transmission; hepatitis
B vaccination; prestorage leukoreduction to decrease febrile
transfusion reactions; ultraviolet irradiation and leukoreduc-
tion to decrease immunogenicity; development of synthetic
storage solutions; new methods to eliminate viable pathogens
by damaging their DNA; and attempts to define better the most
appropriate platelet count at which to transfuse platelets (see
Chapter 64).23, 47–58 Ironically, one of the most important
advances related to platelet transfusion therapy had nothing
to do with the platelet product, but rather concerned the recog-
nition that aspirin inhibits platelet function and thus aspirin
should not be used as an antipyretic or analgesic in patients
who are thrombocytopenic.59
Although attempts to develop substitutes for fresh platelets
that could secure hemostasis in thrombocytopenic patients have
Foreword xvii
been ongoing for more than 50 years,18, 60–71 this goal has not yet
been achieved, despite the advances in our understanding of
platelet physiology. The need for such an agent(s) grew more
apparent in the 1980s and 1990s as a result of the dramatic
increase in the demand for platelet transfusions, especially with
the growth of bone marrow and stem cell transplantation and
autologous stem cell reconstitution, reaching 8,330,000 units
of platelets transfused in the United States in 1992.72 Since then
the growth rate has slowed substantially, with just over 9,000,000
units transfused in both 1997 and 1999,73 and 10,388,000 units
transfused in 2006.74 The number of units transfused per year
more recently has shown a downward trend as more restrictive
transfusion guidelines have been broadly adopted.75
There is also an increased appreciation of the seriousness of
platelet transfusion-transmitted bacterial infections, a conse-
quence of room temperature platelet storage, and estimated
to cause life-threatening sepsis in one in 100,000 recipients
in 2004.76 As a result, new methods to prevent bacterial con-
tamination, including diversion of the first blood drawn, and
to detect bacterial contamination before releasing platelets
for transfusion have been implemented, but their efficacy is
not absolute.77 Photochemical pathogen-reduction methods
have been introduced to lower the risk of transmitting infec-
tion, but current evidence suggests that such treatments may
increase the risk of developing refractoriness to platelet transfu-
sions and increase the number of platelet units transfused.78
Storage of platelets at 4°C would dramatically reduce the risk
of bacterial contamination, but it has been known for more
than 50 years, since the studies of Zucker and Borelli,79 that
exposure to cold temperature affects platelet morphology. Sub-
sequent studies demonstrated that exposure to cold results in
platelet activation,80,81 seriously compromising in vivo survival
and hemostatic effectiveness of the platelets.82 More recently,
basic studies led by Hoffmeister provided insights into the
mechanism responsible for the rapid clearance of platelets
incubated in the cold, identifying the lectin-like interaction
between integrin αMβ2 on hepatic macrophages and β-N-
acetylglucosamine residues on clustered platelet glycoprotein
(GP) Ib molecules as playing a central role in platelets sub-
jected to acute cooling and the interaction between exposed
galactose residues and hepatocyte asialoglycoprotein
(Ashwell-Morell) receptors in platelets refrigerated for
48 hours.83 Refrigeration also increases the binding of von
Willebrand factor to GPIb and, in animal models, preventing
this binding improves in vivo survival of the platelets.84 Loss
of sialic acid during normal platelet aging can also lead to plate-
let clearance via Ashwell-Morell receptors and may stimulate
thrombopoietin production (see Chapter 4).85
At present, a number of different factors are influencing the
field of platelet transfusion therapy (see Chapter 64), includ-
ing: the tendency to transfuse platelets only at lower platelet
counts and with smaller numbers of platelets than in the
past53,59,86; the questioning of the need for prophylactic plate-
let transfusions87; the emergence of cord blood as a source of
stem cells since megakaryocyte reconstitution is often delayed
with this source of stem cells88–90; the development of new con-
ditioning regimens for stem cell reconstitution that produce
much less myelosuppression than in the past91; the availability
of agents that can activate the thrombopoietin receptor (mpl)
(see Chapter 61)92,93; attempts to expand hematopoietic stem
cells ex vivo to increase the likelihood and speed of megakaryo-
cytic engraftment after transplantation with cytokines, growth
factors, and a small molecule CD34+ expander, as well as extra-
cellular proteins in the hematopoietic bone marrow niche, such
as fibronectin94–100; the increasing concern about the compli-
cations of platelet transfusion therapy, including thrombosis,
transfusion-related acute lung injury (TRALI), and immuno-
modulation101; the renewed emphasis on ABO blood group
xviii Foreword
Fig. 7 The clinical course of the patient reported by Duke.12 The transfusion was performed at 2 a.m. on May 12. (From Duke,12 with permission.)
matching102; and continued attempts to develop platelet
substitutes.60–71,103
The advances in understanding stem cell biology, the basic
mechanisms of megakaryocyte differentiation, and platelet
production offer the potential to improve stem cell replace-
ment therapy and platelet transfusion therapy (see
Chapter 66). Megakaryocytes have been generated from both
mouse and human embryonic stem cells when cultured
in vitro under various conditions, but the final step, platelet
production, has been difficult to achieve with high efficiency
or yield.104–109 The technology of induced pluripotent stem
cells (iPS) has been applied to developing human megakaryo-
cytes from human dermal fibroblasts, with demonstrated
success in obtaining platelets that both circulate in immunode-
ficient mice for more than 24 hours and participate in throm-
bus formation.110 Several groups are now actively engaged in
developing large-scale production of platelets from iPS cells,
cord blood, or other sources for transfusion using a variety of
ingenious devices to simulate the megakaryocyte bone marrow
microenvironment, including the shear forces experienced by
megakaryocytes as they extend into bone marrow sinu-
soids.108,111–121 Transfusion of megakaryocyte precursors is
an alternative approach that avoids the need to accomplish
the final, and most technically difficult, steps in producing
platelets in vitro.120–123 Platelets produced from a potential
recipient’s iPS cells provide hope of eliminating the immuno-
logic responses that make some patients refractory to platelet
transfusions, although recent studies in other systems have
raised questions about the immune recognition of iPS cells.124
There is good reason for platelets to lead the way in the use of
iPS-derived cells, since the concerns about the teratogenicity of
iPS cells can be eliminated by transfusing anucleate, irradiated
platelets. The search for an artificial platelet substitute will cer-
tainly continue, and this quest may be significantly advanced if
nanotechnology is able to develop small biocompatible matri-
ces, since the platelet’s small size is an important feature of its
biology, reducing the shear force it experiences relative to large
cells at comparable shear rates.
 Foreword xix

Incidence of hemorrhagic manifestations Almost 150 years later, the association between purpura and
in Japanese radiation casualties at thrombocytopenia was made by Krauss in 1883 and Denys
Hiroshima and Nagasaki in 1887, and confirmed by Hayem in 1890–91, who also noted
that the remaining platelets (which he termed “hematoblasts,”
believing they were primitive erythrocytes), were large and did
Hiroshima Nagasaki not support clot retraction normally.15,16 In 1907, Cole pro-
Exposure duced an antiserum to platelets that did not react with erythro-
Group* cytes, thus demonstrating fundamental immunologic
No. Percent No. Percent
differences (and presumably different lineages) between plate-
lets and erythrocytes.126 Later, similar antisera were used to
A 550 96 248 66 produce animal models of immune thrombocytopenia.127 In
B 485 43 610 42 1916, Kaznelson, a medical student in Prague, proposed that
C 227 12.5 385 24 immune thrombocytopenia, like hemolytic anemia, may be
D 137 8.5 93 13 due to platelet removal by the spleen.128 As a result, he sug-
gested splenectomy as a therapy to his tutor, Professor Schlof-
E 52 7.0 36 6
fer, who followed his suggestion. The first patient so treated
enjoyed an increase in her platelet count from 0.6  109/L to
* A to E represents decreasing exposure to the nuclear radiation. 500  109/L (Fig. 9)!15,128,129
Fig. 8 Risk of developing hemorrhagic purpura after exposure to the The pioneering and courageous studies of Harrington and
atomic bomb explosions at Nagasaki and Hiroshima as a function colleagues, in which blood components from patients with
of distance from the explosion.44 (From Cronkite et al.,44 with immune thrombocytopenia were infused into volunteers,
permission.) including Harrington himself, demonstrated that the agent
producing thrombocytopenia in idiopathic thrombocytopenia
(ITP) could be passively transferred with plasma (Fig. 10).130
Harrington’s group then proceeded to define many important
aspects of immune thrombocytopenia, including the crucial
IMMUNE THROMBOCYTOPENIA role of antibody, and the beneficial effects of splenectomy
Clinical descriptions of purple skin discoloration due to hem- and glucocorticoid therapy on platelet clearance.131
orrhage (“purpura”) extend back to Hippocrates, but Werlhof, Immune mechanisms also underlie HIV-associated throm-
who was physician to King George II of England in the German bocytopenia132–134 (see Chapter 39) and many of the drug-
States, and Behrens independently described cases of what was induced thrombocytopenias (see Chapter 40),135,136 including
most likely immune thrombocytopenia in 1735.15,19,28,125 the uniquely dangerous heparin-induced thrombocytopenia

Platelet transfusion (1910)

Splenectomy (1916)
ACTH/Cortisone (1951)
Prednisone (1956)
thrombocytopenia

Azathioprine (1966)
6-Mercaptopurine (1966)
Immune

Cyclophosphamide (1971)
Vincristine (1974)
Intravenous gammaglobulin (IVIgG) (1981)
Danazol (1983)
Anti-D immunoglobulin (1986)
Rituximab (2000)
Eltrombopag (2008)
Romiplostin (2008)
Oseltamivir (2009)

Aspirin (1940s, 1980s)

Ticlopidine (1991)
Abciximab (1994)
Clopidogrel (1997)
Eptifibatide (1998)
Antiplatelet

Tirofiban (1998)
therapy

Bivalirudin (1999)
Cilostazol (1999)
Aspirin/Dipyridamole (1999)
Prasugrel (2009)
Ticagrelor (2011)
Vorapaxar (2014)
Cangrelor (2015)

1900 1920 1940 1960 1980 2000 2020

Fig. 9 Dates of introduction or U.S. FDA approval of selected therapies for immune thrombocytopenia and to inhibit platelet function.
xx Foreword
Fig. 10 Data from Harrington et al.130 demonstrating that infusion of
plasma from patients with idiopathic thrombocytopenic purpura
could decrease the platelet count in normal individuals. (From
Harrington et al.,130 with permission.)
when it is associated with life-threatening thrombotic compli-
cations (see Chapter 41).136,137 The association of immune
thrombocytopenia with Helicobacter pylori infection, and the
clinical improvement enjoyed by some patients when H. pylori
is eradicated, stimulated new thinking about the pathophysiol-
ogy and therapy of immune thrombocytopenia, even though
the therapeutic results have not been consistent (see
Chapter 39).134,138–140 Although steroids and splenectomy
remain among the most important treatments for immune
thrombocytopenia (Fig. 9), the introduction of intravenous
IgG (a major scientific and technical advance, given the many
challenges to manufacturing a product that would not produce
severe systemic reactions)141,142 and later anti-Rh D therapy
(for patients fortunate enough to be Rh D positive)143 provided
additional effective agents (see Chapter 39).144 Knowledge of
the role of antiplatelet antibodies in the pathophysiology of
the disorder provided the rationale for the use of the anti-B cell
(anti-CD20) monoclonal antibody derivative rituximab, thus
opening a new therapeutic chapter by producing sustained
responses in a sizable percentage of patients refractory to other
treatments (see Chapter 39) (Fig. 9).145 Initial data on attempts
to target a low affinity Fcγ receptor thought to be responsible
for clearing antibody-coated platelets were promising,146 but
this strategy was not developed further. A number of new
approaches to treating immune thrombocytopenia include
inhibiting Syk kinase, since Syk signaling appears to be required
for macrophage clearance of antibody-coated platelets,147 and
inhibiting the neonatal Fc receptor, which recycles antibodies.
As we continue to learn more about the immune response,
additional rational targets will undoubtedly be identified,
offering hope for even better therapy.
The decision to test the efficacy and safety of pro-
thrombopoietic agents in treating patients with refractory
thrombocytopenia was based on a number of findings, includ-
ing that patients with immune thrombocytopenia do not have
very high levels of thrombopoietin,148–151 the improvement in
the platelet count of a single patient with immune thrombocy-
topenia in association with an elevation in thrombopoietin in
response to exposure to high altitude,152 and studies showing
that many patients have evidence of decreased platelet produc-
tion.153,154 The latter finding was surprising, since rapid plate-
let clearance was presumed to be the major cause of
thrombocytopenia, and so it required overwhelming evidence
to convince the scientific community that decreased platelet
production also contributed to the pathophysiology. The suc-
cess of the two currently available thrombopoietic agents in
increasing the platelet count in the majority of patients with
refractory thrombocytopenia is extremely impressive, although
the need for ongoing therapy thereafter in most patients to sus-
tain the increased platelet count is a serious limitation (see
Chapters 39 and 61) (Fig. 9).155–158 Recognition that immune
thrombocytopenia seriously compromises patients’ quality of
life provides a medically meaningful endpoint to assess the
impact of therapy, augmenting data about hemorrhage.145
ALLOANTIGENS, SINGLE NUCLEOTIDE
POLYMORPHISMS, AND ALLOIMMUNE
THROMBOCYTOPENIAS
Interest in single nucleotide polymorphisms (SNPs) has grown
rapidly over the past 20 years because of the remarkable pro-
gress made in sequencing the human genome and the recogni-
tion that identifying such polymorphisms is useful in assessing
disease predisposition and response to medications.159–162
Studies of platelets have made major contributions to this field.
The clinical syndrome of neonatal alloimmune thrombocyto-
penia, in which maternal antibody to fetal platelet antigens
inherited from the father cross the placenta and induce
immune-mediated thrombocytopenia in the fetus, was recog-
nized as early as 1959, and post-transfusion purpura, also
due to alloantigens, was first described soon thereafter (see
Chapter 45).24,163–166 The pioneering studies of Shulman
and colleagues produced a systematic characterization of
important platelet alloantigens at the serologic level.164,165
Later, in landmark studies, Newman and his colleagues applied
the polymerase chain reaction to the trace amounts of messen-
ger RNA present in platelets.167 This new technology very rap-
idly led to the identification of the SNPs that result in the
alloantigens on platelet glycoproteins, thus establishing the
molecular biologic basis of these disorders (see
Chapter 45).168 Furthermore, recognition of the dramatic
HLA-DR restriction in making alloimmune antibodies to cer-
tain polymorphisms has provided important molecular
insights into the immune response, buttressed by the crystal
structure of HLA-DRA/DRB3*0101 in complex with the peptide
from the integrin β3 subunit that contains the Leu/Pro dimor-
phism responsible for the HPA-1 (PIA1) antigens.24,169–173 Novel
approaches to therapy include the development of antibodies or
antibody fragments that will outcompete the alloantibodies and
thus prevent platelet clearance.174
In fulfillment of some of the hoped-for benefits in defining
exomic SNPs and expression quantitative trait loci (eQTLs),
reports of associations between platelet SNPs and differences
in platelet count, size, and function, as well as predispositions
to hemorrhagic and thrombotic diseases, have begun to
emerge, although differences in results from study to study
indicate considerable complexity (see Chapter 5).24,175–195 Fur-
ther, SNPs have been reported to have pharmacogenetic signif-
icance in defining the response to aspirin196–198 and
clopidogrel (see below). As large numbers of individuals
undergo whole exome or whole genome DNA sequencing,
we are beginning to identify the frequency of variants at the
population level. For example, analysis of the genes coding

for the αIIb and β3 subunits of the integrin receptor αIIbβ3
(ITG2A and ITGB3) indicates that 1% of the population
carries a variant for one or the other gene, with 50% of these
variants likely to be deleterious.199 Analysis of the frequency of
variants also provides insights into evolutionary selection pres-
sures related to gene function.199
PLATELET PHYSIOLOGY, ASSAYS OF PLATELET
“ADHESIVENESS,” ADP, PLATELET AGGREGATION,
MYOCARDIAL INFARCTION, AND P2Y12
ANTAGONISTS
The fundamental features of platelet physiology began to be
characterized at the molecular level by a small group of inves-
tigators from the late 1940s to the mid-1960s.17–19 These
included studies of platelet serotonin and vasospasm, the
release reaction and viscous metamorphosis (the loss of defin-
able boundaries between platelets some time after platelets
form aggregates), clot retraction, and the effects of thrombin
and collagen on platelet clumping. Attempts to quantify plate-
let “adhesiveness” resulted in the development of a large num-
ber of techniques, but the method of Hellem200 deserves
singling out, since his glass bead platelet retention assay led
him to discover an association between hematocrit and platelet
adhesiveness. Hellem therefore hypothesized that erythrocytes
contain a factor that can activate platelets. He then went on to
isolate such a factor from erythrocytes and found that its activ-
ity was stable even when boiling water was added to the red
cells. In 1961, Gaarder et al. identified this substance as aden-
osine diphosphate (ADP).201 Modified versions of the glass
bead platelet retention test provided other crucial insights into
platelet physiology, including the definition of the abnormal-
ities in platelet function in uremia, afibrinogenemia, Glanz-
mann thrombasthenia, Bernard-Soulier syndrome, and von
Willebrand disease.202–204 Moreover, the fundamental discov-
ery that high shear conditions accentuate the abnormality in
von Willebrand disease was first made using this assay.205
The test was also extremely important in early attempts
to purify von Willebrand factor, since at that time no other
in vitro assay of von Willebrand factor activity was avail-
able.40,206 Standardization of the platelet retention assay,
however, proved very difficult40,207 and thus it is now primarily
of historical significance.
The need for more quantitative and robust methods of
assessing platelet function led O’Brien and Born independently
to develop turbidometric platelet aggregometry in 1962.208–210
In this technique, the transmission of light through a cuvette
containing stirred platelets at 37°C is continuously recorded.
When aggregates form after the addition of an aggregating
agent, there is an increase in light transmission; when large
aggregates form and move into and out of the light path, the
increase in light transmission is accompanied by wide excur-
sions of the recording (see Chapter 34). This technique revealed
that different agonists produce different patterns of aggrega-
tion. ADP, for example, produces a double wave of platelet
aggregation at certain doses; the second wave depends on the
induction of the release reaction, wherein platelets release the
contents of their granules, including the platelets’ own storage
pool of ADP (see Chapter 34).
In vivo animal studies with ADP later provided a crucial link
between platelet aggregate formation and myocardial infarc-
tion. Thus, building on clinical observations of platelet micro-
emboli traversing the retinal vasculature during transient
ischemic attacks and autopsy data demonstrating platelet
aggregates in the distal coronary artery circulation in patients
suffering myocardial infarctions (reviewed in 36), Jørgensen
and other members of the group led by Mustard demonstrated
Foreword xxi
Fig. 11 Selected electrocardiographic recordings from pigs infused
with ADP into their left coronary artery. (Adapted from Jørgensen
et al.,211 with permission.)
that infusing ADP into the aorta and/or coronary arteries of pigs
resulted in the development of platelet microthrombi, hypo-
tension, and transient arrest of blood flow in the microcircula-
tion, culminating in gross myocardial infarction, death from
arrhythmia, or electrocardiographic ST-segment changes indic-
ative of ischemia in almost 90% of animals (Fig. 11).211 The
causal role of platelets in the phenomenon was established
by showing protection from cardiac dysfunction by inducing
thrombocytopenia in the pigs using 32P before infusing ADP.
Flow chamber studies further supported a role for platelets in
arterial thrombosis,30,34,212 as did Folts’ development of a
dog and primate model of unstable angina,213 and Gold’s
development of a dog model of reocclusion of coronary arteries
after successful thrombolysis.214
In parallel with the studies demonstrating a potential role
for ADP-induced platelet activation/aggregation in coronary
artery thrombosis, the drug ticlopidine, originally isolated in
1974 in a screen for antiplatelet and anti-inflammatory
agents,215 was shown selectively to inhibit ADP-induced plate-
let aggregation and prevent fibrinogen binding to integrin
αIIbβ3.216 Ticlopidine was later found to have greater antith-
rombotic efficacy than aspirin in a wide variety of indications,
including secondary prevention of vascular events after stroke,
transient ischemic attacks, and unstable angina,217 leading to
initial U.S. Food and Drug Administration (FDA) approval in
1991 (Fig. 9). In 1996 the combination of aspirin and ticlopi-
dine was shown in a landmark study to protect against throm-
botic complications of coronary artery stenting better than
anticoagulation, the prevailing standard of care.218 Ticlopidine,
however, had serious albeit uncommon complications, includ-
ing neutropenia and thrombotic thrombocytopenic purpura
(TTP).219,220 As a result, when clopidogrel, another thienopyr-
idine with a better safety profile,221 was found to be effective in
the secondary prevention of vascular events in patients under-
going percutaneous coronary interventions with stents,222 it
became one of the most commonly prescribed drugs in the
world (see Chapter 51) (Fig. 9).222
In 2001, Hollopeter et al., building on studies by other
groups,223,224 reported both the cloning of the elusive P2Y12
ADP receptor, which is the target of ticlopidine and clopidogrel,
and a mutation in the P2Y12 gene in a patient with a mild
bleeding disorder associated with a lack of platelet response
to ADP.225 They also identified the extracellular Cys groups
in P2Y12 as logical targets for the thiol-containing active metab-
olite of clopidogrel, providing a molecular explanation for the
prolonged effects of the drug, since the Cys reaction would
likely be irreversible.
In 2002–03, J€aremo et al. and then Gurbel et al. reported that
the recommended doses of clopidogrel produced very variable
antiplatelet effects when administered to different
xxii Foreword
patients,226,227 and in 2004 Matetzky et al. reported that the
variability in response to clopidogrel correlated with clinical
outcomes in patients undergoing coronary artery stenting.228
Many groups confirmed these results and in 2006, Hulot
et al. identified interindividual variations in the clopidogrel
cytochrome P450 activation process as a major source of the
variability.229 A genome-wide association study among the
Amish population by Shuldiner et al. identified CYP2C19 as
a major contributor to the variable response to clopidogrel,
but it accounted for only 12% of the variability.230 Clinical
studies then identified CYP2C19 variants as correlates of clin-
ical outcomes.231,232 Similarly, several assays of platelet func-
tion correlated with the risk of developing thrombotic events
after coronary stenting.233–235 These data raised the possibility
of improving antiplatelet therapy using functional and/or
genetic assays, and several small randomized studies demon-
strated that function test-guided therapy can improve clinical
outcomes in patients with poor antiplatelet responses to clopi-
dogrel.236–238 However, there is poor correlation between the
different functional assays, and several large randomized stud-
ies failed to demonstrate a benefit of modifying therapy based
on the functional data.239–241 Thus, the role of guided therapy
remains controversial.
Prasugrel, a third thienopyridine approved by the FDA, was
designed so that the gastrointestinal esterases that ordinarily
inactivate 85% of ingested clopidogrel catalyze the first step
in the activation of prasugrel (Fig. 9).242,243 Prasugrel achieves
greater platelet inhibition than clopidogrel and is essentially
unaffected by the CYP2C19 variants that impair clopidogrel’s
activation. In clinical studies, it demonstrated both increased
antithrombotic efficacy and increased risk of bleeding relative
to clopidogrel (see Chapter 51).242,244
Ticagrelor, an oral, non-thienopyridine P2Y12 inhibitor, has
a rapid onset and offset of action (Fig. 9). It demonstrated anti-
platelet effects in patients who did not respond to clopidogrel
and greater overall antithrombotic efficacy than clopidogrel in
a pivotal study (see Chapter 51).245,246 Ticagrelor is similar to
clopidogrel in the risk of bleeding.244 Cangrelor, a reversible
intravenous P2Y12 antagonist, is a modified ATP derivative
with extremely rapid onset and offset of action.247 It was
approved by the FDA in 2015 as an adjunct to coronary artery
stenting (Fig. 9) (see Chapter 51).
ASPIRIN, ARACHIDONIC ACID, PROSTAGLANDINS,
EICOSANOIDS, NITRIC OXIDE, ENDOTHELIAL ECTO-
ADPase (CD39), ANGIOGENESIS, MICROPARTICLES,
AND LYMPHANGIOGENESIS
Gastrointestinal bleeding was linked to aspirin ingestion as
early as 1938,248 and prolongation of the bleeding time by
aspirin ingestion, especially in patients with von Willebrand
disease or telangiectasias, was demonstrated in the 1950s
and early to mid-1960s.22 In fact, excessive prolongation of
the bleeding time by aspirin (“aspirin tolerance time”) was pro-
posed by Quick as a method to enhance the sensitivity of diag-
nosing von Willebrand disease.249 Nonetheless, there was no
widespread appreciation that aspirin specifically interfered
with platelet function until 1967 when Morris reported
decreased “platelet stickiness” using a modified glass bead
assay2 and Weiss, noting his own increase in bleeding from
razor nicks when on aspirin (personal communication,
2001), and building on his previous studies of patients with
abnormalities in platelet ADP release and the exciting new
observations by Bounameaux,38 Hovig,35,250 and Spaet and
Zucker251 that exposure of platelets to connective tissue results
in platelet release of ADP,30 demonstrated with Aledort that
aspirin inhibits platelet aggregation initiated by connective
tissue.252 Very soon thereafter, the inhibitory effects of aspirin
on the second wave of platelet aggregation induced by ADP and
on other platelet functions were reported.253–256 These observa-
tions ushered in a new era in platelet research that was followed
by exciting findings by investigators in many fields related to
the important role of arachidonic acid in cell biology, and
the discovery of prostaglandins, thromboxanes, and the
cyclo-oxygenase enzymes.22,257–261
Translation of the demonstrable antiplatelet effects of aspi-
rin into effective antithrombotic therapy for cardiovascular dis-
ease progressed in a saw-toothed fashion (Fig. 9). Thus, in the
late 1940s, long before the in vitro effects of aspirin on platelets
were defined, two astute clinicians, Paul Gibson in the United
Kingdom and William Craven in the United States, indepen-
dently noted increased bleeding after treating patients with
aspirin and each proposed using aspirin for coronary artery dis-
ease.2,262–264 Gibson began using aspiring to treat coronary
thrombosis and Craven started prescribing aspirin for his male
patients at high risk of developing myocardial infarctions as a
prophylactic measure. In 1956, Craven reported remarkable
success in thousands of men,262 but neither Gibson’s nor his
results influenced the field. In fact, it would take a number
of painful false starts265 before aspirin was convincingly shown
to reduce the mortality of myocardial infarction by almost one-
quarter, the risk of subsequent vascular events after a first event
by about one-third, and the ischemic complications of percuta-
neous coronary interventions by at least one-half (see
Chapter 50).266–269 In addition to the medical benefits of aspi-
rin treatment, the economic benefits of this discovery probably
exceed many billions of dollars each year in both lower health
care costs and increased economic productivity.
Marcus, who was the first to describe the extraordinarily
high content of arachidonic acid in platelet phospholipids,270
also described the complex transcellular eicosanoid metabo-
lism that occurs between platelets, endothelial cells, and leuko-
cytes.271 Studies by Moncada and colleagues and Weksler and
colleagues demonstrated that endothelial cells synthesize pros-
taglandin I2 (PGI2, prostacyclin),272,273 providing the first bio-
chemical evidence that the lack of platelet reactivity of normal
endothelium is an active process, not as had previously been
supposed, simply a function of the inert nature of the surface
of the endothelium (see Chapter 17). This theme was rein-
forced by the subsequent discovery that the nitric oxide
(NO) synthesized by endothelial cells (originally termed
endothelial-derived relaxation factor, EDRF, because of its
effects on smooth muscle cells) is a potent inhibitor of platelet
function and acts synergistically with prostacyclin.274–276
Finally, endothelial cells were found by Marcus and colleagues
to have an ecto-ADPase (CD39) activity capable of rapidly
metabolizing the platelet activator ADP to the platelet inhibitor
adenosine in concert with CD73 (see Chapter 17).277–279 Thus,
endothelial prostacyclin, NO, and CD39 are part of a highly
coordinated active system designed to inhibit platelet activa-
tion that works in concert with endothelial thrombomodulin,
which regulates the level and proteolytic specificity of the
potent platelet activator thrombin.280 The precise contribu-
tions of each of these elements to basal platelet function and
platelet function after vascular injury probably varies in differ-
ent vascular beds.281
Platelets were first reported to play an important role in sup-
porting the endothelium almost 50 years ago.282 Subsequently,
the interactions between platelets and the endothelium in
health and disease were documented by many investigators,
with a particular focus on angiogenesis, especially in the tumor
vasculature (see Chapters 24 and 30).283–287 Platelet micropar-
ticles may play a particularly important role in angiogenesis
(see Chapter 22).288–290 Most recently, platelets have been
shown to have an important role in lymphangiogenesis, with

CLEC-2-podoplanin-dependent platelet thrombi at the junc-
ture of the vascular and lymphatic systems crucial in separating
the system (see Chapters 11 and 24).291–293
CLOT RETRACTION, GLANZMANN
THROMBASTHENIA, GPIIb/IIIa RECEPTOR, AND
αIIbβ3 (GPIIb/IIIa) ANTAGONISTS
The observation that clots formed by shed blood undergo
retraction within minutes to hours probably extends back to
antiquity. Hewson, who discovered fibrinogen in 1770, under-
stood the importance of fibrin in clot retraction,294 but Hayem
in the 19th century is credited with ascribing a central role to
platelets in clot retraction.17 Ultimately, studies of clot retrac-
tion resulted in the discovery by Bettex-Galland and L€ uscher
in 1959 that platelets contain large amounts of the contractile
proteins actin and myosin, which they termed thrombosthenin
(“the strength of the clot”).37,295 This was the first time that
these “muscle” proteins were isolated from a non-muscle cell,
a discovery with profound implications for understanding the
role of these proteins in cell motility in many other non-muscle
cells. Ultimately, the identification of non-muscle myosin type
IIA in platelets paved the way for the discovery of mutations in
the gene for this protein (MYH9) as contributing to a group of
autosomal dominant giant platelet disorders, including May-
Hegglin anomaly and the Fechtner, Sebastian, Epstein, and
Alport-like syndromes (see Chapter 46).296,297 More immedi-
ately, however, the early recognition of the contribution of
platelets to clot retraction provided a platelet function assay
that could be used to diagnose both qualitative and quantita-
tive disorders of platelets and to monitor platelet transfusion
therapy.45
Thus, in 1918 when the Swiss pediatrician Eduard Glanz-
mann studied a group of patients with a bleeding diathesis
and found them to have normal platelet counts but poor clot
retraction, he termed the disorder thrombasthenia (“weak
platelet”).298 Subsequent studies by groups led by Zucker18,299
and Caen300 defined the platelet defect in Glanzmann throm-
basthenia as an inability to aggregate in response to the usual
platelet agonists such as ADP and epinephrine. The deficiency
in platelet fibrinogen found in these patients299 ultimately led
to the recognition that platelets aggregate in vitro by binding
fibrinogen to their surface, with the fibrinogen acting as a bridg-
ing molecule.301–305 The molecular basis of Glanzmann throm-
basthenia was revealed in pioneering studies by groups led by
Nurden and Caen and by Phillips that demonstrated abnor-
malities in two surface glycoproteins termed GPIIb and GPIIIa
(later renamed αIIb and β3 in integrin terminology) based on
their electrophoretic mobility (see Chapters 12 and 48).306,307
Additional studies performed by many outstanding labora-
tories demonstrated that these two glycoproteins form a com-
plex that acts as a receptor for fibrinogen and for a number of
other adhesive glycoproteins, including von Willebrand
factor, that contain arginine-glycine-aspartic acid (RGD)
sequences.301–304 Moreover, as the cloning and sequencing of
many different receptors progressed, it became apparent that
the αIIbβ3 receptor is a member of a large family of receptors
termed integrins that extend back in evolution to Drosophila
and are involved in cell adhesion and aggregation, as well as
protein trafficking and bidirectional signaling, thus playing
important roles in development, immunity, angiogenesis,
bone resorption, and other physiological and pathological pro-
cesses.308–310 Molecular biologic analysis of the defects in
αIIbβ3 causing Glanzmann thrombasthenia have provided
important information linking structure to biogenesis and
function (reviewed in 311 and Chapters 12 and 48). Mice defi-
cient in β3, and thus lacking both αIIbβ3 and αVβ3 receptors,
Foreword xxiii
have many of the clinical and laboratory features characteristic
of Glanzmann thrombasthenia.312 They are also protected
from developing thrombosis.313 These mice have provided
important new insights into the roles of αVβ3 and/or αIIbβ3
in a variety of different phenomena, including tumor angiogen-
esis, wound healing, osteoclast bone resorption, and αIIbβ3-
mediated signal transduction.314–318 They also provide a
model for testing gene therapy of Glanzmann thrombasthenia
(see Chapter 67).319 Mice deficient in αIIb have provided infor-
mation on the impact of selective loss of αIIbβ3 on early hema-
topoiesis, atherosclerosis, and cerebral ischemia-reperfusion
models.320–323
The development in the 1980s of monoclonal antibodies to
αIIbβ3 and the ability to analyze patient DNA via reverse poly-
merase chain reaction (PCR) of platelet RNA has translated into
direct patient benefit in the form of new methods for carrier
detection and prenatal diagnosis in families with Glanzmann
thrombasthenia.305,324–327 Moreover, improved understand-
ing of ligand binding to αIIbβ3 led to the development in
the 1990s of drugs that inhibit the αIIbβ3 receptor (Fig. 9)
(see Chapter 52). The latter, which include a chimeric mono-
clonal antibody fragment (abciximab) and low molecular
weight molecules patterned after the RGD and related
sequences (eptifibatide and tirofiban), have proved efficacious
and safe in preventing ischemic complication of percutaneous
coronary interventions and unstable angina.328–333 These drugs
represent the first rationally designed antiplatelet therapies and
thus mark an important milestone in moving from serendipity
to purposeful drug development based on a molecular under-
standing of platelet function. Mapping the epitope on β3 for
the chimeric monoclonal antibody drug has also provided
valuable insights into ligand binding.334
Another one of the monoclonal antibodies to αIIbβ3 that
was shown to prevent dissociation of αIIb from β3335 proved
useful in studies of the crystal structure of αIIbβ3 since it stabi-
lized the αIIbβ3 headpiece complex during purification and
crystallization.336 The resulting high resolution structure has
provided detailed information on the ligand binding pocket,
the structural basis of the specificity of the low molecular weight
drugs eptifibatide and tirofiban for αIIbβ3, and the likely con-
formational changes associated with receptor activation.336 The
crystal structure of the entire αIIbβ3 ectodomain showed a bent,
closed conformation similar to that of αVβ3,337 and a three-
dimensional reconstruction of the full length αIIbβ3 in a nano-
disc showed a bent receptor with the head partially tilted
upward from the membrane.338 In addition, crystal structure
analysis has defined the binding mechanism for the fibrinogen
γ-chain dodecapeptide339 and the intermediate steps in the
transition of the receptor from its inactive, bent conformation
to its extended, high-affinity ligand binding conformation.340
Nuclear magnetic resonance, biochemical studies, and molecu-
lar dynamics simulations have provided important insights
into the mechanism by which signal transduction initiates
inside-out signaling of αIIbβ3, resulting in the conformational
changes in the ectodomain associated with high affinity ligand
binding and outside-in signaling.341–346
Attempts to develop oral αIIbβ3 antagonists patterned on
the RGD cell recognition sequence failed because when used
chronically, they were associated with increased bleeding, occa-
sional thrombocytopenia, and paradoxically, a variably
increased risk of thrombosis.347,348 These undesirable effects
have been hypothesized to result from the agents inducing con-
formational changes in the receptor leading to the high affinity
ligand binding state. High throughput screening coupled with
medicinal chemistry modifications have identified new αIIbβ3
antagonists that have less ability to induce changes in the β3
subunit, but their clinical utility and safety have not yet been
assessed.349–352
xxiv Foreword
BERNARD-SOULIER SYNDROME, THE GPIb-IX-V
COMPLEX, von WILLEBRAND FACTOR, SHEAR,
ADAMTS-13, AND THROMBOTIC
THROMBOCYTOPENIC PURPURA (TTP)
In 1948, Bernard and Soulier reported on two children of con-
sanguineous parents with a mucocutaneous bleeding disorder,
giant-sized platelets, and thrombocytopenia.21,353 Subsequent
studies conducted in many laboratories identified abnormali-
ties in platelet adhesion due to an inability of patient platelets
to interact with von Willebrand factor.354–357 In 1971, Howard
and Firkin, while trying to develop an animal model of throm-
bocytopenia, discovered that the antibiotic ristocetin, which is
akin to vancomycin but was removed from clinical use because
it caused thrombocytopenia in humans, could induce platelet
aggregation, but only in the presence of von Willebrand fac-
tor.358,359 Later, Brinkhous and colleagues made parallel find-
ings about the snake venom botrocetin.360 These extremely
important discoveries provided crucial tools that facilitated
the in vitro study of the interaction between platelets and
von Willebrand factor. They also permitted the practical devel-
opment, first by Weiss, of functional assays for plasma von
Willebrand factor that have been crucial in the diagnosis and
therapy of von Willebrand disease.361
Building on the pioneering studies by Nurden and Caen that
identified a defect in platelet GPIb as a cause of Bernard-Soulier
syndrome,362 many other investigators provided important
insights into the structure and function of the receptor complex
made up of GPIbα, GPIbβ, GPIX, and GPV, all of which are
members of the leucine-rich repeat family (see
Chapter 10).363–367 Patients have been identified with defects
in GPIbα, GPIbβ, and GPIX, but not GPV (see
Chapter 48).367,368 Thus, the identification of the molecular
defect in Bernard-Soulier syndrome made an important contri-
bution to defining the central role of von Willebrand factor
binding to GPIbα in platelet adhesion. Studies with monoclo-
nal antibodies to GPIbα that inhibited both von Willebrand
factor binding and ristocetin-induced platelet agglutination
confirmed the functional importance of this interaction and
provided tools for rapidly diagnosing Bernard-Soulier syn-
drome.369,370 These GPIbα-specific monoclonal antibodies
also provided insights into the region of GPIbα involved in
von Willebrand factor binding,371 and these inferences were
elegantly confirmed by atomic resolution structural data
derived from X-ray crystallographic studies of isolated regions
of GPIbα and von Willebrand factor.372–375 In turn, the struc-
tural data provided new opportunities to develop novel antith-
rombotic therapeutics that interfere with these interactions376
but, as of this writing, none has received regulatory approval.
In addition, the availability of mouse models of von Willeb-
rand disease, GPIb deficiency, and GPV deficiency377–379
opened exciting new paths for understanding the biologic roles
of the molecular interactions between GPIb and von Willeb-
rand factor, the pathophysiology of Bernard-Soulier syndrome,
and the interactions between thrombin and platelets
(see below).
As indicated above, the discovery of the shear dependence
of the interaction between von Willebrand factor and the plate-
let GPIb can be traced back to early observations using
the glass bead platelet-retention test, in which the defect in
von Willebrand disease was found to be accentuated by increas-
ing the speed of blood flow.205 The importance of shear in
understanding platelet function was defined by many laborato-
ries366,380–385 and helped to explain the importance of the very
large size of von Willebrand factor and its multimeric compo-
sition.380,386–394 The effect of shear on von Willebrand factor
structure was also found to be crucial for the cleavage of
ultra-high molecular weight multimers of von Willebrand fac-
tor by the enzyme ADAMTS-13.375,393–396
The discovery of ADAMTS-13 reads like a detective story,
starting with the original clinical descriptions of TTP by Mosch-
cowitz in 1924397 and inherited relapsing hemolytic-uremic
syndrome by Schulman in 1960398 and Upshaw in 1978,399
with the groups led by Moake, Furlan, Tsai, Sadler, and Ginsburg
all making vital contributions (see Chapter 42).31–33, 400
Building on careful clinical observations, the key clues included:
(1) the 1982 report by Moake et al. of increased amounts of
unusually large von Willebrand multimers in the plasma of
patients with TTP401; (2) the identification and purification in
1996 of a plasma metalloproteinase that could cleave von Will-
ebrand factor multimers, especially under denaturing or high
shear conditions402,403; (3) the discovery in 1997–98 that most
patients with TTP have autoantibodies to the enzyme that
cleaves von Willebrand factor multimers404,405; and (4) the iden-
tification in 2001 of mutations in ADAMTS-13 that cause the
inherited form of the disorder.406
Clinically, for more than 50 years after Moschcowitz’s
description of TTP there was no effective therapy to prevent
the usually rapid progression to death that resulted from the
combination of thrombocytopenia, hemolytic anemia, and
ischemic organ damage, with the latter especially affecting
the brain, kidney, and heart.397,407 Although corticosteroids
produced variable responses,407,408 the first major ray of hope
came from reports in 1976–77 by several groups that exchange
transfusion, plasma infusion, or cryoprecipitate-poor plasma
infusion could produce striking responses.407 Plasma exchange
utilizing the then new continuous flow devices quickly became
the treatment of choice and dramatically improved the progno-
sis, although unpredictable relapses were found to be fre-
quent.409 As the data on autoantibodies to ADAMTS-13 in
TTP emerged, the autoimmune nature of the disorder became
apparent, leading to the introduction of rituximab therapy410
and the substitution of high dose versus low dose corticoste-
roid therapy.411 Although the precise trigger(s) of individual
episodes remains elusive,393 it is possible that in the absence
of ADAMTS-13 activity, cytokines and/or hormonal changes,
perhaps modulated by HDL levels, stimulate endothelial pro-
duction of long strings of von Willebrand factor multimers that
can attract platelets, break loose from the endothelial cell under
shear, and initiate microvascular thrombosis, perhaps abetted
by complement activation.393,412,413 Understanding the patho-
physiology of the disorder opens new avenues for therapy,
including suppressing antibody formation, using the reducing
agent N-acetylcysteine, developing inhibitors of the von Will-
ebrand factor A1 domain interaction with GPIb, and possibly
using recombinant ADAMTS-13 that is engineered so as to
not be recognized by the autoantibody or is stored in platelets
(see Chapter 42).414
The evolution of our understanding of von Willebrand dis-
ease also reads like a detective story, starting with Erik von Will-
ebrand’s 1926 description of a bleeding disorder in a 5-year-old
girl in the Åland Islands, along with 65 other members of her
family.415–417 This disorder was easily differentiated from
hemophilia since it was inherited as an autosomal rather than
an X-linked trait, mucocutaneous symptoms predominated,
and patients had prolonged bleeding times and normal coagu-
lation times. Studies in the 1950s demonstrated that patients
have a partial deficiency of factor VIII,418–422 that the bleeding
time defect could be corrected by infusing a specific fraction of
plasma from normal individuals, and that such infusions led to
a paradoxically delayed and exaggerated increase in factor
VIII.418,419,423 In 1971, Ted Zimmerman in Oscar Ratnoff’s
lab discovered that most von Willebrand patients have reduced
levels of a protein present in normal and hemophiliac
plasma,424 but it required detailed biochemical studies to

establish unequivocally that factor VIII and von Willebrand fac-
tor are separate proteins that circulate as a complex in
plasma.417,425 The pace of discovery then increased rapidly,
with immunologic, biochemical, and molecular biologic stud-
ies in the 1970s and 1980s defining von Willebrand factor’s
subunit structure, remarkable multimeric structure, association
with factor VIII, ability to bind to collagen, GPIbα, and αIIbβ3,
cleavage by ADAMTS13, and translation in association with a
propiece that had previously been identified as von Willebrand
disease antigen II.426–431 Studies of von Willebrand factor car-
bohydrate defined functional variants432 and the important
role of blood group carbohydrates in its in vivo survival, with
the latter correlating with plasma levels of von Willebrand fac-
tor and the risk of both hemorrhage and thrombosis.433–436
The difficulties in standardizing the platelet retention test
limited its utility in assaying von Willebrand factor activity
in vitro.40 Thus, it was fortuitous that, as described above, in
1971 Howard and Firkin discovered that the antibiotic ristoce-
tin could cause platelet agglutination/aggregation only in the
presence of von Willebrand factor. This then led to the ristoce-
tin cofactor assay, which greatly facilitated diagnosing von Will-
ebrand disease and purifying von Willebrand factor; it also
established the greater platelet agglutinating activity of the
higher molecular weight multimers.358,359,361,417,437 Later
studies by Brinkhous’ group demonstrated that the snake
venom protein botrocetin could also agglutinate platelets in
the presence of von Willebrand factor,438 providing an alterna-
tive method for assessing von Willebrand factor function.
Structural biologic and single molecule biophysical studies
have provided exciting new information at atomic resolution of
von Willebrand factor assembly and packaging in helical
tubules in Weibel-Palade bodies, unfurling during secretion,
elongation force-regulated exposure of the A1 domain to plate-
let GPIb, and elongation force-unfolding of the A2 domain as a
prelude to ADAMTS-13 cleavage.375,393,394,439,440 Collectively,
these advances have permitted ever more sophisticated classifi-
cation of von Willebrand disease, allowing for targeted therapy
based on the molecular abnormality.441
THROMBIN-INDUCED PLATELET ACTIVATION
Thrombin was shown to aggregate platelets more than 60 years
ago,18,442 but the precise mechanisms involved remained mys-
terious for a long time because, despite being able to identify
platelet proteins that could bind thrombin with variable affin-
ities, deciphering which interactions actually led to signal trans-
duction was a challenging task.443 Thrombin appeared to act
both as an enzyme (since enzymatic activity was required to ini-
tiate aggregation) and as a ligand (since catalytically inactive
thrombin could enhance the response to catalytically active
thrombin).444 Phillips and Agin established that thrombin
cleaved platelet GPV,445 and platelets from patients with
Bernard-Soulier syndrome, which lack functional GPIb and
GPV molecules, had decreased platelet aggregation in response
to thrombin.446 This raised the possibility that thrombin acti-
vated platelets via GPV cleavage, but the time course of GPV
cleavage did not correlate well with platelet activation.447,448
The landmark studies of Coughlin and colleagues in 1991
using a functional expression cloning strategy opened a new
era by identifying a novel group of seven transmembrane recep-
tors (protease-activated receptors or PARs) that could be acti-
vated by cleavage of the amino-terminal region and creation
of a new “tethered ligand” that could then bind to another site
on the receptor to initiate activation (see Chapter 13).449 Com-
pelling support for the tethered ligand hypothesis came from
studies demonstrating that short peptides from the tethered
ligand region could themselves activate platelets.449 Subse-
quent studies identified the presence of at least two such
Foreword xxv
receptors on human platelets that have thrombin cleavage sites
(PAR-1 and PAR-4), while parallel studies in mice failed to
identify a PAR-1 homologue, but did identify a receptor
homologous to PAR-4 and another receptor from the same
family, PAR-3.450–452 Phillips’ group established that the plate-
lets of mice deficient in GPV, but not normal mice, could aggre-
gate in response to proteolytically inactive thrombin and that
this response depended on binding of thrombin to GPIb.453
Similar responses could be obtained after thrombin cleaved
platelet GPV from normal platelets. Thus, one possible expla-
nation for the dual nature of thrombin-induced platelet aggre-
gation emerged in which thrombin cleavage of the PAR
receptors and GPV, followed by thrombin-initiated signaling
through binding to GPIb, each plays a role. These mechanisms
may be linked since binding of thrombin to GPIb may acceler-
ate hydrolysis of PAR-1.454 The precise binding mode of throm-
bin to GPIb remains uncertain since different crystal structures
have been reported.455,456 As thrombin plays a central role in
thrombosis, thrombin receptors became logical therapeutic tar-
gets and the PAR-1 antagonist, vorapaxar was approved for
human use in 2014 to reduce the risk of myocardial infarction
and stroke (Fig. 9) (see Chapter 53).457–461
PLATELETS, METASTASES, SIALIC ACID, PLATELET
AGING, SEPSIS, AND PLATELET SURVIVAL
Approximately 50 years ago, Gasic and colleagues, working on
the hypothesis that sialic acid residues on the glycoproteins of
tumor cells and/or endothelial cells were important in metas-
tasis formation, injected neuraminidase into mice and demon-
strated a marked reduction in metastasis formation when TA3
ascites tumor cells were subsequently injected.462–464 Further
studies, however, demonstrated that the neuraminidase injec-
tion caused thrombocytopenia (presumably by altering surface
glycoproteins and/or decreasing platelet surface charge465) and
that thrombocytopenia produced by alternative methods also
protected against metastasis (Fig. 12).462 Additional studies
demonstrated that platelets can interact directly with some
tumors, and that some tumors can induce or enhance platelet
aggregation.466–469 In some experimental models, inhibiting
the αIIbβ3 receptor could also decrease metastasis forma-
tion,466,470 and platelet-derived lysophosphatidic acid was
implicated as a tumor mitogen and promoter of bone metasta-
ses.471 Since both platelets and some tumors can facilitate
thrombin formation, and since thrombin, either directly or
indirectly, may have both growth-promoting and growth-
inhibiting effects, the potential mechanisms involved are
complex.466,472,473 The discovery that platelets contain high
concentrations of vascular endothelial growth factor (VEGF),
a factor implicated in tumor angiogenesis, and can release it
when activated by agonists or tumor cells, added yet another
dimension to this evolving story (see Chapters 24 and
30).474–476 Whether antiplatelet therapy can be exploited to
decrease metastasis formation in humans remains an unan-
swered, but intriguing, question.464,469 Of concern, however,
are data showing that gene targeting of the β3 integrin in mice
did not protect against the growth and metastasis of mammary
carcinoma, and was actually associated with enhanced tumor
growth using human tumor cells injected into mice.477,478
Gasic’s linking of thrombocytopenia to neuraminidase
injection (Fig. 12) was followed by Mustard’s group’s demon-
stration in the 1970s that removing sialic acid from platelets
in vitro with neuraminidase resulted in a dramatic decrease
in platelet survival in rabbits.465 They went on to demonstrate
that two viruses with neuraminidase activity (influenza and
Newcastle) could reduce platelet sialic acid content and also
dramatically reduce platelet survival in rabbits, raising the
xxvi Foreword
9.0
8.0
7.0
Mean number of metastases
6.0
5.0
4.0
3.0
2.0
Fig. 12 Effect of neuraminidase on
circulating platelets and lung
1.0
metastases. The number of lung
metastases resulting from intravenous
injection of 100 units of neuraminidase. 0
10
Lung metastases were counted 14–
16 days after tumor cell inoculation. (From
Gasic et al.,462 with permission.)
possibility that at least a part of the thrombocytopenia associ-
ated with these viral infections may be due to loss of platelet
sialic acid.479 They also assessed the distribution of sialic acid
on density-separated platelets, and concluded that the densest
platelets (which are the largest and presumably youngest) have
the most sialic acid and the least dense platelets (which are the
smallest and presumably oldest) have the least.480 At about the
same time, Karpatkin’s group demonstrated that infusion of
desialylated platelets into rabbits stimulates the production
of new platelets by an unknown mechanism.481
The results of these early studies were intriguing but lacked
information on the molecular mechanism(s) involved. Almost
30 years later, Hoffmeister’s group renewed the study of the
association between platelet survival and glycoprotein carbo-
hydrates, spurred by an interest in extending the shelf life
and decreasing the likelihood of bacterial contamination of
platelets for transfusion by refrigeration.482,483 They ultimately
determined that loss of sialic acid with platelet aging due to
endogenous neuraminidase activity results in platelet clearance
via the hepatocyte Ashwell-Morell receptors that recognize the
penultimate mannose residues that become exposed with the
loss of sialic acid.484 Moreover, they found that clearance of
platelets lacking sialic acid by the hepatocyte Ashwell-Morell
receptors stimulated thrombopoietin production.485 Together,
these data provide credible molecular mechanisms to explain
the previous studies.
Most importantly, the development of the neuraminidase
inhibitor drug oseltamivir to treat influenza opened the possi-
bility of assessing whether loss of platelet sialic acid contributes
to the development of thrombocytopenia in a range of disease
states. In 2009, a Turkish patient with chronic immune throm-
bocytopenia developed H1N1 influenza, and soon after she
was treated with oseltamivir for influenza, her platelet count
increased transiently from under 40 x 109/L to more than
500 x 109/L.486 The authors proposed that oseltamivir may
be a new therapy for immune thrombocytopenia even in the
absence of influenza (Fig. 9). Thereafter, platelet desialylation
was proposed as a mechanism of thrombocytopenia in associ-
ation with anti-GPIb antibodies in an animal model, indepen-
dent of Fc-mediated clearance.487 Shao et al. then reported a
sustained increase in platelet count after only a 5-day course
900
Metastases
Platelets 800
Standard error
700
Number of platelets
600
X 103/mm3
500
400
300
200
100
20 30 40 50 60 70 80 90 100
Hours between treatment and tumor inoculation
or platelet counting
of oseltamivir in a patient with chronic immune thrombocyto-
penia with an antibody to GPIb, but no responses in an unspe-
cified number of patients with antibodies to αIIbβ3.488
Additional studies suggest that CD8(+) T cells may contribute
to the desialylation of platelets in immune thrombocytope-
nia489 and that Kupfer cells, in addition to hepatocytes, may
clear desialylated platelets from the circulation via CLEC4F.490
In parallel with studies in immune thrombocytopenia,
investigators proposed that clearance of desialylated platelets
via the Ashwell-Morrell receptor also contribute to the throm-
bocytopenia associated with pneumococcal sepsis.491,492 A
subsequent randomized controlled study of treatment with
oseltamivir in 106 patients with sepsis showed a significant
increase in platelet count and shortened time to recovery in
treated patients.493 Thus, a new treatment for thrombocytope-
nia in association with a variety of pathologic states may be on
the horizon.
REFLECTIONS ON THE PAST AND THOUGHTS
ABOUT THE FUTURE
Staggering progress has been made in understanding platelet
physiology and the pathophysiology of disorders of platelet
number and function in the past 130 years, but most particu-
larly in the past 50 years. Advances in the broad disciplines
of biochemistry, molecular biology, structural biology, and
the high throughput “omics” techniques have made possible
an increasingly detailed understanding of the complex systems
and pathways that control the fundamental cellular responses
as well as how the molecular interactions occur at the atomic
level. Next-generation sequencing is revolutionizing the evalu-
ation of patients with platelet function disorders494–496 and
RNA-seq, coupled with platelet proteomics, has revolutionized
our understanding of how megakaryocytes and platelets
respond to disease states and their environment (see Chapters
5–8).497–504 RNA-seq analysis of platelets may also have diag-
nostic potential in cancer and perhaps other disorders.505 These
technologies will also provide exciting new information about
platelet adhesion and aggregation, signal transduction, granule
secretion, and cytoskeletal organization. Gene therapy and
gene editing using CRISPR or other methods for platelet

disorders is no longer science fiction, since important proof of
concept experiments have already been performed successfully
(see Chapter 67).319,506–509 The profound advances in defining
the genetic variations underlying differences in platelet func-
tion and the response to antiplatelet therapy are already provid-
ing valuable pharmacogenetic information.510,511 The optimal
integration of genetic risk assessment, clinical risk assessment,
and platelet function data remains to be established on a
drug-by-drug basis, but the potential for personalizing and
thus improving antiplatelet therapy is clear (see Chapters 5
and 36).511
Transgenic mice and mice in which genes have been
“knocked out” have revolutionized platelet research, providing
the opportunity to assess the role of single genes, or combina-
tions of genes, on platelet function (see Chapter 5).512–516 The
identification of similarities between human platelets and zeb-
rafish thrombocytes opens up even greater opportunities for
identifying the role of individual genes in platelet function
via mutagenesis screens,517 and this model system has already
helped to identify NBEAL2 as the causative gene in gray platelet
syndrome518 and to define the role of several new proteins in
platelet function.519 These approaches have the added benefit
of permitting assessment of platelet function in intact animals,
thus providing confirmation of in vitro and ex vivo findings.
However, as with all animal models, cautions are advisable
since, for example, mouse platelets are known to differ from
human platelets in a number of fundamental ways, including
platelet size, platelet number, and the PAR receptor reper-
toire.512 Moreover, humans and mice have profoundly differ-
ent cardiac physiology as demonstrated by the mouse’s 600
beat per minute heart rate! Nonetheless, studies of fundamen-
tal aspects of platelet function are quite reassuring with regard
to the similarities between human and mouse platelets, and so
there is reason to be optimistic that many (but perhaps not all)
observations made in mice will be extrapolatable to humans.
The development of well-defined and robust mouse models
of thrombosis and hemostasis, as well as complex biologic phe-
nomena related to platelet function, such as vascular injury,
atherosclerosis, metastasis formation, and inflammation, are
especially important.520 New advances in imaging, especially
dynamic imaging (see Chapter 20), are providing extraordinary
insights into phenomena such as platelet formation from
megakaryocytes,521,522 the effects of antiplatelet therapy on
models of atherosclerosis,523 and platelet thrombus forma-
tion.524–530 It is both fitting and ironic, therefore, that intravital
microscopy, the same technique that Bizzozero used to make
his landmark discoveries about platelets,5 after a long period
of dormancy is once again cherished as a most powerful
method to reveal the platelet’s contributions to hemostasis
and disease.
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 PART
I Platelet Biology
The Evolution of Mammalian Platelets
1 Jack Levin
Departments of Laboratory Medicine and Medicine, University of California School of Medicine, San Francisco, CA, United States
INTRODUCTION 1
INVERTEBRATES 1
NONMAMMALIAN VERTEBRATES 3
COMPARATIVE HEMOSTASIS 6
A COMPARISON OF HUMAN PLATELETS AND LIMULUS
AMEBOCYTES 6
THE EVOLUTION OF HEMOSTASIS AND BLOOD
COAGULATION 11
MEGAKARYOCYTES AND MAMMALS 13
Marine Mammals 15
Order Monotremata 15
Order Marsupialia 16
Platelet Levels 17
A Comparison of Nonplacental and Placental Mammals 18
CONCLUSIONS 18
ACKNOWLEDGMENTS 19
REFERENCES 20
INTRODUCTION
The mammalian platelet is derived from the cytoplasm of
megakaryocytes, the only polyploid hematopoietic cell. Poly-
ploid megakaryocytes and their progeny, nonnucleated plate-
lets, are found only in mammals. In all other animal species,
cells involved in hemostasis and blood coagulation are nucle-
ated. The nucleated cells primarily involved in nonmamma-
lian, vertebrate hemostasis are designated thrombocytes to
distinguish them from nonnucleated platelets.
In many invertebrates, only one type of cell circulates in the
blood (or hemolymph), and this single type of cell is typically
involved in multiple defense mechanisms of the animal,
including hemostasis. These cells are capable of aggregating
and sealing wounds. This process is probably the earliest cell-
based hemostatic function. A comparison of amebocytes (the
only type of circulating cell in the hemolymph of the horseshoe
crab, Limulus polyphemus) with human platelets provides a basis
for understanding the many nonhemostatic functions of plate-
lets.1–4 Platelets appear to possess many of the multiple capa-
bilities that characterize “primitive” circulating amebocytes,
only one of whose functions is hemostasis. For example, plate-
lets possess rudimentary bactericidal and phagocytic activity
(see Chapter 29). They have been shown to interact with bac-
teria, endotoxins, viruses, parasites, and fungi. Platelets not
only are important for the maintenance of hemostasis but also
are inflammatory cells (see Chapter 28). Despite these overall
Platelets. https://doi.org/10.1016/B978-0-12-813456-6.00001-1
Copyright © 2019 Elsevier Inc. All rights reserved.
similarities in function, there remains no proof that inverte-
brate blood cells evolved into platelets. Furthermore, the fact
that nonnucleated platelets and their polyploid megakaryocyte
progenitors in the bone marrow are present only in mammals
suggests that some important feature of mammalian physiol-
ogy benefits from this unique mechanism for the production
of anucleate cells from the cytoplasm of a larger cell, for the
apparently major purpose of supporting hemostasis. However,
because both monotremes, which are egg-laying mammals,
and marsupials, which have a nonplacental pregnancy, possess
megakaryocytes and platelets, it is apparent that neither live
birth nor the presence of a placenta accounts for the evolution
of platelets in mammals. Therefore, the biological advantage
gained from the generation of nonnucleated platelets by poly-
ploid megakaryocytes remains unidentified (Figs. 1.1 and 1.2).
INVERTEBRATES
In many marine invertebrates, only one type of cell circulates in
the blood or is present in the coelomic fluid. This single cell
type plays multiple roles in the defense mechanisms of the ani-
mal, including hemostasis. Such cells are capable of aggregating
and sealing wounds. Although the biochemical basis for adhe-
sion of these cells is not understood, the participation of cell
aggregation in invertebrate hemostasis suggests that this pro-
cess is perhaps the earliest cell-based hemostatic function
(Figs. 1.1 and 1.2).1–4 The hemocytes of the ascidian Halo-
cynthia roretzi, for example, aggregate after removal from the
hemolymph (i.e., the circulating body fluid, equivalent to
blood, in animals with open circulatory systems).3 Aggregation
depends on divalent cations and is inhibited by ethylene
diamine tetraacetic acid (EDTA). Similar to the response of
mammalian platelets to vascular trauma, repeated sampling
of hemolymph from the same area of an individual H. roretzi
results in hemocytes that are increasingly activated, as mea-
sured by aggregometry (Fig. 1.3). Furthermore, the aggregated
hemocytes release a factor into the plasma that induces addi-
tional aggregation. The hemocytes of the California mussel
Mytilus californianus rapidly aggregate and adhere to foreign sur-
faces after the removal of blood from the animal.8 Aggregation
in vitro was shown to be a two-step process and distinct from
adhesion. Adhesion is Ca++- or Mg++-dependent.8 The parallels
with mammalian platelet function are evident.
The enormous range of types of cell-based coagulation in
insects has been described by Gr
egoire.9 The hemolymph of
many insects contains coagulocytes, which on contact with a
foreign surface extrude long, straight, threadlike processes that
may contain cytoplasmic granules. These cytoplasmic exten-
sions mesh with similar processes from other coagulocytes, cre-
ating a hemostatic plug. Examples of coagulocyte aggregation
and cytoplasmic expansions are shown in Figs. 1.4 and 1.5.
Thrombocytoids, a type of circulating hemocyte in the
1
2 PART I Platelet Biology

Fig. 1.1 Aggregation and alteration in the shape of Limulus amebocytes during cellular clotting on a glass surface. Note the prominent
nucleus and the variety of shapes that occur after activation. The cells have also become degranulated (see also Figs. 1.20, 1.22, and 1.23).
Magnification
200 (upper) and
320 (lower). (From Levin and Bang,1 with permission.)

hemolymph of the blowfly Calliphora erythrocephala tend to
fragment, resulting in “anucleated cytoplasmic fragments”
(Fig. 1.6).10,11 Their fragments then aggregate and form net-
works which are believed to promote hemostasis and seal
wounds.10,11 Each cell fragment remains surrounded by an
intact plasma membrane. The authors conclude that “The
thrombocytoid of this insect in some respects resembles the
megakaryocyte of mammals. The transformations exhibited
by these cells..., such as increased fragmentation and agglutina-
tion, are analogous to the process of agglutination of blood
platelets in mammals giving rise to the white thrombus.”
In some invertebrates, hemostasis is provided entirely by
cell aggregation at the site of a wound.12–14 In others, the hemo-
cytes contain coagulation factors or clottable proteins that are
released after activation and/or aggregation of the cells.15–18 In
the Arthropoda, as in other invertebrate classes, mechanisms of
hemostasis vary widely. In decapod crustaceans (e.g., Homarus
americanus, the American lobster), hyaline cells, a type of hemo-
cyte, initiate coagulation when they lyse.19 In some arthropods,
circulating amebocytes release a coagulase that activates a clot-
table protein already in the plasma.20 Plasma from the hemo-
lymph of L. polyphemus, the American horseshoe crab, normally
lacks coagulation factors.16,17 However, after activation, ame-
bocytes, the only type of circulating blood cell in Limulus,
release a cascade of coagulation factors that result in coagula-
tion of the plasma.21,22 Activation of Limulus amebocytes not
only results in initiation of coagulation, but also is associated
with the initiation of other defense mechanisms, including
 The Evolution of Mammalian Platelets 3

Fig. 1.3 Activation of hemocyte aggregation in Halocynthia roretzi.

Point A represents the point on the tunic of the animal through which the
hemolymph was repeatedly taken; point B represents a different point on
the tunic. The time between the initial collection of hemolymph (0min) and
subsequent collections is indicated above each tracing. The bar
represents 5 min. The extent of light scattering is shown on the ordinate in
arbitrary units. (From Takahashi et al.,3 with permission.)

Fig. 1.2 Appearance of long filamentous processes after the

activation of Limulus amebocytes. These processes are often
connected with those of other amebocytes. Magnification
200 (upper)
and
320 (middle and lower). (From Levin and Bang,1 with permission.)

wound healing, activation of a primitive complement system,

and release of antibacterial factors.23

NONMAMMALIAN VERTEBRATES
Nonmammalian vertebrates have nucleated, often spindle-
shaped thrombocytes, the first cells to evolve that specialize
in hemostasis.12,15,24 Thrombocytes are found in fish, and in
some species multiple types of thrombocytes have been
described.25,26 However, some reports of multiple types of
Fig. 1.4 Dytiscus marginalis L. (Coleoptera). Clustering of
thrombocytes may have inadvertently described technical arti-
coagulocytes around a fragment of cuticle stimulates the reaction of
facts of the methods of blood collection, which resulted in cell
hemolymph at wound sites. Magnification
800. (From Gr
egoire,9 with
activation and morphological alterations of some of the throm-
permission.)
bocytes. Thrombocytes have been variously described as spin-
dle shaped (fusiform), spiked, spherical, oval, or teardrop in
appearance, with a few but variable number of cytoplasmic In addition, size differences may exist between immature and
granules.27 There are a central round or elongated nucleus mature thrombocytes.28 Quantification of fish thrombocytes
and a rim of cytoplasm. The nuclear: cytoplasmic ratio is greater has been made difficult by the often-described resemblance
than that of the nucleated red blood cells. Periodic acid-Schiff between fish thrombocytes and lymphocytes, and the tendency
(PAS)-positive cytoplasmic granules are sometimes described. of thrombocytes to clump.25 The most extensive quantitative
4 PART I Platelet Biology
Fig. 1.5 Erodius tibialis (Coleoptera).
The threadlike cytoplasmic processes are
shown, carrying along the granules
produced by six coagulocytes (asterisks).
Magnification
960. (From Gre
goire,9 with
permission.)
Fig. 1.6 Ultrathin section of a thrombocytoid with numerous deep
invaginations. The cytoplasm is described as demonstrating rough
surfaced endoplasmic reticulum with cysternae whose profiles are
parallel and characteristically curved. Orig. magnification

12,000. (From Zachary et al.,10,11 with permission.)
study, based on Wright-stained blood smears, has reported that
52% (mean; range, 46%–61%) of white blood cells in 121 spe-
cies of marine fishes are thrombocytes.29 In that study, throm-
bocytes were overall the most common type of white blood
cell. Cytochemical analysis has been successful in distinguish-
ing thrombocytes from lymphocytes in fish.30,31 Thrombocytes
of the piracanjuba Brycon orbignyanus30 and the Murray cod
Maccullochella peelii peelii31 were PAS-positive, but negative
for a wide range of other cytochemical markers (Fig. 1.7). In
contrast, the thrombocytes of seven other species of fish were
positive for both PAS and acid phosphatase.32,33 The thrombo-
cytes of the relatively primitive Australian lungfish Neoceratodus
forsteri contain, in addition to PAS, variable amounts of acid
phosphatase, gamma-naphthyl acetate esterase (ANAE), and
AS-D chloroacetate esterase (AS-D).34 Examples of thrombo-
cytes in cartilaginous fish are shown in Fig. 1.8.35 A single
von Willebrand factor transcript that encodes a simpler protein
compared with higher vertebrates has been identified in the
jawless Atlantic hagfish, Myxine glutinosa (Fig. 1.9).36 It is pre-
sent in both thrombocytes and endothelial cells.
Some species of estuarine cyprinodontiform fishes have
been described as having a seasonal variation in the ratio of
mature to immature circulating thrombocytes.28 Immature
thrombocytes reached their highest levels during July and
August, which the authors interpreted as indicating an
increased rate of thrombopoiesis. Fish thrombocytes contain
bands of microtubules33,37 and in at least some species are
described as phagocytic.33,38–40 In contrast to platelets, throm-
bocytes are not typically aggregated by adenosine diphosphate
(ADP), or epinephrine.12,41,42 Zebrafish (Danio rerio) thrombo-
cytes have been shown in vivo to play a role in the develop-
ment of arterial thrombi.43 However, avian thrombocytes
demonstrated a much lesser ability to occlude an experimen-
tally damaged carotid artery than did murine platelets, perhaps
because the density of αIIbβ3 receptors on human platelets is at
least 18–25 times greater than on chicken thrombocytes.44
Avian thrombocytes are similar in appearance to the throm-
bocytes in fish and are believed to be produced by mononu-
clear precursors in the bone marrow.45 Four developmental
stages of thrombocyte precursors have been described in the
chick embryo, and collectively account for 0.6–2.4% of nucle-
ated cells in the bone marrow.46 PAS-positive cytoplasmic gran-
ules were present in all stages of the thrombocytic lineage. An
extensive ultrastructural study of six species of domestic birds
concluded that although thrombocytes are similar in size to
lymphocytes, thrombocytes have a denser nucleus and a very
highly vacuolated cytoplasm.47 Flow cytometry has been used
to discriminate between lymphocytes and thrombocytes in
chickens48 and ducks.49 The developmental stages of the
thrombocyte lineage in chicken are shown in Fig. 1.10.50
Phagocytosis has been demonstrated.51 As in fish, thrombo-
cytes are the most common white blood cell in the chicken48
and ducks.49 Similar to platelets, avian thrombocytes contain
serotonin (5-hydroxytryptamine)52,53 and release what appears
to be β-thromboglobulin during the release reaction.54
The thrombocytes of at least some birds, amphibians, rep-
tiles, and fish have a membrane system referred to as the
surface-connected canalicular system (SCCS).37,55–59 This sys-
tem is also a feature of mammalian platelets and has been linked
to their derivation from the cytoplasm of megakaryocytes.60
However, the presence of the SCCS in nonmammalian throm-
bocytes indicates that this system reflects an important function
of blood cells that play a major role in hemostasis, and that the
SCCS need not be derived from the demarcation membrane sys-
tem (DMS) of megakaryocyte cytoplasm. Bovine61 and African
elephant (Loxodonta africana)62 platelets are apparently excep-
tions in that they do not contain a SCCS, although bovine mega-
karyocytes have a DMS.60 Importantly, the platelets of the
African elephant also lack microtubules.62 Elegant studies of
the thrombocytes of the dogfish Mustelus canis demonstrated
that the series of cytoskeletal changes that occurred following
exposure to thrombin paralleled those of platelets.63 Further-
more, the authors concluded that the responsiveness of nucle-
ated fish thrombocytes to mammalian thrombin indicated
the presence of evolutionarily conserved signal transduction
pathways. Presumably, this conclusion can be applied to the
thrombocytes of other nonmammalian vertebrates.
Thrombocytes from an alligator (a reptile) and a bullfrog
(an amphibian) are shown in Fig. 1.11. Multiple examples of
the thrombocytes of other nonmammalian vertebrate species
are shown in Fig. 1.12.64–68 Cytochemical studies of the throm-
bocytes of the giant lizard Gallotia simonyi (a reptile) failed to
detect any of the substrates for the six cytochemical stains uti-
lized, including PAS and four enzymes.69 However, the throm-
bocytes of two species of Australian crocodiles were
PAS-positive.70 Serotonin has been detected immunochemi-
cally in seagull, alligator, and sea turtle thrombocytes
 The Evolution of Mammalian Platelets 5

Peroxidase ACP ALP b-glu 1

Lymphocyte
Thrombocyte

NCE NAE NBE

Lymphocyte
Thrombocyte

SBB PAS
Lymphocyte
Thrombocyte

Fig. 1.7 Murray cod (Maccullochella peelii peelii) lymphocytes and thrombocytes. Light microscopy of blood smears. Top: enzymatic staining
for nonesterases. Peroxidase (A,B); acid phosphatase (ACP) (C,D); alkaline phosphatase (ALP) (E,F); and β-glucuronidase (β-glu) (G,H). Middle:
enzymatic staining for esterases. Naphthol AS chloroacetate esterase (NCE) (A,B); naphthyl acetate esterase (NAE) (C,D); and α-naphthyl butyrate
esterase (NBE) (E,F). Bottom: nonenzymatic staining: Sudan black B (SBB) (A,B); and periodic acid Schiff (PAS) (C,D). In contrast to lymphocytes,
thrombocytes were positive only for PAS, which indicated the presence of glycogen (bottom panel, D). Magnification
100. Bar ¼ 10 μm. (Modified
from Shigdar et al.,31 with permission.)

(Fig. 1.13) and its presence in alligator and sea turtle thrombo-
cytes confirmed with HPLC (Fig. 1.14).53 Serotonin has not
been detected in the thrombocytes of the American bullfrog
Rana catesbeiana53 or the tortoise Geoclemys reevesii.71 Phyloge-
netic insights have been gained from an analysis of the presence
of serotonin in the thrombocytes and platelets of vertebrates
and selected mammalian orders, respectively (Fig. 1.15).53
Studies of the African clawed frog, Xenopus laevis, using an
antibody against thrombocytes, identified cells in the liver
and spleen that expressed CD41 and had higher ploidy levels
than the nucleated, tetraploid (4 N) erythrocytes.72 Hepatic
thrombocytes had higher DNA levels that did circulating
thrombocytes (Fig. 1.16).72 Circulating thrombocytes had a
mean ploidy level of 8 N and were acetylcholinesterase positive,
in contrast to erythrocytes. When the hepatic cells of X. laevis
were cultured in the presence of recombinant X. laevis thrombo-
poietin (TPO), the cells differentiated into polyploid CD41-
expressing cells73 (Fig. 1.17). The subsequent culture of these
cells after removal of TPO resulted in the production of mature,
spindle shaped thrombocytes, which were activated by throm-
bin (Fig. 1.18). Therefore, these cells were considered to have
features similar to mammalian megakaryocytes. It will be
instructive to determine the ploidy levels of the precursors of
thrombocytes in other nonmammalian vertebrates.
6 PART I Platelet Biology

Fig. 1.8 Transmission electron micrographs of thrombocytes in cartilaginous fish (Chondrichthyes). Left: dogfish (Squalus acanthias)
thrombocyte. Note the single nucleus in the lower region of the cell. A group of peripheral microtubules, sectioned lengthwise, are present in the upper left
part of the cell. Right: skate (Raja eglanteria) thrombocytes after their incubation with skate thrombin. Four thrombocytes have aggregated, with a loss of
distinct cytoplasmic boundaries. The aggregation and fusion of nucleated thrombocytes after exposure to thrombin resembles the response of human
platelets to thrombin. In contrast to platelets, adenosine diphosphate does not cause aggregation of thrombocytes. (From Lewis,34 with permission).

Fig. 1.9 Hagfish (Myxine glutinosa) von

Willebrand Factor (VWF) is expressed in
peripheral blood. Fluorescence microscopy
images of hagfish blood processed for
immunofluorescence staining of VWF, using
polyclonal antihuman VWF antibody. Left image (i)
shows 4 VWF-positive cells (arrows), which are
smaller than neighboring VWF-negative
erythrocytes (One of these is outlined in white). Right
image (ii) is a higher power view showing the
punctate staining pattern of a VWF-positive cell
surrounded by VWF-negative erythrocytes and
spindle cells. (From Grant et al.,36 with permission.)

COMPARATIVE HEMOSTASIS following experiment. ... There is a curious change produced in

An overview of comparative hemostasis is provided in
Table 1.1.74 Because of the great heterogeneity in types of blood
cells and coagulation mechanisms in invertebrates, any attempt
to summarize the characteristics of hemostasis in these animals
cannot avoid oversimplification and inaccuracy. Overall, how-
ever, it is clear that when cells are present in the invertebrate cir-
culation or coelomic fluid, they always play a role in
hemostasis.12,75
Among the extensive original studies of the blood by Wil-
liam Hewson (1739–1774) is a highly instructive plate that
illustrates the “red particles of the blood” in a wide variety of
animals76 (Fig. 1.19). The following statements in Hewson’s
text accompany this plate:
their shape by being exposed to the air, for soon after they are
received on the glass they are corrugated, or from a flat shape are
changed into irregular spheres. This change takes place so rapidly,
that it requires great expedition to apply them to the microscope
soon enough to observe it.76 (pp. 233–234).
Hewson (and his colleague Magnus Falconar) were actually
observing the hemocytes of lobsters becoming activated after
removal from the animal, changing shape, and then aggregat-
ing. Note the obviously altered appearance of the hemocytes,
in contrast to the multiple examples of genuine “red particles
of the blood” (in Fig. 1.19, an original Hewson plate; compare
his Figs. 11 and 12).

In the blood of some insects the vesicles [blood cells] are not red,
but white, as may easily be observed in a lobster (which Linnaeus
calls an insect), one of whose legs being cut off, a quantity of a
clear sanies flows from it; this after being some time exposed to the
air jellies, but less firmly than the blood of more perfect animals.
When it is jellied it is found to have several white filaments; these
are principally the vesicles concreted, as I am persuaded from the
A COMPARISON OF HUMAN PLATELETS AND
LIMULUS AMEBOCYTES
L. polyphemus, the horseshoe crab, is the last surviving member
of the class merostomata, which included marine spiders. The
Limulus amebocyte, which is the only type of circulating blood
cell in the hemolymph, has probably been the most intensely
 The Evolution of Mammalian Platelets 7

357 358 359

360 361 362

Fig. 1.10 Camera lucida drawings of the

stages of maturation of the thrombocyte
lineage in the bone marrow of the white
leghorn chicken. Thromboblasts (357, 358);
early immature thrombocytes (359–362);
midimmature thrombocyte (363); late immature
thrombocyte (364); mature
thrombocyte (365). (From Lucas and Jamroz,50
363 364 365 with permission.)

Fig. 1.11 Transmission electron micrographs of thrombocytes. Left: alligator (Alligator mississippiensis) thrombocytes. The three thrombocytes
demonstrate a spindle shape, large nuclei, and a fine open canalicular system. Right: bullfrog (R. catesbeiana) thrombocytes. Two of the thrombocytes
contain large nuclei. The inclusion in the lowest cell may be a phagocytosed red blood cell. (From Lewis,35 with permission.)

studied of the invertebrate blood cells involved in hemostasis
and blood coagulation.1,77 The concentration of amebocytes
in the blood of adult limuli is approximately 15
109/L. Ame-
bocytes are nucleated cells that are approximately the size of
mammalian monocytes. Their cytoplasm is packed with
granules (Fig. 1.20).5 After activation on a foreign surface (or
by bacterial endotoxins), amebocytes spread and degranulate
(Fig. 1.20, right panel). Degranulation is associated with exocy-
tosis.16,78–80 The amebocytes, which are normally discoid (like
platelets), develop pseudopodia and microspikes.81 These cells
8 PART I Platelet Biology

Fig. 1.12 Thrombocytes of the common lizard (Podarcis S. sicula Raf.), dogfish (Scyliorhynus stellaris L.), electric ray (Torpedo
marmorata), red-legged partridge (Alectoris rufa rufa L.), eagle (Aquila rapax), and Brujn’s echidna (Zaglossus bruijni). Top panel: A.
group of lizard thrombocytes (May Gru €nwald Giemsa). B. Lizard thrombocytes positively stained for acid phosphatase. C. Dogfish thrombocyte with
granules stained for β-glucuronidase. Lower left panel, A–C: A. torpedo thrombocyte (T) positively stained and basophilic erythroblast (Eb)
negatively stained for platelet factor 4 (PF4). [Inset: Thrombocyte (left) positive and eosinophilic granulocyte (E) negative for PF4]. B. Red-legged
partridge thrombocytes positively stained for α-naphthylacetate esterase. C. Red-legged partridge thrombocytes positively stained for acid
phosphatase. Lower right panel, D: Eagle thrombocytes (T), in a field that contains a heterophile (right), basophile (left), and many nucleated red blood
cells. In this figure, the relative sizes of the thrombocytes in the species shown are not to scale. Note the tendency of thrombocytes to clump (top panel:
A and B; lower left panel: B and C). The cleft that sometimes appears in the nucleus of thrombocytes has been described in many reports (top panel: C).
Lowest right panel, F: variably shaped thrombocytes in Bruijn’s echidna. (From Pica et al.,64–66 D’Ippolito et al.,67 and Jain,68 with permission.)

are also capable of retraction (Fig. 1.21),82 apparently similar to
the process of clot retraction induced by mammalian platelets.
Amebocytes have a Toll-like receptor and have been shown to
bind bacterial endotoxin.83,84 These cells are also capable of
phagocytosis.85
Fig. 1.22 6 shows the ultrastructure of a Limulus amebocyte.
The cytoplasm contains at least two types of granules
(Fig. 1.23)7 that are biochemically different and contain all
the components of the blood coagulation mechanism.7,21,77
Marginal microtubule bands are present (Fig. 1.24).
Mammalian platelets are appropriately considered as func-
tioning primarily to support a range of hemostatic mecha-
nisms, both by maintaining the integrity of blood vessels
and by contributing to the process of blood coagulation. How-
ever, platelets have many other capabilities, although often
rudimentary, that appear to be unrelated to their hemostatic
function. Platelets have been shown to be bactericidal for B.
subtilis and E. coli (but not S. aureus), but only in the presence
of thrombin and a heat-labile plasma protein fraction
>100 kDa.86 The bactericidal mechanism has not been not
 The Evolution of Mammalian Platelets 9

established. Despite approximately 450 million years of evolu-

Fig. 1.13 Immunocytochemical evidence for serotonin in human
platelets and in seagull and alligator thrombocytes. Platelets or
thrombocytes were incubated with a specific serotonin antibody and a
fluorescently labeled secondary antibody. The morphology of human
platelets (A), seagull thrombocytes (C), and alligator thrombocytes (E) is
shown. Leukocytes and erythrocytes are also present in (C) and (E). The
bright spots indicate the presence of serotonin-specific fluorescent
labeling in granules in platelets (B), and in seagull (D) and alligator (F)
thrombocytes. Bar ¼ 10 μm. (From Maurer-Spurej,53 with permission.)
1000
Human
800
Alligator
Voltage (mV)
tion,87 mammalian platelets retain many of the functions of 1
Limulus amebocytes (and of many other comparable inverte-
brate blood cells). This phenomenon may have resulted from
the retention of multiple functions previously found in a single,
“all-purpose” circulating cell type, such as the Limulus amebo-
cyte, only one of whose functions was hemostasis. Studies of
the thrombocytes of the rainbow trout Oncorhynchus mykiss
concluded that these cells had both hemostatic and phagocytic
functions. The authors speculated that “some aspects of this
dual functionality observed in thrombocytes may have been
lost with the evolution of the anucleate platelet.”88
The multiple characteristics shared by mammalian platelets
and Limulus amebocytes are summarized in Table 1.2. Platelets
have rudimentary bactericidal and some phagocytic or
phagocytic-like activity.86,90–93 However, an ultrastructural
study of interactions between human platelets and various-size
particles concluded that platelets were not true phagocytes, and
that the uptake of bacteria involved the channels of the open
canalicular system (OCS).94 Nevertheless, an exhaustive review
of studies that described the interactions of thrombocytes and
platelets with particulate materials or bacteria presented con-
siderable data that appeared to support the conclusion that
both thrombocytes and platelets are capable of phagocytosis.95
Platelets contain endotoxin-binding substances96 and have
been shown to interact with bacteria, endotoxins, viruses, and
fungi.97–101 Murine102 and human103 platelets and chicken
thrombocytes104 have been shown to express a functional
Toll-like receptor-4 (TLR4), the receptor for bacterial endotoxin
(LPS).102 The extensive role of platelets in antimicrobial host
defense101,105 is reviewed in Chapter 29. Staphylococcus can
stimulate human platelets to undergo the release reaction.106
Microbicidal proteins polymorphic protein-1 (PMP-1) and
PMP-2 are small cationic peptides released by rabbit platelets,
which disrupt the membrane of S. aureus and cause cell
death.107 It has been demonstrated that staphylococci have a
receptor for the Fc fragment of immunoglobulin G (IgG) that
provides a mechanism for aggregation of human platelets by
the formation of a complex composed of bacteria, IgG, and
platelets.108 Furthermore, it has been shown that platelet bind-
ing by S. aureus is mediated, at least in part, by direct binding of
clumping factor A (ClfA) to a novel 118-kDa platelet mem-
brane receptor.109 Pneumococcus (S. pneumoniae) and vaccinia
virus can induce the release of serotonin from human plate-
lets.110,111 Platelets may preferentially carry oral streptococci
to damaged endocardium due to their serine-rich repeat adhe-
sins, which target platelet sialoglycans.112 Fibrinogen in con-
junction with other unidentified plasma factors was required
for platelet aggregation and secretion.113 The aggregation of
human platelets by S. viridans, S. pyogenes, and S. sanguis has
been demonstrated.114–116
Human platelets have been reported to be cytotoxic for par-

600 asites by a mechanism involving an IgE receptor on the platelet

Sea turtle surface.117,118 Platelets also have been shown to play a role in
400 the excretion of Schistosoma mansoni in the stool of mice.119
Other studies have demonstrated that platelets enhance the
200 adherence of schistosome eggs to endothelial cells, and that
Fresh water turtle, interleukin (IL)-6, produced by activated monocytes, markedly
frog, tuna, salmon increases the cytotoxicity of platelets against schistosomula.120
0
Under certain circumstances, platelets demonstrate a chemo-
tactic response and migrate,121–123 as do amebocytes.124 It
4 6 8 10 12 14 16
has been concluded that the thrombocytes of the turbot Psetta
Time (min) maxima are also capable of movement.33 Recent studies have
Fig. 1.14 Quantitative determination of serotonin with HPLC. convincingly documented this capability of platelets by dem-
Serotonin levels in isolated platelets or thrombocytes were determined onstrating that they are able to function as cellular scavengers,
chromatographically. Human platelets and alligator and sea turtle and collect bacteria from the vascular surface.125 The data indi-
thrombocytes contain serotonin, but the thrombocytes of the fresh water cate that platelets can function as mechano-sensors and inter-
turtle, frog, tuna, or salmon do not. (From Maurer-Spurej,53 with acted importantly with neutrophils and the immune system.
permission.) Direct involvement of αIIbβ3 integrin in platelet migration
10 PART I Platelet Biology

4000 3000 2000 1000 900 800 700 600 500 400 300 200 100 90 80 70 60 50 40 30 20 10 1 Million years ago
Fossil time
Humans
Circulating serotonin
present
Gorillas
Monkeys
Cats, dogs

Pigs
310
Cattle
Birds

Living reptiles
Fishes
Vertebrates
Protists
Origin of eukaryotes
Prokaryotes

Fig. 1.15 Phylogenetic comparison of species with and without circulating serotonin. Fossil records were used for the comparison of species
divergence times. Time estimates were based on previously published studies. The time scale is linear only within the ranges 10–100, 100–1000, and
1000–5000 million years. Serotonin appears in the circulation in the American alligator and leatherback turtle, reptiles that evolved approximately 310
million years ago. Avian and mammalian blood also contains serotonin. (From Maurer-Spurej,53 with permission.)

4N 4N
PB T12+ Spleen T12+
T12− T12−
Count

Count

0 0
101 102 103 104 105 101 102 103 104 105
DNA contents DNA contents

4N
T12+ PB T12+
Liver
T12− Spleen T12+
Fig. 1.16 DNA content analysis Liver T12+
of polyploid T12+ cells (African
clawed frog, Xenopus laevis
Count

Count

thrombocytes) in the peripheral

blood, spleen, and liver. (A–C)
Each panel shows the DNA
histograms of cells labeled with T12
and Hoechst 33342; T12+ (black
line) and T12 (gray fill) DNA
histograms. (D) White, black, and
gray areas of the histogram indicate 0 0
peripheral blood, spleen, and liver
101 102 103 104 105 101 102 103 104 105
T12+ cells, respectively. (From
Tanizaki et al.,72 with permission.) DNA contents DNA contents
Another random document with
no related content on Scribd:
their main contentions—and justify the Kaiser’s lordly contempt of
the scrap of paper, are of a piece with every manifestation of the
political cult which has become one of Germany’s holiest
possessions. And it is because the British nation as a whole
obstinately refused to listen to those who apprised them of this
elemental movement, and of the dangers it concealed, that they
dispensed with a large land army, slackened the work of
shipbuilding, and trusted to a treaty which they are now surprised to
see dealt with as a mere scrap of paper.
In like manner the British people at first smiled sceptically at the
narratives of Belgians who witnessed and described the killing of
unarmed men, women, and children, the finishing of the wounded on
the battlefield, the living shields of women and girls with which they
protected their soldiers, the taking and shooting of hostages, and
other crimes against humanity. After all, it was argued, the Germans
are not quite so unlike ourselves as these stories would have us
believe. They, too, are men who have left wives, sisters, mothers,
and children at home, and the wells of human pity are not dried up
within them. They are incapable of such savagery. Those tales
evidently belong to the usual class of fiction which sprouts up on all
battlefields.
Yet, whatever the truth might be—and since the fiendish
passions of the soldiery were let loose against Louvain, Malines, and
Rheims we know that some of the narratives were based on
gruesome facts—the ground at first taken up was untenable. Nobody
possessing even a superficial acquaintance with Prussian history
had grounds for asserting that the German army was incapable of
such diabolical deeds. Its recorded doings in seasons of peace
demonstrated its temper. That the officers and the rank and file are
obedient to their commanders will not be gainsaid. To their Kaiser
they are, if possible, still more slavishly submissive. Well, the Kaiser,
when his punitive expedition was setting out for China, addressed
them thus: “When you encounter the enemy you will defeat him. No
quarter shall be given, no prisoners shall be taken. Let all who fall
into your hands be at your mercy. Just as the Huns a thousand years
ago, under the leadership of Etzel (Attila), gained a reputation in
virtue of which they still live in historical tradition, so may the name of
Germany become known in such a manner in China that no
Chinaman will ever again even dare to look askance at a German.”
The monarch who gave utterance to those winged words was not
conscious of saying aught that might shock or surprise his people.
His false conscience felt no qualms. The principle underlying this
behest was the foundation-stone of Prussian culture. And the
Kaiser’s wish is now realized. The name of Germany, whose love of
wanton destruction, delight in human torture, and breach of every
principle of manly and soldierly honour are now become proverbial,
will henceforward be bracketed in history together with that of the
Huns.
How British people who read and stigmatized these barbarous
behests, emphatically issued by the supreme ruler of the German
nation and the supreme head of the German Church, should have
held him who uttered or the troops that executed them incapable of
the crimes laid to their charge in Belgium is a mystery. Terrorism in
occupied countries has always been part of the Prussian method of
waging war. It is such an excellent substitute for numbers! The
examples of it given in the years 1814 and 1815 are still
remembered. Since then it has been intensified. During the Boxer
movement in China I witnessed illustrations of it which burned
themselves in my memory. The tamest of all was when the German
troops arrived in Tientsin. The nights were cool just then, and a knot
of soldiers were dismayed at the prospect of spending a night
without blankets. I happened to know where there was an
untenanted house with a supply of blankets, and out of sheer
kindness I took them to it. With a smile of gratitude the officer in
command set the blankets on one side. Every portable article of
value was next seized and appropriated. And then the soldiers took
to smashing vases, statues, mirrors, the piano, and other articles of
furniture. They laughed at my remonstrances, and reminded me of
the Kaiser’s orders. All at once they abandoned the spoil, and
rushed down to the courtyard to shoot some Chinese who were said
to be there. As luck would have it, however, the newcomers were
their own comrades, so there were no executions that first evening.
But the Kaiser’s men made up for it later.
 Germany’s necessity, as defined by her War Lord or any of her
high officials, knows no law. Stipulations and treaties are for non-
German States, which must be held strictly to their obligations. To
Teutons the Treaty of Bucharest and the neutrality of Belgium were
meaningless terms. But only to Teutons. The Japanese are to be
made to respect the neutrality of China. For the chosen people are a
law unto themselves. That is, and has long been, the orthodox
doctrine of the Pan-German Church. What more natural than its
application to the treaty of 1839, which Bismarck confirmed in writing
in the year 1870, and which the Kaiser and Herr von Bethmann
Hollweg, with the hearty approval of the whole articulate German
nation, have recently spoken of contemptuously as a scrap of paper?
If any doubt could be entertained as to the extent to which this
German theory of morality has spread, it will have been dispelled by
the body of eminent German theologians who have just issued their
appeal to Evangelical Christians abroad. They, at any rate, have no
fears that their eloquent appeal will be treated as a mere scrap of
paper. It is the word of their “good old God.”
 CHAPTER I
THE CAREFULLY LAID SCHEME

Europe’s tremendous tragedy, the opening scenes of which are now

unfolding themselves to horrified humanity, is no ordinary conflict
arising out of a diplomatic quarrel which timely concessions and soft
words might have settled with finality. In its present issues it is the
result of a carefully laid scheme of which the leaders of the German
people are the playwrights and the Kaiser the chief actor. It was
cleverly thought out and patiently prepared. The manifold forces let
loose by the Berlin Government for the purpose of leading up to a
coup de théâtre which involves the existence of cultured Europe had
long since got beyond the control even of those who were employing
them. All that was still possible was the choice of the moment for
ringing up the curtain and striking the first fell blow. And, sooth to
say, judging by the data in the hands of the Berlin Foreign Office, no
conjuncture could have been more propitious to Germany’s designs
than the present. For circumstance had realized most of the desired
conditions, and the Kaiser, without hesitating, availed himself of his
good fortune. It is useless to dissemble the fact that the copious
information accumulated in the Wilhelmstrasse warranted the belief
that there could not have been a more auspicious moment for the
realization of the first part of the Kaiser’s programme than the
present. If Germany be indeed set apart by Providence as the
people chosen to rule Europe and sway the world, the outcome of
the present conflict should be to sanction this inscrutable decree of
Fate. Certainly the hour has struck for which she has been waiting
and keeping her powder dry during the past forty years. It is now or
never.
 Of this ingeniously conceived scheme the Achilles tendon was its
diplomatic aspect. And here Prussian clumsiness asserted itself
irrepressibly, as is its wont. A worse case with which to go before the
world than that of Germany in the present struggle it would be hard
to imagine. She has deliberately brought about a crude, naked
might-struggle, in which war-lust and brute force are pitted against
the most sacred and imprescriptible rights that lie at the very roots of
organized society. And she calls on God to help her to effect her
purpose.
The British nation is loath to think evil of its neighbours. It
generously credits them with the best—or at any rate the least
wicked—motives, and, even when the evidence on the other side is
overwhelming, gives them the benefit of the doubt. How strong the
evidence was in this case I pointed out over and over again. In 1911,
for instance, I wrote: “Since Europeanism was killed at Sedan and
buried at Frankfurt-on-the-Main, over forty years ago, international
treaties have been steadily losing their binding force. Their
significance has been gradually transformed into that of historic
souvenirs, symbolizing a given political conjuncture. To-day they are
nothing more. The unique, solid foundation of peace that remains is
readiness on the part of the peace Powers to defend it on the
battlefield.”
Optimists in this country objected that the German people and
their Chancellor were peacefully disposed, and utterly averse to
letting loose the horrors of an unparalleled war. And I replied that
even if in a certain sense the optimists were right, the attitude of the
German nation was beside the question. Nobody ever wants war, but
only the spoils it brings. “Germany,” I explained, “having spent
fabulous sums of money and human labour in creating an army
greater in numbers and more formidable than that of any of her
rivals, would consider the military superiority which this weapon
bestows upon her as a title-deed to property belonging to her
competitors. She would, accordingly, demand a return for her outlay,
would call for the neighbour’s territory she coveted, and expect to
receive it as a propitiatory sacrifice. War would not be her main
object, but only the fruits of war, extorted by threats which are more
than mere words. She would virtually say to France, Belgium, or
Holland, ‘I have it in my power to take what I want from you, and to
ruin you over and above. But I trust I may receive amicably from your
sagacity what I should be forced to wrest violently from your
shortsightedness.’ That is at bottom a modified form of the line of
action pursued by the bandit barons of mediæval Germany, a robust
survival into the twentieth century.” And it is exactly what has since
happened. The White Paper tells the story of the German Kaiser’s
attempt to induce our Government to connive at the seizure of
France’s colonies, which Germany needed for her enterprising
people.
But although for years I and some few others had been
preaching the imminence of this danger which no diplomatic
arguments could exorcize, the bulk of the British nation hoped on,
refusing to impute to the German people the motives or the aims
3
which we knew it entertained. In the Contemporary Review I was
attacked by the celebrated Professor Hans Delbrück for affirming, as
I have done for over twenty years, that Germany was concentrating
all her efforts on the coming struggle between herself and this
country, and the learned Professor did me the honour to say that so
long as I was allowed to express my views on foreign politics in the
Contemporary Review there would and could be no entente between
Great Britain and Germany. “As long as Mr. Dillon is permitted,” this
German Professor and successor of Treitschke wrote, “to set forth in
the Contemporary Review his fantastic views, engendered by hatred
and suspicion, about German policy, all those will be working in vain
who believe that peace between our nations can be secured by
4
arbitration treaties.” I then summed up my opinions as follows:

When I read the smooth-tongued, plausible panegyrics on

Germany’s politics, which are served up to us here in England
every year, and contrast them with the systematic
aggressiveness which everybody with open eyes and ears sees
and hears in Berlin, I behold Germany rise before me in the form
of a cuttlefish, with many lasso-like arms, ever ready to seize
their unsuspecting prey, and also ready, when itself is in danger,
 to shed an ink-like fluid which blackens the water and hinders
effective pursuit.

Everything that has come to pass since then offers a pointed

illustration of that presentment. The attempt to obtain without a war a
return for her outlay on her army and navy by calling for coveted
territory as a propitiatory sacrifice was energetically made during the
Morocco crisis. But the spring of the Panther failed of its purpose.
Germany’s further experiences during the London Conference were
likewise discouraging. The loose ranks of the Entente Powers closed
up at the approach of herself and her ally, and Albania proved a
mere torso. Then the supreme effort was put forth a few weeks back,
and the Berlin Government, alive to the possibility of a like unfruitful
result, determined to abide by and prepare for the extreme
consequences, which, sooth to say, appeared to them less
formidable than they really were.
Congruously with this resolve every precautionary measure that
prudence prompted or circumstance suggested was adopted
betimes, some secret, others public.
For the behoof of the European public the former were flatly
denied, and the latter glibly explained away.
Method characterized all these preparations, towards which the
British nation was particularly indulgent. Foremost among them was
the increase of the German army and the levy of the non-recurring
war-tax. Now, if Russia had had recourse to a measure of this kind,
all Europe would have clamoured for explanations. Germany was
allowed to have her way unquestioned. Honi soit qui mal y pense.
And yet the German Chancellor dropped a hint of his real purpose
which ought to have been sufficient to put Europe on its guard. He
spoke of the coming conflict between the Teutons and the Slavs. And
in truth that was the keynote to the situation. In Russia it was heard
and understood. Whether it was also taken to heart and adequately
acted upon there is another matter. In these islands most people
listened, smiled, and went their way unheeding. Yet this was the first
step towards tackling the Entente Powers one by one, which
constituted the alpha and omega of the Kaiser’s policy.
Another of the timely precautions taken by Germany, who was
resolved to make ready for every contingency, however improbable
—and a general European war seemed even to her statesmen most
improbable—was the purchase of horses. She despatched agents to
Great Britain, and especially to Ireland, in search of mounts suitable
for cavalry service, and also draught-horses. And during the months
of March, April, and May large numbers of these animals were
exported from the four provinces of Ireland to Hamburg without
exciting protest or occasioning comment. For the British are a
trusting people. And now the French army is obliged to make an
effort to acquire a fresh supply of mounts, and may encounter very
serious difficulties. Corn was also laid in, and heavy shipments of it
went to Hamburg for the troops.
The German banking manœuvres were begun later. Enormous
sums of gold were garnered in by German financial institutions
through their influential agents in England, of whom several enjoyed
the friendship, but, one hopes, not the confidence, of some of our
eminent public men. And even since the war began large batches of
cheques and bills endorsed to London bankers by financial houses
of Sweden, Denmark, Holland, Portugal, Italy, have been forwarded
to London for discount and collection. Indeed, Germany appears to
have been paying for foodstuffs drawn from these neutral countries
in cheques and bills which, strange to say, were still being
discounted here. For in this respect, too, the British are a trusting
people. Even mobilization would seem to have been commenced
secretly long before the crisis had become acute. We learn from the
newspaper press that among the papers found on a captured
German general is a service letter disciplining him for not
immediately answering an order for mobilization dated July 10th,
when no one outside of Germany had a suspicion that war was
impending. This date enables us to gauge the sincerity of the
Kaiser’s efforts to “moderate” Austria’s “impetuosity.”
Whoever wishes to have an inkling of Germany’s method of
opening the diplomatic chess-game which preceded the war, and
was intended to “localize” it as far as seemed conducive to her
interests, must endeavour to get a glimpse of the action of the
smaller hidden wheels within the wheels of official diplomacy. For the
Berlin Foreign Office worked on various lines, keeping official, semi-
official, and absolutely secret agents, diplomatic and journalistic,
hard at work all the time. Thus in Russia there was the titular
Ambassador, Count Pourtalès, over whose head the Military
Ambassador, a German officer who had access to the Tsar, and was
kept posted about everything that was going on in Russia, was wont
to despatch messages direct to the Kaiser. And this personage was
better informed of what was being done, neglected, and planned by
the Russian Government than some of the Russian Secretaries of
State. He had direct access to the highest society, and indirect to
every local institution in the Empire. To my knowledge, this German
Aide-de-Camp in the suite of the Russian Emperor despatched
detailed reports about the intrigues which were spun to oust the
present War Minister, Sukhomlinoff, from his post, and have the
Assistant War Minister appointed in his place. And I am able to add a
piquant detail: in one of these reports he assured his chief that
although the Assistant Minister, Polivanoff, is in his opinion the better
man, his appointment at the then conjuncture would throw things
military out of gear for a considerable time in Russia. But the Tsar
was not to be tempted. General Sukhomlinoff, who is undoubtedly
the right man in the right place, remained at his post.
It is hardly an exaggeration to say that Russia had no secrets
whatever from the agents, diplomatic and military, of the German
Government. Every intrigue that was woven, every scheme that was
laid before the various State departments in Petrograd, every casual
remark dropped by the Tsar in the intimacy of private life to a
courtier, every real or supposed weakness in the Imperial defences,
was carefully reported, with all the local anecdotic embroidery, and
duly taken cognizance of in Berlin. Among high officials there were
some who, without evil intent, but solely in virtue of what they
honestly but foolishly regarded as the privilege of private friendship,
were wont to unburden themselves of momentous State secrets to
certain representatives of the Empire with which Russia is now at
war. These representatives were made aware of the advice tendered
to the Tsar by his Majesty’s trusted advisers in various critical
emergencies, and they announced it to their chiefs, the Tsar’s
present enemies. There was, for instance, a few years ago, one
influential Russian statesman without whose assent the Government
would undertake nothing of real importance, a patriot whose leanings
towards Austria and Germany were natural and frankly proclaimed.
In the interests of his country, which he identified with the triumph of
his own particular party, this Russian laid bare many matters to the
Austrian Ambassador, then Baron Aehrenthal, who, being himself an
Austrian of the same political school of thought, warmly sympathized
with his friend, and also took due note of his friend’s confidences.
That, it is asserted, was the main source of Aehrenthal’s spirited
policy. He believed he knew Russia’s weak points, and relied on their
handicapping the diplomacy of the Tsar. And then his countrymen
ascribed to military weakness the concessions which the Russian
Government made for the sake of European peace.
I can affirm that certain State documents, which I could, if
necessary, describe, were in this way conveyed to the future enemy,
and that one of these, together with all the facts and figures adduced
therein as proofs, contributed materially to Germany’s decision to
present her ultimatum to Russia, by convincing her that that Empire
would not venture to take up the challenge. I make this statement
with first-hand knowledge. Thus Russian ingenuousness and
candour have played their part—certainly a material part—in bringing
down a frightful calamity on that nation.
European and Asiatic Russia is positively weevilled with
Germans. Most of the foreign trade there is carried on through the
intermediary of German agents, almost every one of whom is in
touch with the German Consulate of the provincial chief town. In the
railway administration, too, there were numerous public servants,
some of whom, by education, tradition, religion, language, and
sympathy, are as German as Herr Bassermann or Admiral von
Tirpitz. And all these channels of information were so many
tributaries of the great stream which flowed unceasingly between the
Singers’ Bridge and the Wilhelmstrasse.
 For in the Berlin War Office they were informed of three matters
of supreme moment, which weighed heavy in the scales when war
and peace trembled in the balance. First, that the vaunted Russian
gold reserve had been immobilized, and was therefore not available
for war; second, that the army was unready; and third, that the Tsar,
for dynastic reasons, would on no account embark on another war.
In the Wilhelmstrasse and in the German War Office reports had
been received setting forth in detail that the Russian land forces had
been uniformly neglected in the interests of a short-sighted economy,
and that the wear and tear of the army during the Japanese
campaign had never been made good, could not, indeed, be made
good without an enormous outlay, whereas only a few paltry million
roubles had been spent on current needs in lieu of the milliards
without which reorganization was not feasible. Russia, therefore,
was not to be feared. And this inference was duly communicated to
the German Ambassador in Vienna, M. von Tschirschky, who worked
really hard and successfully to bring about the present conflict,
without, however, foreseeing its extent.
The other documents turned upon Russian finances. But the
burden of their message was the same. The line of reasoning and
the sequence of allegations was this: Russia’s gold reserve was
indeed large, but had been spirited away. For the State Bank had
lent out vast sums to the private banks, most of which are financed
by German institutions. And these loans had been given, not, as in
France and Berlin, for a maximum term of two months, but for six,
eight, twelve, fourteen months. The private banks in turn, thirsty for
profits, had distributed the money thus borrowed among private
individuals, who employed it in wild speculation. And the result was
that the gold reserve in Russia could not be made liquid in time
should hostilities break out this year; consequently a war in the year
1914 would entail a financial crash of unconceived dimensions. As
for the Russian money deposited in Berlin, it, too, was locked up
there, and would be commandeered by the German Government
were Russia to be forced into an armed conflict. The shock which
this revelation is supposed to have given the Tsar was also
described for the benefit of the Wilhelmstrasse. And the revelation
itself constituted another of the elements which decided Germany to
cross the Rubicon.
In France the Germans were nearly as much at home as in
Russia, one marked difference being that a larger percentage of
State secrets there was to be found in the newspapers. But whatever
the periodical prints failed to divulge was ascertained without
difficulty and reported without delay. It is a curious fact, but it is a
fact, that Germans had ready access to almost every man of mark in
the Republic, and statesmen there who would hum and haw before
receiving well-known Russian or British publicists were prepared to
admit them on the recommendation of Germans and Austrians who
made no secret of their nationality. I heard this statement in Paris,
and naturally hesitated to credit it. But as it was worth verifying, I
verified it. And this is what I found. Some eminent men in Paris had
refused to see a certain public man of European note, some on the
ground that they were too busy just then, others because it was
against their custom. The foreigner was advised to renew his
application at once, but through a private individual, a citizen of one
of the Powers now at war with the Republic. And he did. The result
was amazing. Within three days the doors of them all were thrown
open to him. But the quintessence of the irony lies in one piquant
detail: one of these French statesmen said to the intermediary who is
now inveighing against France and the French: “Let me see. Is not
that friend of yours a contributor to a periodical which is strongly pro-
German? If so, I had rather not meet him at all.” “By no means,” was
the answer. “He is very anglophile, and, of course, a great friend of
France.” “Ah, very well then, he can come.”
 CHAPTER II
THE MANY-TRACKED LINES OF GERMAN
DIPLOMACY

German diplomacy never contented itself with its one natural

channel. All its lines were many tracked. The Ambassador’s reports
were checked over his head by those of his secretaries, of the
consular agents, of the military and commercial attachés, of the
heads of great financial institutions and big business firms, who
enjoyed and abused the hospitality of Great Britain, France, and
Russia, and by the secret communications of professional spies and
the disclosures made by unwitting betrayers of secrets. During the
Morocco crisis the German Foreign Secretary, von Kiderlen
Waechter, was in direct and continuous telegraphic contact with the
first Secretary of the German Embassy in Paris, von Lanken, over
the head of the Ambassador, von Schoen. And here in London
Prince Lichnowsky, like his colleague Pourtalès in St. Petersburg,
shrank during the period of the crisis preceding the war to a mere
figure-head of the Embassy. Herr von Kuhlmann was the
Ambassador. His information was treated as decisive. His views
were listened to with respect. For he always strove and generally
contrived to repair to the source himself. Thus it was he who was
asked to visit Ireland and send in a report to the Wilhelmstrasse on
the likelihood of civil war breaking out there, and its probable
duration and general effect upon the country and the Government.
Herr von Kuhlmann’s communication, which was checked by the
accounts of German correspondents and of a number of spies who
were despatched independently to Belfast and other parts of Ulster,
made a profound impression on the Kaiser and his official advisers.
From the gist of it they derived their conviction, which was still strong
during the week that ended on July 30th, that England’s neutrality
was a foregone conclusion. For a time Herr von Kuhlmann’s
judgment was categorical. He had no misgivings. According to him
the die had already been cast, and the effect of the throw could not
be altered. The British Cabinet was bound hand and foot by the
sequel of its Home Rule policy. But even had it been otherwise, it
was committed to peace on other grounds. The Asquith Government
and the party it represented were firmly resolved not to be drawn into
a Continental war, whatever its origin or its issues. That was the
motive which had restrained Sir Edward Grey from contracting any
binding obligations towards France.
And so unhesitatingly was this view adopted in Berlin that when
on July 29th the German Ambassador terminated one of his
despatches with the expression of his personal impression—
founded, he confessed, on nothing more tangible than the manner,
intonation, looks of Sir Edward Grey—that if France were dragged
into war Great Britain would not remain neutral, his timid warning
failed to modify the accepted dogma that England was resolved to
stand by inactive and look on at the shock of mighty armies on the
Continent, satisfied to play the part of mediator as soon as victory
and defeat should have cleared the way for the readjustment of the
map of Europe.
This amazing misjudgment can be explained without difficulty.
Paradoxical though it may sound, the German Government suffered
from a plethora of information. It was too well informed of what was
going on in Russia, France, and Britain, and too little qualified to
contemplate in correct perspective the things revealed. Take, for
example, Russia. Every one of the influences to which the Tsar was
supposed to be accessible, every one of the alleged weak points of
the General Staff, the War Ministry, the Railway administration, the
Finances, were all entered in the records and weighed among the
motives for action. To the Austrian Foreign Office they were
communicated by the German Ambassador, von Tschirschky, with
whose own preconceived opinions of Russia’s inertness they
dovetailed to perfection. All these data were at the fingers’ ends of
the responsible leaders of the respective Governments, all the
inferences drawn were set down as highly probable, and the final
conclusion to which they pointed was that Russia would not fight
under present circumstances, even if from a military point of view
she could take the field, and that in any case she was sufficiently
aware of her impotence to recognize her inability and bend before
she was broken.
It is easy, in the light of recent events, to laugh at these
deductions and to deride the naïveté of German omniscience. But on
analysing the materials which Berlin statesmen had for a judgment,
one discerns the reasons which led them to believe that a good
prima facie case had been made out for its accuracy. One
characteristic and clinching argument was advanced with an air of
triumphant finality. These data, it was urged, are not theoretic
assumptions formed in Germany. They are the deliberate views of
competent Russians, arrived at in the conscientious discharge of
their duty and uttered for the welfare of their own country. Is not that
guarantee enough for the correctness of the facts alleged and the
sincerity of those who advance them?
The truth is, the Berlin authorities were too well supplied with
details, while lacking a safe criterion by which to measure their
worth. German diplomacy is many sided, and admirably well served
by a variety of auxiliary departments such as journalism, commerce,
educational establishments abroad, and espionage of a discreet and
fairly trustworthy character. But congruously with the tyrannical spirit
of system which pervades everything German, this paramount
organon for supplying the directors of the Empire’s policy with data
for their guidance and goals for their many converging movements
deals too exclusively in externals. Prussian diplomatists and
statesmen possess a vast body of information respecting the social
and political currents abroad, the condition of national defences and
party governments, the antagonisms of political groups, and other
obvious factors of political, military, naval, and financial strength and
weakness. But these facts nowise exhaust the elements of the
problem with which statesmanship is called upon to cope. There are
other and more decisive agencies which elude analysis and escape
the vigilant observation of the Prussian materialist. This superficial
observer is bereft of a sense for the soul-manifestations of a people,
for the multitudinous energies and enthusiasms stored up in its inner
recesses, for those hidden sources of strength which the wanton
violation of truth and justice set free, and which steel a nation to the
wrenches of real life and nerve it for a titanic struggle for the right.
Above all, he takes no account of a nation’s conscience, which,
especially in Anglo-Saxon peoples, is in vital and continuous contact
with their modes of feeling, thought, and action. He is a self-centred
pedant, capable indeed of close and thorough research and of
scrupulous loyalty to his own creed, but bringing to his work nothing
but the materialistic maxims of a cynically egoistic school,
impassioned by narrow aims, dissociated from humanity, blinded by
stupid prejudices, and bereft of innate balance. It is system without
soul.
Of the Russian army the Staffs of Berlin and Vienna thought
meanly. “A mob in uniform,” was one description. Less
contemptuous was this other: “A barracks of which only the bricks
have been got together, the cement and the builders being still
lacking.” Others there were—and these were the most serious
appraisers—who held that in another five or six years the Russian
land forces might be shaped into a formidable weapon of defence
and possibly of offence. But this opinion was urged mainly as an
argument against waiting. I once heard it supported tersely in the
following way. The army depends upon finances rather than
numbers. Without money you cannot train your soldiers. Ammunition
and guns, which are essential conditions to good artillery fire, involve
heavy expenditure. So, too, does rifle firing. Well, Russia’s army has
had no such advantages during the years that have elapsed since
her campaign against Japan. During all that time the salient trait of
her financial policy has been thrift. Grasping and saving, the State
has laid by enormous sums of money and has hoarded them miserly.
One effect of these precautions has been the neglect of the army
and the navy. At the close of the war Russia’s navy was practically
without ships and her diplomacy without backbone. And since then
little has been done to reinforce them.
 Two hundred and fifty millions sterling were borrowed by Russia
at the close of the war with Japan, it was argued. That sum may be
taken roughly to represent the cost of the campaign. But it did not
cover the wear and tear of the war material, the loss of the whole
navy, the destruction of fortresses, barracks, guns, private property,
etc., which would mount up to as much again. What was needed to
repair this vast breach in the land and sea forces was another loan
of at least three hundred millions sterling more. And this money was
not borrowed. Consequently the rebuilding of the damaged defences
was never undertaken. Only small annual credits, the merest
driblets, were allotted by the Finance Ministry to the War Office and
the Admiralty, and with these niggardly donations it had been
impossible to repair the inroads made by the war on the two imperial
services. But the Tsar’s Government, it was added, are about to turn
over a new leaf. Large war credits have been voted by the Duma.
Far-reaching reforms are planned for the army. Russia, awakened by
Germany’s preparations and warned by the Chancellor’s allusion to
the struggle between Slavs and Teutons, will make a strenuous effort
to fashion her vast millions into a formidable army. This work will
take at least from three to five years. We cannot afford to accord her
this time, nor can we blink the fact that she will never be less
redoubtable than she is to-day.
That was the theoretical side of the case. It was reinforced by
considerations of a concrete nature, the criticisms of Russian experts
of high standing and long experience whose alleged utterances were
said to bear out the conclusion that a war waged by Russia against
Germany, or even against Austria, at the present conjuncture would
be suicidal. Never before, it was urged, was the Tsardom less ready
from any point of view for a campaign than at the present moment.
And this, it was reiterated, is the ripe judgment of Russian competent
authorities whose names were freely mentioned. These men, it was
stated, had strongly urged the Tsar’s Government and the Tsar
himself to bear well in mind this deplorable plight of the army when
conducting the foreign business of the Empire.
That the Russian Government was aware of the view thus taken
in Berlin and Vienna may safely be assumed. For Russia kept her
eyes open and knew more about German machinations and the
assumptions on which they hinged than was supposed. Having had
an opportunity of picking up ideas on the subject, she had not let it
pass unutilized. Respecting one scheme she knew every detail; I
allude to the intention of Austria and Germany to declare the Treaty
of Bucharest a mere scrap of paper. Ever since that treaty was
signed, it had been the inflexible resolve of Austria and Germany to
upset it. I write this with first-hand knowledge. But even had I not had
this knowledge, it might have been taken for granted on a priori
grounds. The Balkan equilibrium as established by that instrument
was deemed lacking in stability. Count Berchtold admitted this to the
British Ambassador during the critical days. Its Servian elements
were particularly obnoxious to Austria, who had refrained from
annexing Turkish territory on the assumption that she would be
amply repaid for her self-restraint by political and economical
influence in the Peninsula.
Now, this assumption had been belied by events. Salonica was
under the dominion of Greece, whose leanings towards France and
Great Britain were notorious and fixed. Servia had waxed great, and
was striving to add further to her power and territory at Austria’s
expense. Bulgaria was sullen, and might become rebellious.
Roumania, estranged from the Dual Monarchy, had seemingly
moved within the political orbit of Russia. And even Turkey,
abandoned to herself among these prospective enemies of the
Teutonic Powers, was amenable to their suasion and to the pressure
of France and England. Such a state of affairs could not be brooked
by Austria-Hungary, who beheld her Slav possessions threatened in
Bosnia, Herzegovina, and Dalmatia, nor by Germany, who feared
that her road to the sea and to Asia Minor would be blocked.
Accordingly the two allies decided to apply the scrap of paper
doctrine to the Treaty of Bucharest, to cut up Greater Servia, bribe
Bulgaria with the Macedonian provinces which King Ferdinand had
lost by the treacherous attack on his allies, deprive Greece of the
islands and throw them as a sop to Turkey, win over Roumania by
intimidation and cajolery, and constrain her to make a block with
Bulgaria and Turkey against Servia and Greece.
 This preconcerted scheme had been questioned by easy-going
optimists in Great Britain before the outbreak of the war. But it has
been virtually acknowledged since then not only by the Austrian
Government but also by the “cream of Germany’s intelligence” in a
pamphlet entitled “Truth About Germany.” This statement of our
enemy’s case was drawn up for American consumption by a
committee which includes among its members Prince von Bülow,
Herr Ballin, Field-Marshal von der Goltz, Herr von Gwinner,
Professor Harnack, the theologian, Prince Hatzfeldt, Herr von
Mendelssohn, Professor Schmoller, and Professor Wundt. In the
chapter dealing with the last Balkan war as one of the causes of the
present conflict, these gentlemen argue that the outcome of that
struggle was a humiliation for the Habsburg Monarchy, and that it
had been so intended by the Ministers of the Tsar. And then comes
their important admission that ever since the Treaty of Bucharest, the
two Teutonic allies had been diligently preparing for war.

As soon as the Balkan troubles began (they write), Austria-

Hungary had been obliged to put a large part of her army in
readiness for war, because the Russians and Serbs had
mobilized on their frontiers. The Germans felt that what was a
danger for their ally was also a danger for them, and that they
must do all in their power to maintain Austria-Hungary in the
position of a great Power. They felt that this could only be done
by keeping with their ally perfect faith and by great military
strength, so that Russia might possibly be deterred from war and
peace be preserved, or else that, in case war was forced upon
them, they could wage it with honour and success. Now, it was
clear in Berlin that, in view of the Russian and Servian
preparations, Austria-Hungary, in case of a war, would be
obliged to use a great part of her forces against Servia, and
therefore would have to send against Russia fewer troops than
would have been possible under the conditions formerly
prevailing in Europe. Formerly even European Turkey could
have been counted upon for assistance, but that, after her recent
defeat, seemed very doubtful. These reasons and
considerations, which were solely of a defensive nature, led to
 the great German military Bills of the last two years. Also
Austria-Hungary was obliged to increase its defensive strength.

These preparations, America is informed, “were merely meant to

protect us against, and to prepare us for, the attacks of Moscovite
barbarism.” But Russia’s incipient army reorganization—which
cannot have been very thorough, seeing that in spite of it the
German Government regarded the Russian army as incapable of
5
taking the field—is cited as evidence of malice prepense.
Disingenuousness could hardly go further.
Any experienced European statesman would have divined this
plan even without a concrete clue. I knew it, and exposed it in the
columns of the Daily Telegraph.