ARTICULO SOBRE FISIOLOGIA Y DESARROLLO EN HEMOSTASIA

1. INVITED REVIEW
Historical perspective and future direction of coagulation
research
H. SAITO,* T. MATSUSHITA and T. KOJIMAà
*Nagoya Medical Center, Nagoya, Department of Transfusion Medicine, Nagoya University Hospital, Nagoya, and àDepartment of Medical
Technology, Nagoya University School of Health Sciences, Nagoya, Japan
To cite this article: Saito H, Matsushita T, Kojima T. Historical perspective and future direction of coagulation research. J Thromb Haemost 2011; 9
(Suppl. 1): 352–363.
Summary. Over the past 100 years, remarkable advances have
been made in our understanding of the mechanisms of blood
coagulation. Starting with the early clinical observations of rare
patients with hereditary clotting disorders, our knowledge has
increased in keeping pace with the introduction of new
technologies: from simple laboratory tests to protein chemistry,
to DNA technology, and to gene targeting technology.
Advances in basic research have been successfully translated
into improved methods for the diagnosis of bleeding disorders
as well as thrombosis, and the development of recombinant
clotting factors for replacement therapy in patients with
haemophilia. New promising anticoagulants have also been
developed for the treatment of thrombotic disorders. Based on
the unique nature of blood coagulation research the close
interactions and collaborations between basic scientists and
clinicians have played a major role in these developments. It is
anticipated that blood coagulation research will continue to
play a leading role in promoting better care of the patients with
bleeding disorders or thromboembolism.
Keywords: bleeding, blood coagulation, gene therapy, haemo-
stasis, knock-out mouse, thrombosis.
Introduction
Under physiological conditions blood maintains fluidity and it
circulates unimpeded within blood vessels. When a blood
vessel is ruptured, however, blood clots promptly at the site of
vessel injury. Blood also clots rapidly in vitro when it is placed
in a glass tube. The mechanism by which blood clots in vivo
and in vitro has attracted the attention of numerous investi-
gators. Extensive research has been performed in an effort to
understand the mechanisms of both the maintenance of blood
fluidity within blood vessels and the prompt clotting upon
injuries to blood vessels. Clinical observations of the patients
with bleeding or thrombotic tendency have provided impor-
tant insight into the process of blood clotting. In 1905
Morawitz [1] proposed the so called Ôthe classic theory of
blood coagulationÕ by summarising the knowledge that had
been available at that time. According to his theory only four
substances are involved in coagulation: thrombokinase
derived from damaged tissue, prothrombin, fibrinogen, and
calcium.
Remarkable advances have been made in our understanding
of the mechanisms of blood coagulation in the past 100 years.
Our knowledge increased as new technologies were introduced
into coagulation research over time (Fig. 1). The clinical
observation of patients coupled with simple laboratory tests
was the only approach available until the 1960s. Protein
chemistry was introduced in the 1970s and it became possible to
isolate and characterise many clotting factors and inhibitors
from plasma. Determination of the primary structure of large
plasma proteins by amino acid sequence analysis was still a
formidable, time-consuming task, but the application of DNA
technology in the 1980s made it feasible to elucidate the
structure of large proteins such as factor VIII and von
Willebrand factor. Crystallographic analysis of some clotting
factors and inhibitors also became possible. Thus, the primary,
secondary and, in some cases, tertiary structures of proteins
involved in blood clotting were clarified. This knowledge was
helpful to promote the development of new anticoagulants.
Furthermore, recombinant DNA technology was applied to
synthesise recombinant clotting factors, including factors VIII
and IX, leading to a safer replacement therapy for patients with
haemophilia. Another dramatic advance was brought about by
the application of gene targeting technology in the 1990s, which
facilitated exploration of the in vivo function of individual
clotting factors and inhibitors of blood coagulation by the
study of knock-out mice. More recently new methodologies of
regenerative medicine such as induced pluripotent stem cells
(iPS cells) have become available; certainly these will facilitate
blood coagulation research in the near future.
We will attempt to review the history of coagulation research
during the past 100 years and to evaluate how the advances in
basic research have been translated into clinical care of patients.
We will also discuss the future direction of coagulation
Correspondence: Hidehiko Saito, 4-1-1 Sanno-Maru, Naka-Ku,
Nagoya 460-0001, Japan.
Tel.: +81 52 951 1111; fax: +81 52 951 0664.
E-mail: hi.saito@nnh.hosp.go.jp
Journal of Thrombosis and Haemostasis, 9 (Suppl. 1): 352–363 DOI: 10.1111/j.1538-7836.2011.04362.x
Ó 2011 International Society on Thrombosis and Haemostasis

2. research. It should be stressed that as all of the numerous
important contributions cannot be detailed due to length
constraints, we will cover only selective aspects of blood
coagulation research. We must admit that our selection may be
biased.
Clinical observations: discovery of rare patients with
bleeding symptoms or thrombosis
Many coagulation factors were initially discovered as agents
functionally deficient in the plasma of patients with certain
hereditary bleeding disorders. In 1936 Patek and Stetson [2]
reported that there was a substance in normal plasma and
markedly deficient or unavailable in haemophilic plasma,
which in small amounts effectively, shortened the clotting
time of haemophilic blood, both in vitro and in vivo. This
agent was later termed antihaemophilic factor (AHF),
antihaemophilic globulin (AHG), or factor VIII (Fig. 2).
Around the same time Dam discovered vitamin K through
his observations on a bleeding tendency of chicks fed with an
ether-extracted diet [3].
During the 1940s and 1950s many new coagulation factors
were reported by a number of investigators, including factors V
[4], VII [5], IX [6–9], X [10,11], XI [12] and XIII [13]. Some
clotting factors were discovered as agents functionally deficient
in individuals with a prolonged coagulation time but without
any bleeding tendency. These include Factor XII [14], Fletcher
factor [15] and Fitzgerald factor [16]. New factors were
originally described by their own names, yet in some cases
the same factor was assigned with multiple names by different
investigators. Therefore a chaotic situation arose. To settle the
confusion, an international committee (The International
Committee on the Nomenclature of Blood Coagulation
Factors) was established in 1954 and a system of using Roman
numerals was adopted to identify each coagulation factor.
Human plasma contains many agents (natural anticoagu-
lants) that inhibit the activity of activated clotting factors.
Although the presence of antithrombin was suspected since the
early 1900s [17], it was in 1965 that a familial thrombotic
tendency was found to be associated with antithrombin
deficiency [18]. Similarly, protein C and protein S were
identified in plasma [19,20] before the discovery of a hereditary
thrombotic disorder due to a deficiency of each factor [21,22].
Thus, the orders of the discovery of natural anticoagulants and
their deficient states in man were different from those of
clotting factors. Yet, the physiologic relevance of natural
anticoagulants is strongly underscored by the high incidence of
venous thromboembolism in individuals with a congenital
deficiency. Inherited resistance to activated protein C, another
familial thrombophilia [23] was found to be due to a point
mutation in the factor V gene (factor V Leiden) [24].
Interestingly, factor V Leiden is a major risk factor for deep
vein thrombosis (DVT) in Caucasians but it was not present in
Asians, which is consistent with the fact that DVT is much
more common in Caucasians than in Asians.
Studies of rare patients with a bleeding or thrombotic
tendency strongly influenced the development of our concepts
concerning blood coagulation. Without these hereditary disor-
ders, our knowledge of the highly complex reaction consisting
of many trace plasma proteins would still have been at a
primitive stage. It is the unique nature of blood coagulation
research that promotes close interactions and cross-talk
between basic scientists and clinicians.
Advances in basic research that promoted our
understanding of physiology and the pathology of
coagulation
Evolution of knowledge on blood coagulation including the
natural anticoagulant system
The development of simple laboratory tests such as QuickÕs
one-stage prothrombin time (PT) [25] and the partial throm-
boplastin time (PTT) [26] were essential in facilitating the
screening and diagnosis of coagulation abnormalities (Fig. 2).
Based on these tests, specific assays for the activities of
individual clotting factors were developed using factor deficient
plasmas and these assays were used to purify various clotting
factors from plasma. In addition to the practical roles, the PT
and PTT have played an important conceptual role in our
Technology 1960 1970 1980 1990 2000 2010
Protein chemistry
DNA technology
Gene targeting
Regenerative medicine
Clinical observation with
simple laboratory tests
Fig. 1. Advances of technology used in coagulation research. Introduction of various technologies was illustrated over time.
Past, present and future of coagulation research 353
Ó 2011 International Society on Thrombosis and Haemostasis

3. understanding of blood coagulation in vitro regarding the
distinction of the intrinsic pathway and the extrinsic pathway.
As new clotting factors were discovered, modification of the
classic theory was required to incorporate the new information.
For example it was difficult to incorporate antihaemophilic
factor (AHF, factor VIII), a missing factor in classic haemo-
philia, into the coagulation scheme of Morawitz, because the
prothrombin time of haemophilic plasma was normal. Fur-
thermore, the classic theory was unable to explain the
mechanism through which blood clots upon surface contact
in the absence of tissue thromboplastin: the intrinsic pathway.
It was not clear how, and in which order, factors XII, XI, X,
IX, and V interacted to activate prothrombin to thrombin.
Many investigators have attempted to study the sequences of
blood coagulation reactions in test tubes by using crude
preparations of coagulation factors and, based on those
studies, several schemes of blood clotting, some of which are
very complicated, were proposed. The waterfall [27] or cascade
[28] hypothesis, separately proposed in 1964, conceives that the
blood coagulation reaction for the intrinsic pathway consists of
a series of sequential activations of clotting factors. When
blood comes into contact with a foreign surface, factor XII is
activated. Activated factor XII in turn activates factor XI, the
next factor in line. Activated factor XI then converts factor IX
to activated factor IX, leading to the ultimate generation of
thrombin. The waterfall-cascade hypothesis was outstanding in
its simplicity and represented a major advance in our under-
standing of blood coagulation mechanism.
The waterfall-cascade hypothesis was later modified as the
function of the clotting factors was better defined. For example,
factor V and VIII are activated by thrombin [29], not the
proteins above them in the cascade; factors V and VIII function
as co-factors rather than enzymes [30], and factor IX is also
activated by a tissue factor-factor VIIa complex [31]. Further-
more, thrombin activates factor XI in the presence of high
molecular weight kininogen (HMWK) and a negatively
charged surface [32,33]. Thus, an alternative mechanism for
the activation of factor XI independent of factor XII existed.
This finding is relevant to in vivo haemostasis, as patients with a
deficiency of factor XII, prekallikrein or HMWK have no
bleeding tendency whereas patients with factor XI deficiency
suffer from bleeding. The contact phase of blood coagulation is
unique in that it appears to participate in the generation of not
only thrombin but also fibrinolytic activity and kinin under
certain in vitro conditions; this subject has been reviewed
recently [34]. The intrinsic pathway and the extrinsic pathway
were originally considered to join at the level of factor X.
However, the activation of factor IX by the extrinsic pathway
and the activation of factors V, VIII and XI by thrombin make
the distinction of the two pathways less clear-cut than was
initially thought.
A deficiency of a coagulation factor that mediates platelet
adhesion also leads to a severe bleeding tendency. The
pathophysiology of von Willebrand disease (VWD) first
described by a Finnish physician Erik von Willebrand [35],
was initially characterised by low plasma levels of factor VIII,
Classic theory of blood
coagulation (Morawitz,
‘05) [1]
Quick’s one-stage prothrombin
time (PT) (’35) [25]
Discovery of vitamin K
(’35) [3]
Partial thromboplastin
time (PTT) (’53) [26]
Roman numeral to
identify coagulation
factors (International
Committee on the
Nomenclature of
Blood Coagulation
Factors) (’54)
VK epoxide
reductase
(’78) [119]
Factor IX is
activated by TF-
VIIa (’77) [31]
VK-dependent
carboxylase (’75) [118]
Waterfall [27] or cascade [28] hypothesis
(’64)
Thrombin activates factor XI
in the presence of HMWK
(’91) [32, 33]
Antithrombin
(’18) [17]
Normal plasma shortened clotting time
of hemophilics (AHF, Patek and
Stetsonn) (’36) [2]
Factor V (’47) [4]
Factor XII (’55) [14]
Factor XI (’53) [12]
Factor VII (’51) [5]
Factor IX (’47-’52) [6-9]
Factor X (’56-7) [10,11]
Factor XIII (’60) [13]
Fletcher factor (’65) [15]
Fitzgerald factor (’75) [16]
Protein C (’76) [19]
Protein S (’77) [20]
TFPI (’87) [44]
Heparin cofactor II (’82) [43]
Thrombomodulin (’82) [40,41]
ADAMTS13 (’01)
[61,62]
LMAN1 (’98) [63] MCFD2 (’03) [64]
1900 1920 1940 1950 1960 1970 1980 2000 2010
Fig. 2. Timeline for the evolution of knowledge on coagulation. The upper half of the Figure depicts key theories regarding blood coagulation and
important laboratory tests over time. Two ubiquitous enzymes involved in production of vitamin-K dependent proteins are also aligned in this part.
The lower half shows the discoveries of coagulation factors (below a dotted line) and natural anticoagulants (above a dotted line). LMAN1 and
MCFD2 are responsible genes for development of combined FV and VIII deficiency and are below a dotted line of this part. The number in ()
denotes the year of the report and that in [] is the reference number.
354 H. Saito et al
Ó 2011 International Society on Thrombosis and Haemostasis

4. but differed from classic haemophilia in that the VWD
symptoms were corrected by transfusion of a concentrate
prepared from the plasma of patients with severe haemophilia
[36]. In 1971, Zimmerman and Ratnoff found that antiserum
against human AHF also reacted with plasma from patients
with classic haemophilia. This ÔAHF-likeÕ antigen, however,
was found in decreased amounts in the plasma of patients with
von Willebrand disease [37]. This provided the first evidence for
a haemostatic Ôvon Willebrand factorÕ distinct from factor VIII
and other clotting factors in the blood.
Isolation and characterisation of antithrombin were
achieved from late 1960 to early 1970 [38,39]. Heparin
accelerates the neutralisation of thrombin by antithrombin by
500 fold. Heparin appears to bind to the lysyl residues on
antithrombin, thereby leading to a conformational change of
antithrombin. This conformational alteration makes the reac-
tive site arginine more accessible to the active serine centre of
thrombin [39]. In the presence of heparin, antithrombin also
inhibits the activities of factor IXa, Xa, XIa, and XIIa,
exhibiting a powerful control over the activation of the blood
coagulation cascade.
Another anticoagulant system that plays a major role in
maintaining blood fluidity and controlling haemostasis is the
protein C – protein S – thrombomodulin pathway. Protein C
was isolated in 1976 as a new vitamin K-dependent plasma
protein [19]. Thrombomodulin was isolated as a cofactor for
thrombin-catalysed protein C activation in 1982 [40,41].
Thrombomodulin is located on the endothelial cell surface
and serves as a thrombin receptor. Along with protein S,
another vitamin K-dependent protein [20], protein C and
thrombomodulin are now recognised to be very important in
controlling not only haemostasis but also inflammation [42].
The discovery and elucidation of the protein C-thrombomod-
ulin pathway is a major breakthrough in blood coagulation
research.
Other natural anticoagulants include heparin co-factor II
[43] and tissue factor pathway inhibitor (lipoprotein-associated
coagulation inhibitor) [44]. TFPI is the major inhibitor of the
tissue factor-Xa complex. The physiologic functions of these
inhibitors are less well understood.
Isolation, characterisation and cloning of clotting factors and
inhibitors
The majority of blood clotting factors, except for fibrinogen,
are trace proteins that are not easy targets for purification and
structural analysis. The primary structure of fibrinogen was
delineated in the 1970s with amino acid sequencing by the
effort of several investigators [45,46]. The introduction of DNA
technology in the 1980s revolutionised the determination of the
primary structure of many clotting factors. The first coagula-
tion factor that was cloned and sequenced was factor IX in
1982 [47,48], followed by other factors [49–54]. DavieÕs group
played a major role in the endeavour of the application of
DNA technology to coagulation research. Without DNA
technology it would have been impossible to delineate the
primary structure of tissue factor, factor VIII and von
Willebrand factor. But it should be pointed out that DNA
technology alone is not sufficient to elucidate the complete
primary structure of proteins as post-translational modifica-
tions such as carbohydrate attachments are not reliably
deduced from cDNA.
Elucidation of the molecular basis of inherited coagulation
disorders and thrombotic tendency
Once cDNA of the clotting factors are cloned and sequenced,
it is possible to explore DNA abnormality underlying
hereditary bleeding disorders and thrombotic tendencies.
Molecular genetic analysis of haemophilia A and B, von
Willebrand disease and other disorders disclosed a variety of
mutations and the list of mutations in each disorder was
compiled in databases, many sponsored by the ISTH (http://
hadb.org.uk/, http://www.kcl.ac.uk/ip/petergreen/haemBdat-
abase.html, http://www.vwf.group.shef.ac.uk/) [55,56]. A re-
cent study found a specific mutation in the F9 gene in
the recovered DNA extracted from bone fragments of the
Russian TsarÕs family, thereby identifying the cause of the
Royal Disease of Queen VictoriaÕs descendants as haemo-
philia B [57]. In some cases the significance of the mutation
was confirmed by an in vitro expression study, and the
pathogeneses of the disorders were delineated at the molec-
ular and cellular level [58,59].
Familial thrombotic thrombocytopenic purpura (TTP) is a
rare life-threatening disorder presenting with haemolytic
anaemia, thrombocytopenia, renal failure, fever and neuro-
logic abnormality. The pathogenesis of this disorder was
unknown until recently, when the disease locus was mapped
to chromosome 9q34 by a linkage analysis of families with
TTP. The responsible gene was identified as ADAMTS13,
which encodes a novel metalloprotease, ADAMTS13 [60].
Prior work that identified ADAMTS13 as the VWF-cleaving
protease [61,62] implicated the role of VWF in the patho-
genesis of this disorder.
Another example illustrating the power of molecular
biology is the study on the pathogenesis of the combined
factor V and VIII deficiency, a very rare hereditary bleeding
disorder. The genetic locus of this disorder was mapped to
chromosome 18q, and LMAN1 (ERGIC-53) was unexpect-
edly identified as the gene responsible for the disorder with
mutations of this gene found in patients [63]. This gene
encodes LMAN1, a component of the ER-Golgi intermedi-
ate compartment protein, suggesting that the combined
deficiency results from a defect in the intracellular transport
of factor V and factor VIII. The same group also identified
disruption of another gene, MCFD2, as the cause of this
disorder in other patients [64].
Studies with knock-out mice
Genetic manipulations in mice greatly facilitated our under-
standing of the blood coagulation system in vivo. The late 1990s
Past, present and future of coagulation research 355
Ó 2011 International Society on Thrombosis and Haemostasis

5. and early 2000s have seen an explosion in the number of papers
reporting mouse models of gene deletion or overexpression of
procoagulants and anticoagulants. Valuable information that
had not been possible to obtain in patients with hereditary
coagulation abnormalities was procured in experiments using
mice models. Furthermore, it is possible to produce double-
knock out mice to evaluate the effect of simultaneous deletion
of two different genes on the phenotype.
In terms of procoagulants, a variation in the effect of gene
deletion was observed; knock-out of factors II (prothrombin),
V, VII, X or tissue factor resulted in embryonic lethality or fatal
neonatal bleeding [65–72], whereas those of factors VIII, IX,
XI, XII and XIII survived beyond a neonatal period [73–78]. It
is of note that mice lacking fibrinogen are born normal in
appearance and in spite of a bleeding tendency; long-term
survival is possible, consistent with afibrinogenemia in humans
[79]. These findings suggest that some coagulation factors such
as tissue factor, and factors II, V, VII and X play an important
role in foetal development of the mouse. Knock-out mice of
natural anticoagulants such as antithrombin, protein C,
thrombomodulin, and TFPI [80–83] all lead to embryonic or
perinatal lethality. Even when knock-out mice are born and
grow normal without apparent abnormality, it is possible to
explore the physiologic significance of a deleted factor by
challenging mice with insults that induce bleeding or throm-
bosis and examining the consequences.
Recent studies of knock-out mice of the contact factors
unexpectedly revealed that factors XII and XI, and HMWK
play some role in the protection from arterial thrombosis.
Factor XII-deficient mice were shown to have a defect in
occlusive thrombus formation in response to ferric chloride
injury [84]. Similarly, factor XI-deficient mice failed to form a
thrombus with ferric chloride [85]. Inhibition of factor XI by
antisense therapy produced potent antithrombotic activity in
various venous and arterial thrombosis models [86]. Mice
deficient in plasma kininogen are also protected from arterial
thrombosis induced by vascular injury [87]. These findings are
intriguing in the light of the fact that mice deficient in factors
XII, XI or kininogen do not display a prolonged bleeding time,
suggesting that haemostasis at the site of vascular injury
appears to be normal. It is important, however, to keep in mind
that there may be a species difference between humans and mice
and the findings in mice may not be extrapolated into humans.
Application to clinical medicine
The progress in some basic research has been effectively
translated into clinical medicine resulting in improved patient
care. Selected topics on the diagnosis and treatment of bleeding
disorders and thrombosis will be reviewed.
Diagnosis
Synthetic substrates for clotting factor assay The
knowledge of the primary structure of clotting factors and
the identification of the cleavage sites has allowed development
of synthetic substrates of small molecular weight, enabling the
application of photometry in coagulation analysis [88]. The
traditional manual clotting time assays using hands and stop
watch have been replaced by automated assays using
chromogenic substrates with high specificity, sensitivity and
accuracy. Automated assays using chromogenic substrates are
widely utilised not only in the clinical laboratory but also in the
pharmaceutical industry for high-throughput screening of
anticoagulants.
DNA technology into carrier detection and prenatal
diagnosis of haemophilia and hereditary thrombotic
tendency In the early 1970s the detection of a female carrier
ofhaemophiliaAbecamepossiblewiththepredictionrateof70–
90% by employing the combined measurements of factor VIII
and von Willebrand factor [89]. When the F8 gene was cloned,
this method was replaced by DNA technology based on either
the use of a restriction fragment length polymorphism located
withintheF8gene[90]ordirectmutationdetection[91].Prenatal
diagnosis may also be performed with foetal DNA extracted
from chorionic villi [92]. The importance of ethical issues and
genetic counselling can not be overemphasised in the above
situations.Thechoiceofaproperpolymorphismisimportant,as
the incidence of common polymorphisms of some genes may be
variable among different ethnic groups [93]. Similarly, DNA
diagnosisofhereditarydeficiencyofantithrombin,proteinCand
protein S, and factor V Leiden and prothrombin gene variant
became possible, as the underlying DNA abnormality of each
disorder had been elucidated.
Biomarkers A number of biomarkers to detect
hypercoagulable states have been developed, including
fibrinopeptides, thrombin-antithrombin complex,
prothrombin fragments and D-dimer [94–97]. These tests are
useful for the diagnosis of deep vein thrombosis/pulmonary
embolism and disseminated intravascular coagulation (DIC).
The absence of functional vitamin K or the presence of
antagonist of the action of vitamin K, such as warfarin, results
in the appearance in the circulation of abnormal coagulation
factors, which have been termed proteins induced by vitamin K
absence or antagonist (PIVKA) [98], and the abnormal
prothrombin (PIVKAII) appears in a variety of hepatic and
nutritional disorders characterised by impaired hepatic vitamin
K-dependent carboxylation [99]. PIVKAII is now widely used
as a marker of hepatocellular carcinoma.
Treatment
Developmentandclinicalapplicationofplasmaconcentrates
(VIII, IX, prothrombin complexes) and recombinant clotting
factors (VIII, IX, VIIa) Advances in the understanding of
the properties of clotting factors and in the protein
fractionation methods have allowed one to produce plasma-
derived factors for patients with haemophilia. Following the
development of the plasma fractionation method by Cohn, a
fibrinogen fraction rich in factor VIII was used for replacement
356 H. Saito et al
Ó 2011 International Society on Thrombosis and Haemostasis

6. therapy of classic haemophilia in the late 1950s [100,101].
Cryoprecipitates were then introduced, leading to improved
management of bleeding episodes [102]. Factor VIII and factor
IX concentrates of intermediate to high purity derived from
human plasma became available in the 1970s for therapeutic
purposes, and major surgery became possible in patients with
haemophilia in the 1980s. The availability of factor VIII
concentrates has also made possible the home treatment of
haemophiliacs, contributing to the improvement of prognosis
and quality of life of these patients [103]. However, factor
concentrates prepared from a large plasma pool have been
contaminated with blood-borne virus including hepatitis B and
hepatitis C, as well as HIV, which resulted in the unfortunate
spread of HIV infection among haemophiliacs [104]; a tragedy
that will never be forgotten. Efforts have been made to improve
the safety of plasma-derived factors by adopting measures such
as pasteurisation and solvent-detergent that would inactivate
virus during production. Recombinant DNA technology was
then introduced in the 1980s to manufacture recombinant
clotting factors for therapeutic purposes. The safety and
efficacy of recombinant factors VIII and IX have been
demonstrated by clinical experience [105,106]. The problems
of the current recombinant factors include the high cost and
limited availability. Bioengineering techniques are being
applied to further improve the properties of recombinant
factors: increased biosynthesis and secretion, longer half-life,
better functional activity and deduced antigenicity [107].
The management of haemophiliacs who have developed
antibodies to factor VIII or IX represents a serious problem.
Prothrombin complex concentrates containing prothrombin,
factors VII, IX and X have been developed and successfully
used to ÔbypassÕ the site of action of factor VIII inhibitor
[108,109]. There are, however, some concerns about throm-
botic complications. A further step forward was the successful
use of plasma-derived activated factor VII (VIIa) in controlling
bleeding in the inhibitor patients [110]. Recombinant VIIa then
became an important addition to the therapeutic regimen for
inhibitor patients [111]. rVIIa is also being used on off-label
basis in a variety of conditions: trauma, cardiovascular surgery,
thrombocytopenia and liver disease. A recent analysis of
clinical trials found that there was an increased risk for
coronary artery thrombosis among elderly patients who
received high doses of rVIIa [112].
Development and clinical application of various
anticoagulants including natural anticoagulants Until
very recently, heparin and warfarin (a derivative of
dicumarol) were the major anticoagulant drugs in wide
clinical use. As compared with developments of many new
drugs for hypertension or diabetes mellitus in the past 50 years,
development of new anticoagulants has been very slow despite
significant progress in our understanding of blood coagulation
mechanisms.
Both heparin and dicumarol are substances that occur in
nature and were incidentally discovered to have the anticoag-
ulant activity. Heparin was isolated from dog liver and was
shown to retard blood clotting in vitro and in vivo in 1918 [17].
The safety and efficacy of heparin were demonstrated for
patients with deep vein thrombosis and pulmonary embolism
as early as in the late 1930s [113].
Insightful studies of a new haemorrhagic disease in cattle in
Canada in the early 1920s suggested that spoiled sweet clover
contained a haemorrhagic agent [114]. Investigators at the
University of Wisconsin then isolated, characterised and
synthesised the active agent, dicumarol, from spoiled sweet
clover hay in 1941 [115]. Immediately after chemical synthesis,
dicumarol was used to treat patients with post-operative deep
vein thrombosis and pulmonary embolism [116,117]. It seems
remarkable to note how soon both heparin and dicumarol were
clinically applied as anticoagulants following their identifica-
tion and isolation.
The molecular basis of the action of vitamin K and warfarin
have lately been elucidated. Vitamin K-dependent carboxylase
catalyses the posttranslational conversion of glutamyl residues
in the vitamin K-dependent coagulation factors to c-carboxy-
glutamyl (Gla) residues, which are required for the calcium-
dependent interaction in the blood coagulation cascade [118].
The vitamin K is an essential cofactor for carboxylase, and
warfarin exerts its anticoagulant activity by inhibiting the
regeneration of vitamin K through blocking vitamin K epoxide
reductase [119]. One of the limitations of warfarin has been that
there was no accurate way to estimate the proper dose for
individual patients. But it is now feasible to genotype patients
for SNPs of the cytochrome enzymes that control warfarin
metabolism and sensitivity, leading to the pharmacogenetic
algorithm that provides better predictions of the appropriate
dose of warfarin [120].
Heparin and warfarin have many targets in the clotting
cascade: they act on multiple coagulation factors. Warfarin has
been the only oral anticoagulant until very recently, but it has a
number of limitations, including slow onset of action, a narrow
therapeutic window, multiple drug and dietary interactions,
and the need for monitoring. Novel anticoagulants have been
developed that are selective for one specific clotting factor,
possess fewer limitations, and hopefully cause less bleeding
than heparin or warfarin (Fig. 3). The low molecular weight
heparin (LWMH) represented a major advance: it has more
anti-Xa activity to anti-IIa activity ratio, a longer plasma half-
life, and causes less bleeding than heparin. Also, the fact that it
needs no coagulation monitoring is an advantage of LMWH.
The leading question in the development of anticoagulant
drugs has been ÔIs it possible to make a potent anticoagulant
without a bleeding risk?Õ We assume that this aim is almost
impossible to achieve, as the mechanisms underlying intravas-
cular thrombosis and formation of haemostatic plug at the site
of venepuncture are almost indistinguishable and a bleeding
tendency caused by anticoagulants is not a side effect but the
main effect of the drug.
Novel anticoagulants that have been approved or are in
advanced stages of development include direct thrombin
inhibitor, factor Xa inhibitor and natural anticoagulants
(Table 1).
Past, present and future of coagulation research 357
Ó 2011 International Society on Thrombosis and Haemostasis

7. Direct thrombin inhibitors (DTI). The advantage of DTI
over heparin is that DTI inactivates not only fluid-phase
thrombin but also fibrin-bound thrombin. Argatroban, an
arginine derivative, was developed in Japan [121] and it is
approved by FDA for patients with heparin-induced throm-
bocytopenia (HIT). Hirudin is also approved in HIT. Dabig-
atran, an oral thrombin inhibitor, was recently compared with
warfarin in patients with atrial fibrillation and at risk of stroke.
Dabigatran caused fewer haemorrhage and prevented more
strokes than warfarin [122].
Factor Xa inhibitor. Fondaparinux is a chemically synthes-
ised analogue of the pentasaccharide sequence of heparin that
promotes binding of antithrombin to factor Xa. It selectively
blocks the activity of factor Xa in the presence of antithrombin.
Foundaparinux is FDA approved for the prevention and
treatment of venous thromboembolism [123]. The oral factor
Xa inhibitors also appear to show great promise. The efficacy
and safety of rivaroxaban [124] and apixaban [125] have been
recently reported in the treatment of symptomatic DVT and in
thromboprophylaxis following hip replacement, respectively.
Edoxaban has been also shown to be effective in the prevention
of venous thromboembolism after hip replacement [126]. There
has been some debate whether or not factor Xa inhibitors are
superior to DTI in efficacy and safety, since the selective
inhibition of coagulation factors above thrombin appears to be
a more effective strategy. This question will not be settled unless
Fibrinogen
DTI
Prothrombin
Fibrin
Thrombin
Va
Xa
Heparin - AT
Complex
X
IXa
XIa
IX
XI
VIIIa
TF
TFPI
VIIa
Injuries
LMW-heparin
Fondaparinux
Complex
Complex
AT
AT
-
-
Anti-Xa
APC PC
TMThrombin
Warfarin
Anti-Xa
Revaroxaban
Apixaban
Argatroban
Edoxaban
DTI
Hirudin
Dabigatran
NAC
TM: thrombomodulin
APC: activated protein C
AT: antithrombin
TFPI: tissue factor
pathway inhibitor
Fig. 3. Sites of actions of established and novel anticoagulants in the coagulation cascade. The targets of anticoagulants are illustrated in the coagula-
tion cascade. The orange dotted line indicates the sites of action of established anticoagulants (warfarin, heparin, LMWH, and Fondaparinux), while
the pink dotted line the sites of action of novel anticoagulants (DTI, anti-Xa and NAC). DTI, direct thrombin inhibitor; anti-Xa, anti-activated factor X
or factor Xa inhibitor; LMW-heparin, low molecular weight heparin; NAC, natural anticoagulant.
Table 1 Characteristics of new oral anticoagulants
Dabigatran Rivaroxaban Apixaban Edoxaban
Target Flla FXa FXa FXa
Prodrug Yes No No No
Dosing Twice daily Once daily Twice daily Once daily
Coagulation monitoring No No No No
Bioavailability (%) 6 80 50 50
Half-life (h) 12–17 5–9 12 9–11
Renal excretion (%) 80 65 25 35
Antidote None None None None
Drug interactions P-gp* inhibitors Potent inhibitors of
CYP3A4
or P-gp
Potent inhibitors
of CYP3A4
Potent inhibitors
of CYP3A4 or P-gp
Clinical status Approved in Canada and Europe for
VTE prophylaxis after major orthopedic
surgery
Approved in USA and Japan for
stroke prevention in NVAFà
Approved in Canada and
Europe for VTE prophylaxis
after major orthopedic surgery
None Approved in Japan for
VTE prophylaxis after
major orthopedic surgery
*P-gp, P-glycoprotein 1;
CYP3A4, cytochrome P450 3A4; à
NVAF, non-valvular arterial fibrillation.
358 H. Saito et al
Ó 2011 International Society on Thrombosis and Haemostasis

8. a head to head comparison of factor Xa inhibitor and DTI is
made.
Natural anticoagulants. Natural anticoagulants derived
from plasma or produced by recombinant technology have
recently emerged as promising antithrombotic drugs for some
patients with sepsis. Disseminated intravascular coagulation
(DIC) is a serious condition associated with sepsis and it is a
strong predictor of mortality in sepsis patients. Several
randomised clinical trials have been reported on the efficacy
and safety of antithrombin (AT), activated protein C (APC),
thrombomodulin (TM) and TFPI in patients with severe
sepsis with or without DIC. The results of these clinical trials
are variable. There is evidence that AT reduced mortality in
some patients with sepsis [127]. A large scale clinical trial of
recombinant APC demonstrated that treatment with APC
significantly reduced the 28-day mortality rate of the patients
with severe sepsis [128]. Similarly, a clinical trial of the plasma-
derived APC showed that a relatively small dosage of APC
improved the 28-day mortality in DIC patients associated
mostly with haematological malignancy [129]. Recombinant
human soluble thrombomodulin has recently been shown to
more significantly improve DIC and alleviate bleeding symp-
toms in DIC patients, as compared with heparin [130]. In
contrast, recombinant TFPI was shown not to improve the
survival of the patients with severe sepsis and coagulation
abnormality, although TFPI attenuated hypercoagulable
states [131]. It is anticipated that natural anticoagulants will
be used for other indications.
Future directions
Traditionally, blood coagulation research was performed in
cell-free system of plasma or in mixtures of purified clotting
factors, since the experiments are easier to perform and analyse.
However, it became increasingly apparent that the interactions
between procoagulants, anticoagulants and cells such as
platelets, leukocytes, and vascular endothelial cells are impor-
tant to understand the pathogenesis of bleeding as well as
thrombosis. The majority of information concerning the
kinetics of blood coagulation reactions was also obtained in
test tubes under static, non-flow conditions [132]. Flow may
have an effect on the interactions among clotting factors,
platelets, leukocytes and vascular wall. With improved tech-
niques it is now possible to study thrombus formation under
flow conditions in vitro and in live animals in vivo [133,134]. It is
expected that technical advances will allow us to obtain more
clinically relevant information regarding the mechanisms of
bleeding and thrombosis.
Haemophilia has been a good candidate for gene therapy
since the cloning of genes for factors IX and VIII, because
haemophilia is a recessive monogenic disease and the
attainment of circulating factor levels of as little as a few
percent by gene transfer would be expected to substantially
reduce the bleeding risk in patients. However, most clinical
trails to date failed to show efficacy in achieving long-term
expression of therapeutic levels of clotting factors, although
the gene therapy in animal models of haemophilia have
demonstrated some promise [135–137]. More effort will be
needed to overcome several obstacles to the clinical applica-
tion such as low expression and immunogenicity. It should
also be noted that the safety of gene therapy should be
secured in haemophilia in which safe and effective replace-
ment therapy is already available and further attempts to
develop recombinant factors with improved properties are
under way.
Recent advances in the technology of regenerative med-
icine will certainly give a great impetus to blood coagulation
research. Induced pluripotent cells (iPS cells) may be
produced from a skin biopsy of patients with hereditary
clotting disorders and may be analysed in detail to elucidate
the cellular process by which the levels of coagulation
factors are reduced. iPS cells derived from patients may be
modified to introduce normal gene and may be used for
gene therapy.
Although the contact system was considered to be irrelevant
to in vivo haemostasis and neglected, as patients with deficiency
of factor XII, prekallikrein, and HMWK have no bleeding
tendency, now from the recent studies in knock-out mice, the
contact factors are revived as novel targets for anticoagulants
with a minimal bleeding risk [138].
With an aging population the incidence of vascular
diseases is expected to steadily increase. The importance of
the diagnosis and management of thromboembolism cannot
be overstressed. More importantly, the prevention of
thrombosis should become a top priority of research,
thereby leading to reduced morbidity, mortality and medical
cost. Genome-wide association studies will help to yield
important information on the susceptibility genes for
vascular diseases.
Conclusion
We have reviewed selective aspects of blood coagulation
research; certainly it is impressive to see the great progress that
has been made in the past 100 years. Looking back on the
history of blood coagulation research, it is evident that the
main focus has shifted from studies of haemostasis and
haemorrhagic diseases in the early years to those of mainte-
nance of blood fluidity and thrombotic disorders in recent
years. It should be pointed out that most progress was
secondary to intimate interactions and the collaboration of
clinicians of many disciplines and basic scientists. Foundation
of the International Committee of Thrombosis and Haemo-
stasis (ICTH) and the International Society on Thrombosis
and Haemostasis (ISTH) in 1969 played a leading role in the
promotion of basic and clinical research. Particularly, the
Scientific and Standardisation Committee (SSC) has been
instrumental in the definition of the nomenclature and the
standardisation of assay methods in the field. It is evident that
our Society will continue to contribute to the promotion of our
knowledge of blood coagulation, bleeding disorders and
thrombosis.
Past, present and future of coagulation research 359
Ó 2011 International Society on Thrombosis and Haemostasis

9. Disclosure of Conflict of Interests
H. Saito and T. Matsushita have no conflicts of interest.
T. Kojima is a consultant to Bayer HealthCare and
GlaxoSmithKline.
References
1 Morawitz P. Die Chemie der Blutgerinnung. Ergeb Physiol 1905; 4:
307–422.
2 Patek AJ, Stetson RP. Hemophilia. I. The abnormal coagulation of
the blood and its relation to the blood platelets. J Clin Invest 1936; 15:
531–42.
3 Dam H. The antihaemorrhagic vitamin of the chick. Biochem J 1935;
29: 1273–85.
4 Owren PA. Parahaemophilia. Haemorrhagic diathesis due to absence
of a previously unknown clotting factor. Lancet 1947; 1: 446–8.
5 Alexander B, Goldstein R, Landwehr G, Cook CD. Congenital
SPCA deficiency: a hitherto unrecognized coagulation defect with
hemorrhage rectified by serum and serum fractions. J Clin Invest
1951; 30: 596–608.
6 Pavlovsky A. Contribution to the pathogenesis of hemophilia. Blood
1947; 2: 185–91.
7 Aggeler PM, White SG, Glendening MB, Page EW, Leake TB, Bates
G. Plasma thromboplastin component (PTC) deficiency: a new dis-
ease resembling hemophilia. Proc Soc Exp Biol Med 1952; 79: 692–4.
8 Schulman I, Smith CH. Hemorrhagic disease in an infant due to
deficiency of a previously undescribed clotting factor. Blood 1952; 7:
794–807.
9 Biggs R, Douglas AS, Macfarlane RG, Dacie JV, Pitney WR, Mer-
sky C, OÕBrien JR. Christmas disease: a condition previously mis-
taken for haemophilia. Br Med J 1952; II: 1378–82.
10 Telfer TP, Denson KW, Wright DR. A ÔnewÕ coagulation defect. Br J
Haematol 1956; 2: 308–16.
11 Hougie C, Barrow EM, Graham JB. Stuart clotting defect. I. Segre-
gation of an hereditary hemorrhagic state from the heterogeneous
group heretofore called ‘‘stable factor’’ (SPCA, proconvertin, factor
VII) deficiency. J Clin Invest 1957; 36: 485–96.
12 Rosenthal RL, Dreskin OH, Rosenthal N. New hemophilia-like
disease caused by deficiency of a third plasma thromboplastin factor.
Proc Soc Exp Biol Med 1953; 82: 171–4.
13 Duckert F, Jung E, Shmerling DH. A hitherto undescribed congenital
haemorrhagic diathesis probably due to fibrin stabilizing factor defi-
ciency. Thromb Diath Haemorrh 1960; 5: 179–86.
14 Ratnoff OD, Colopy JE. A familiar hemorrhagic trait associated with
a deficiency of a clot-promoting fraction of plasma. J Clin Invest 1955;
34: 602–13.
15 Hathaway WE, Belhasen LP, Hathaway HS. Evidence for a plasma
thromboplastin factor. I. Case report, coagulation studies and phys-
icochemical properties. Blood 1965; 26: 521–32.
16 Saito H, Ratnoff OD, Waldmann R, Abraham JP. Fitzgerald trait.
Deficiency of a hitherto unrecognized agent, Fitzgerald factor,
participating in surface-mediated reactions of clotting, fibrinolysis,
generation of kinins, and the property of diluted plasma enhanc-
ing vascular permeability (PF/Dil). J Clin Invest 1975; 55: 1082–9.
17 Howell WH, Holt E. Two new factors in blood coagulation- heparin
and proantithrombin. Am J Physiol 1918; 47: 328–41.
18 Egeberg O. Inherited antithrombin deficiency causing thrombophilia.
Thromb Diath Haemorrh 1965; 13: 516–30.
19 Stenflo J. A new vitamin K-dependent protein: purification from
bovine plasma and preliminary characterization. J Biol Chem 1976;
251: 355–63.
20 DiScipio RG, Hermodson MA, Yates SG, Davie EW. A comparison
of human prothrombin, factor IX (Christmas factor), factor X (Stuart
factor), and protein S. Biochemistry 1977; 16: 698–706.
21 Griffin JH, Evatt B, Zimmerman TS, Kleiss AJ, Wideman C. Defi-
ciency of protein C in congenital thrombotic disease. J Clin Invest
1981; 68: 1370–3.
22 Comp PC, Nixon RR, Cooper MR, Esmon CT. Familiar protein S
deficiency is associated with recurrent thrombosis. J Clin Invest 1984;
74: 2082–8.
23 Dahlba¨ ck B, Carlsson M, Svensson PJ. Familial thrombophilia due
to a previously unrecognized mechanism characterized by poor anti-
coagulant response to activated protein C: prediction of a cofactor to
activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004–8.
24 Bertina RM, Koeleman BPC, Koster T, Rosendaal FR, Dirven RJ,
de Ronde H, van der Velden PA, Reitsma PH. Mutation in blood
coagulation factor V associated with resistance to activated protein C.
Nature (Lond) 1994; 369: 64–7.
25 Quick AJ, Stanley-Brown M, Bancroft FW. A study of the coagu-
lation defect in hemophilia and in jaundice. Am J Med Sci 1935; 190:
501–11.
26 Langdell RD, Wagner RH, Brinkhous KM. Effect of anti-hemophilic
factor on one-stage clotting tests: a presumptive test for hemophilia
and a simple one-stage anti-hemophilic factor assay procedure. J Lab
Clin Med 1953; 41: 637–47.
27 Davie EW, Ratnoff OD. Waterfall sequence of for intrinsic blood
coagulation. Science 1964; 145: 1310–2.
28 Macfarlane RG. An enzyme cascade in the blood coagulation
mechanism, and its function as a biochemical amplifier. Nature
(Lond) 1964; 202: 498–9.
29 Rapaport SI, Shiffman S, Patch MJ, Ames SB. The importance of
activation of anti-haemophilic globulin and proaccelerin by traces of
thrombin in the generation of intrinsic prothrombinase activity. Blood
1963; 21: 221–36.
30 Nesheim ME, Taswell JB, Mann KG. The contribution of bovine
factor V and factor Va to the activity of prothrombinase. J Biol Chem
1979; 254: 10952–62.
31 Osterud B, Rapaport S. Activation of factor IX by the reaction
product of tissue factor and factor VII: additional pathway for
initiating blood coagulation. Proc Natl Acad Sci USA 1977; 74: 5260–
4.
32 Gailani D, Broze GJ Jr. Factor XI activation in a revised model of
blood coagulation. Science 1991; 253: 909–12.
33 Naito K, Fujikawa K. Activation of human blood coagulation factor
XI independent of factor XII: Factor XI is activated by thrombin and
factor XIa in the presence of negatively charged surfaces. J Biol Chem
1991; 266: 7353–8.
34 Saito H. Studies on Fletcher trait and Fitzgerald trait- A rare chance
to disclose bodyÕs defense reactions against injury. Thromb Haemost
2010; 104: 867–74.
35 von Willebrand EA. Fin. Uber hereditare Pseudohemophilie. Acta
Med Scand 1931; 76: 521–49.
36 Nilsson IM, Blomba¨ ck M, Jorpes E, Blomba¨ ck B, Johansson SA. von
WillebrandÕs disease and its correction with human plasma fraction 1-
0. Acta Med Scand 1957; 159: 179–88.
37 Zimmerman TS, Ratnoff OD, Powell AE. Immunologic differentia-
tion of classic hemophilia (factor VIII deficiency) and von Wille-
brandÕs disease, with observations on combined deficiencies of
antihemophilic factor and proaccelerin (factor V) and on an acquired
circulating anticoagulant against antihemophilic factor. J Clin Invest
1971; 50: 244–54.
38 Abildgaard U. Highly purified antithrombin III with heparin cofactor
activity prepared by disc electrophoresis. Scand J Clin Lab Invest
1968; 21: 89–91.
39 Rosenberg RD, Damus PS. The purification and mechanism of ac-
tion of human antithrombin-heparin cofactor. J Biol Chem 1973; 248:
6490–505.
40 Esmon NL, Owen WG, Esmon CT. Isolation of a membrane-bound
cofactor for thrombin-catalyzed activation of protein C. J Biol Chem
1982; 257: 859–64.
360 H. Saito et al
Ó 2011 International Society on Thrombosis and Haemostasis

10. 41 Comp PC, Jacocks RM, Ferrell GL, Esmon CT. Activation of pro-
tein C in vivo. J Clin Invest 1982; 70: 127–34.
42 Esmon CT. The protein C pathway. Chest 2003; 124: 26S–32S.
43 Tollefsen DM, Majerus DW, Blank MK. Heparin cofactor II: puri-
fication and properties of a heparin-dependent inhibitor of thrombin
in human plasma. J Biol Chem 1982; 257: 2162–9.
44 Broze GJ Jr, Miletich JP. Isolation of the tissue factor inhibitor
produced by HepG2 hepatoma cells. Proc Natl Acad Sci USA 1987;
84: 1886–90.
45 Blomba¨ ck B, Blomba¨ ck M, Henschen A, Hessel B, Iwanaga S,
Woods KR. N-terminal disulphide knot of human fibrinogen. Nature
1968; 218: 130–4.
46 Doolittle RF, Watt KWK, Cottrell BA, Strong DD, Riley M. The
amino acid sequence of the a-chain of human fibrinogen. Nature
1979; 280: 464–8.
47 Kurachi K, Davie EW. Isolation and characterization of a cDNA
coding for human factor IX. Proc Natl Acad Sci USA 1982; 79: 6461–
4.
48 Choo KH, Gould KG, Rees DJ, Brownlee GG. Cloning of the
gene for human anti-haemophilic factor IX. Nature 1982; 299: 178–
80.
49 Sadler JE, Shelton-Inloes BB, Sorace JM, Harlan JM, Titani K,
Davie EW. Cloning and characterization of two cDNAs coding for
human von Willebrand factor. Proc Natl Acad Sci USA 1985; 82:
6394–8.
50 Ginsberg D, Handin RI, Bonthron DT, Donlon TA, Bruns GA, Latt
SA, Orkin SH. Human von Willebrand factor (vWF): isolation of
complementary DNA (cDNA) clones and chromosomal localization.
Science 1985; 228: 1401–6.
51 Jenny RJ, Pittman DD, Toole JJ, Kriz RW, Aldape RA, Hewick
RM, Kaufman RJ, Mann KG. Complete cDNA and derived amino
acid sequence of human factor V. Proc Natl Acad Sci USA 1987; 84:
4846–50.
52 Morrissey JH, Fakhrai H, Edgington TS. Molecular cloning of the
cDNA for tissue factor, the cellular receptor for the initiation of the
coagulation protease cascade. Cell 1987; 50: 129–35.
53 Spicer EK, Horton R, Bloem L, Bach R, Williams KR, Guha A,
Kraus J, Lin TC, Nemerson Y, Konigsberg WH. Isolation of cDNA
clones coding for human tissue factor: primary structure of the protein
and cDNA. Proc Natl Acad Sci USA 1987; 84: 5148–52.
54 Scarpati EM, Wen D, Broze JG Jr, Miletich JP, Flandermeyer RR,
Siegel NR, Sadler JE. Human tissue factor: cDNA sequence
and chromosome localization of the gene. Biochemistry 1987; 26:
5234–8.
55 Reitsma PH, Bernardi F, Doig RG, Gandrille S, Greengard JS, Ire-
land H, Krawczak M, Lind B, Long GL, Poort SR, Saito H, Sala N,
Witt I, Cooper DN. Protein C deficiency: a database of mutations,
1995 update. Thromb Haemost 1995; 73: 876–89.
56 Gandrille S, Borgel D, Sala N, Espinosa-Parrilla Y, Simmonds R,
Rezende S, Lind B, Mannhalter C, Pabinger I, Reistma PH, Form-
stone C, Cooper DN, Saito H, Suzuki K, Bernardi F, Aiach M.
Protein S deficiency: a database of mutations-summary of the first
update. Thromb Haemost 2000; 84: 918–34.
57 Rogaev EI, Grigorenko AP, Faskhutdinova G, Kittler ELW, Moli-
aka YK. Genotype analysis identifies the cause of the ‘‘Royal Dis-
ease’’. Science 2009; 326: 817.
58 Yamamoto K, Tanimoto M, Emi N, Matsushita T, Takamatsu J,
Saito H. Impaired secretion of the elongated mutant of protein C
(protein C-Nagoya): molecular and cellular basis for hereditary pro-
tein C deficiency. J Clin Invest 1992; 90: 2439–46.
59 Matsushita T, Kojima T, Emi N, Takahashi I, Saito H. Impaired
Human Tissue Factor-mediated Activity in Blood Clotting Factor
VIInagoya (Arg304 fi Trp). J Biol Chem 1994; 269: 7355–63.
60 Levy GG, Nichols WC, Lian EC, Foround T, McClintick JN,
McGee BM, Yang AY, Siemieniak DR, Stark KR, Gruppo R,
Sarode R, Shurin SB, Chandrasekaran V, Stabler SP, Sabio H,
Bouhassira EE, Upshaw JD Jr, Ginsberg D, Tsai H-M. Mutations in
a member of the ADAMTS gene family cause thrombotic thrombo-
cytopenic purpura. Nature 2001; 413: 488–94.
61 Fujikawa K, Suzuki H, McMullen B, Chung D. Purification of hu-
man von Willebrand factor-cleaving protease and its identification as
a new member of the metalloproteinase family. Blood 2001; 98: 1662–
6.
62 Gerritsen HE, Robles R, Lammale B, Furlan M. Partial amino acid
sequence of purified von Willebrand factor-cleaving protease. Blood
2001; 98: 1654–61.
63 Nichols WC, Seligsohn U, Zivelin A, Terry VH, Hertel CE, Wheatley
MA, Moussalli MJ, Hauri HP, Ciavarella N, Kaufman RJ, Ginsberg
D. Mutations in the ER-Golgi intermediate compartment protein
ERGIC-53 cause combined deficiency of coagulation factors V and
VIII. Cell 1998; 93: 61–70.
64 Zhang B, Cunningham MA, Nichols WC, Bernat JA, Seligsohn U,
Pipe SW, McVey JH, Schulte-Overberg U, de Bosch NB, Ruiz-Saez
A, White GC, Tuddenham EG, Kaufman RJ, Ginsberg D. Bleeding
due to disruption of a cargo-specific ER-to-Golgi transport complex.
Nat Genet 2003; 34: 220–5.
65 Sun WY, Witte DP, Degen JL, Colbert MC, Burkart MC, Holmba¨ ck
K, Xiao Q, Bugge TH, Degen SJF. Prothrombin deficiency results in
embryonic and neonatal lethality in mice. Proc Natl Acad Sci USA
1998; 95: 7597–602.
66 Xue J, Wu Q, Westfield LA, Tuley EA, Lu D, Zhang Q, Shim K,
Zheng X, Sadler JE. Incomplete embryonic lethality and fatal neo-
natal hemorrhage caused by prothrombin deficiency in mice. Proc
Natl Acad Sci USA 1998; 95: 7603–7.
67 Cui J, OÕShea KS, Purkayastha A, Saunders TL, Ginsburg D. Fetal
haemorrhage and incomplete block to embryogenesis in mice lacking
coagulation factor V. Nature 1996; 384: 66–8.
68 Rosen ED, Chan JCY, Idusogie E, Clotman F, Vlasuk G, Luther
T, Jalbert LR, Albrecht S, Zhong L, Lissens A, Schoonjans L,
Moons L, Collen D, Castellino FJ, Carmeliet P. Mice lacking factor
VII develop normally but suffer fatal perinatal bleeding. Nature 1997;
390: 290–4.
69 Dewerchin M, Liang Z, Moons L, Carmeliet P, Castellino FJ, Collen
D, Rosen ED. Blood coagulation factor X deficiency causes partial
embryonic lethality and fatal neonatal bleeding in mice. Thromb
Haemost 2000; 83: 185–90.
70 Bugge TH, Xiao Q, Kombrinck KW, Flick MJ, Holmba¨ ck K,
Danton MJ, Colbert MC, Witte DP, Fujikawa K, Davie EW, Degen
JL. Fatal embryonic bleeding events in mice lacking tissue factor, the
cell-associated initiator of blood coagulation. Proc Natl Acad Sci
USA 1996; 93: 6258–63.
71 Carmeliet P, Mackman N, Moons L, Luther T, Gressens P, van
Vlaenderen I, Demunck H, Kasper M, Breier G, Evrard P, Mu¨ ller M,
Risau W, Edgington T, Collen D. Role of tissue factor in embryonic
blood vessel development. Nature 1996; 383: 73–5.
72 Toomey JR, Kratzer KE, Lasky Nm, Stanton JJ, Broze GJ Jr.
Targeted disruption of murine tissue factor gene results in embryonic
lethality. Blood 1996; 88: 1583–7.
73 Bi L, Lawler AM, Antonarakis SE, High KA, Gearhart JD,
Kazazian HH. Targeted disruption of the mouse factor VIII gene
produces a model of haemophilia A. Nat Genet 1995; 10: 119–21.
74 Lin H-F, Maeda N, Smithies O, Straight DL, Stafford DW. A
coagulation factor IX-deficient mouse model for human hemophilia
B. Blood 1997; 90: 3962–6.
75 Wang L, Zoppe M, Hackeng TM, Griffin JH, Lee K-F, Verma IM. A
factor IX-deficient mouse model for hemophilia B gene therapy. Proc
Natl Acad Sci USA 1997; 94: 11563–6.
76 Gailani D, Lasky NM, Broze GJ Jr. A murine model of factor XI
deficiency. Blood Coagul Fibrinolysis 1997; 8: 134–44.
77 Pauer HU, Renne´ T, Hemmerlein B, Legler T, Fritzlar S, Adham
I, Mu¨ ller-Esterl W, Emons G, Sancken U, Engel W, Burfeind P.
Targeted deletion of murine coagulation factor XII gene- a model
for contact phase activation in vivo. Thromb Haemost 2004; 92: 503–
8.
Past, present and future of coagulation research 361
Ó 2011 International Society on Thrombosis and Haemostasis

11. 78 Lauer P, Metzner HJ, Zettlmeissl G, Li M, Smith AG, Lathe R,
Dickneite G. Targeted inactivation of the mouse locus encoding
coagulation factor XIII-A: hemostatic abnormalities in mutant mice
and characterization of the coagulation deficit. Thromb Haemost
2002; 88: 967–74.
79 Suh TT, Holmba¨ ck K, Jensen NJ, Daugherty CC, Small K, Simon
DI, Potter S, Degen JL. Resolution of spontaneous bleeding events
but failure of pregnancy in fibrinogen-deficient mice. Genes Dev 1995;
9: 2020–33.
80 Ishiguro K, Kojima T, Kadomatsu K, Nakayama Y, Takagi A,
Suzuki M, Takeda N, Ito M, Yamamoto K, Matsushita T,
Kusugami K, Muramatsu T, Saito H. Complete antithrombin
deficiency in mice results in embryonic lethality. J Clin Invest 2000;
106: 873–8.
81 Jalbert LR, Rosen ED, Moons L, Chan JC, Carmeliet P, Collen D,
Castellino FJ. Inactivation of the gene for anticoagulant protein C
causes lethal perinatal consumption coagulopathy in mice. J Clin
Invest 1998; 102: 1481–8.
82 Healy AM, Rayburn HB, Rosenberg RD, Weiler H. Absence of the
blood-clotting regulator thrombomodulin causes embryonic lethality
in mice before development of a functional cardiovascular system.
Proc Natl Acad Sci USA 1995; 92: 850–4.
83 Huang ZF, Higuchi D, Lasky N, Broze GJ Jr. Tissue factor pathway
inhibitor gene disruption produces intrauterine lethality in mice.
Blood 1997; 90: 944–51.
84 Renne´ T, Pozgaiova´ M, Gruner S, Schuh K, Pauer HU, Burfeind P,
Gailani D, Nieswandt B. Defective thrombus formation in
mice lacking coagulation factor XII. J Exp Med 2005; 202: 271–
81.
85 Wang X, Smith PL, Hsu MY, Gailani D, Schumacher WA, Ogletree
ML, Seiffert DA. Effects of factor XI deficiency on ferric chloride-
induced vena cava thrombosis in mice. J Thromb Haemost 2006; 4:
1982–8.
86 Zhang H, Lo¨ wenberg EC, Crosby JR, MacLeod AR, Zhao C, Gao
D, Black C, Revenko AS, Meijers JC, Stroes ES, Levi M, Monia BP.
Inhibition of the intrinsic coagulation pathway factor XI by antisense
oligonucleotides: a novel antithrombotic strategy with lowered
bleeding risk. Blood 2010; 116: 4684–92.
87 Merkulov S, Zhang WM, Komar AA, Schmaier AH, Barnes E, Zhou
Y, Lu X, Iwaki T, Catellino FJ, Luo G, McCrae KR. Deletion of
murine kininogen gene 1 (mKng1) causes loss of plasma kininogen
and delays thrombosis. Blood 2008; 111: 1274–81.
88 Svendsen L, Blomba¨ ck B, Blomba¨ ck M, Olsson PI. Synthetic chro-
mogenic substrates for determination of trypsin, thrombin and
thrombin-like enzymes. Thromb Res 1972; 1: 267–78.
89 Zimmerman TS, Ratnoff OD, Littell AS. Detection of carriers of
classic hemophilia using immunologic assay of antihemophilic factor
(factor VIII). J Clin Invest 1971; 50: 255–8.
90 Oberle I, Camerino G, Heilig R, Grunebaum L, Cazenave JP, Cra-
panzano C, Mannucci PM, Mandel JL. Genetic screening for
hemophilia A (classic hemophilia) with a polymorphic DNA probe. N
Engl J Med 1985; 312: 682–6.
91 Antonarakis SE, Waber PG, Kittur SD, Patel AS, Kazazian HH Jr,
Mellis MA, Counts RB, Stamatoyannopoulos G, Bowie EJ, Fass
DN, Pittman DD, Wozney JM, Toole JJ. Hemophilia A detection of
molecular defects and of carriers by DNA analysis. N Engl J Med
1985; 313: 842–8.
92 Gitschier J, Lawn RM, Rotblat F, Goldman E, Tuddenham EGD.
Antenatal diagnosis and carrier detection of haemophilia A using
factor VIII gene probe. Lancet 1985; 1: 1093–4.
93 Kojima T, Tanimoto M, Kamiya T, Obata Y, Takahashi T, Ohno R,
Kurachi K, Saito H. Possible absence of common polymor-
phisms in coagulation factor IX gene in Japanese. Blood 1987; 69:
349–52.
94 Nossel HL, Younger LR, Wilner GD, Procupez T, Canfield RE,
Butler VP Jr. Radioimmunoassay of human fibrinopeptide A. Proc
Natl Acad Sci USA 1971; 68: 2350–3.
95 Lau HK, Rosenberg RD. The isolation and characterization of a
specific antibody population directed against the thrombin anti-
thrombin complex. J Biol Chem 1980; 255: 5885–93.
96 Lau HK, Rosenberg JS, Beeler DL, Rosenberg RD. The isolation
and characterization of a specific antibody population directed
against the prothrombin activation fragments F2 and F1+2. J Biol
Chem 1979; 254: 8751–61.
97 Gaffney PJ, Lane DA, Kakkar VV, Brasher M. Characterisation of a
soluble D dimer-E complex in crosslinked fibrin digests. Thromb Res
1975; 7: 89–99.
98 Hemker HC, Veltkamp JJ, Loeliger EA. Kinetic aspects of the
interaction of blood clotting enzymes. III. Demonstration of an
inhibitor of prothrombin conversion in vitamin K deficiency. Thromb
Diath Haemorrh 1968; 19: 346–63.
99 Blanchard RA, Furie BC, Jorgensen M, Kruger SF, Furie B. Ac-
quired vitamin K-dependent carboxylation deficiency in liver disease.
N Engl J Med 1981; 305: 242–8.
100 Blomba¨ ck M, Nilsson IM. Treatment of hemophilia A with human
antihemophilic globulin. Acta Med Scand 1958; 161: 301–21.
101 Kekwick RA, Wolf P. Concentrate of human antihaemophilic factor:
its use in six cases of haemophilia. Lancet 1957; I: 647–50.
102 Pool JG, Shannon AE. Production of high-potency concentrates of
antihemophilic globulin in a closed-bag system. Assay in vitro and in
vivo. N Engl J Med 1965; 273: 1443–7.
103 Rabiner SF, Telfer MC. Home Transfusion for Patients with
Hemophilia A. N Engl J Med 1970; 283: 1011–5.
104 Davis KC, Horsburgh CR Jr, Hasiba U, Schocket AL, Kirkpatrick
CH. Acquired immunodeficiency syndrome in a patient with hemo-
philia. Ann Intern Med 1983; 98: 284–6.
105 White GC 2nd, McMillan CW, Kingdon HS, Shoemaker CB. Use of
recombinant antihemophilic factor in the treatment of two patients
with classic hemophilia. N Engl J Med 1989; 320: 166–70.
106 Roth DA, Kessler CM, Pasi KJ, Rup B, Courter SG, Tubridy KL.
Human recombinant factor IX: safety and efficacy studies in hemo-
philia B patients previously treated with plasma-derived factor IX
concentrates. Blood 2001; 98: 3600–6.
107 Pipe SW. The promise and challenges of bioengineered recombinant
clotting factors. J Thromb Haemost 2005; 3: 1692–701.
108 Kurczynski EM, Penner JA. Activated prothrombin concentrate for
patients with factor VIII inhibitors. N Engl J Med 1974; 291: 164–7.
109 Lusher JM, Shapiro SS, Palascak JE, Rao AV, Levine PH, Blatt PM;
the Hemophilia Study Group. Efficacy of prothrombin-complex
concentrates in hemophiliacs with antibodies to factor VIII. A mul-
ticenter therapeutic trial. N Engl J Med 1980; 303: 421–5.
110 Hedner U, Kisiel W. Use of human factor VIIa in the treatment of
two hemophilic patients with high titer inhibitors. J Clin Invest 1983;
21: 1836–41.
111 Roberts HR, Monroe DM, White GC. The use of recombinant factor
VIIa in the treatment of bleeding disorders. Blood 2004; 104: 3858–64.
112 Levi M, Levy JH, Andersen HF, Truloff D. Safety of recombinant
activated factor VII in randomized clinical trials. N Engl J Med 2010;
363: 1791–800.
113 Murray G. Heparin in thrombosis and embolism. Br J Surg 1939; 27:
567–98.
114 Schofield FW. A brief account of a disease in cattle simulating hem-
orrhagic septicaemia due to feeding sweet clover. Can Vet Rec 1922; 3:
74–8.
115 Stahmann MA, Huebner CF, Link KP. Studies on the hemorrhagic
sweet clover disease. V. Identification and synthesis of the hemor-
rhagic agent. J Biol Chem 1941; 138: 513–27.
116 Bingham JB, Meyer OO, Pohle FJ. Studies on the hemorrhagic agent
3,3¢-methylenebis (4-hydroxycoumarin). I. Its effect on the pro-
thrombin and coagulation time of the blood of dogs and humans. Am
J Med Sci 1941; 202: 563–78.
117 Allen EV, Barker NW, Waugh JM. A preparation from spoiled sweet
clover. [3-3¢ methylene-bis-(4-hydroxycoumarin) which prolongs
362 H. Saito et al
Ó 2011 International Society on Thrombosis and Haemostasis

12. coagulation and prothrombin time of the blood: a clinical study.
JAMA 1942; 120: 1009–15.
118 Esmon CT, Sadowski JA, Suttie JW. A new carboxylation reaction.
The vitamin K-dependent incorporation of H14
Co3
–
into prothrom-
bin. J Biol Chem 1975; 250: 4744–8.
119 Whitlon DS, Sadowski JA, Suttie JW. Mechanism of coumarin ac-
tion: significance of vitamin K epoxide reductase inhibition. Bio-
chemisty 1978; 17: 1371–7.
120 The International Warfarin pharmacogenetics consortium. Estima-
tion of the Warfarin dose with clinical and pharmacogenetic data. N
Engl J Med 2009; 360: 753–64.
121 Okamoto S, Hijikata A, Kikumoto R, Tonomura S, Hara H, Nin-
omiya K. Potent inhibition of thrombin by the newly synthesized
arginine derivative No. 805. The importance of stereostructure of its
hydrophobic carboxamide portion. Biochem Biophys Res Commun
1981; 101: 440–6.
122 Connolly SJ, Ezekowitz MD, Yusuf S, Eikelboom J, Oldgren J,
Parekh A, Pogue J, Reilly PA, Themeles E, Varrone J, Wang S,
Alings M, Xavier D, Zhu J, Diaz R, Lewis BS, Darius H, Diener H-
C, Joyner CD, Wallentin L; RE-LY Steering Committee and Inves-
tigators. Dabigatran versus warfarin in patients with atrial fibrillation.
N Engl J Med 2009; 361: 1139–51.
123 Turpie AG, Bauer KA, Erksson BI, Lassen MR. Fondaparinux
versus enoxaparin for the prevention of venous thromboembolism in
major orthopedic surgery. A meta-analysis of 4 randomized double-
blind studies. Arch Intern Med 2002; 162: 1833–40.
124 EINSTEIN Investigators, Bauersachs R, Berkowitz SD, Brenner B,
Bu¨ ller HR, Decousus H, Gallus AS, Lensing AW, Misselwitz F, Prins
MH, Raskob GE, Segers A, Verhamme P, Wells P, Agnelli G,
Bounameaux H, Cohen A, Davidson BL, Piovella F, Schellong S.
Oral Rivaroxaban for symptomatic venous thromboembolism. N
Engl J Med 2010; 363: 2499–510.
125 Lassen MR, Gallus A, Raskob GE, Pineo G, Chen D, Ramirez LM;
ADVANCE-3 Investigators. Apixaban versus enoxaparin for
thromboprophylaxis after hip replacement. N Engl J Med 2010; 363:
2487–98.
126 Raskob G, Cohen AT, Eriksson BI, Puskas D, Shi M, Bocanegra T,
Weitz JI. Oral direct factor Xa inhibition with edoxaban for throm-
boprophylaxis after elective total hip replacement. A randomized
double-blind dose-response study. Thromb Haemost 2010; 104: 642–9.
127 Kienast J, Juers M, Wiedermann CJ, Hoffmann JN, Ostermann H,
Strauss R, Keinecke HO, Warren BL, Opal SM; KyberSept investi-
gators. Treatment effects of high-dose antithrombin without con-
comitant heparin in patients with severe sepsis with or without
disseminated intravascular coagulation. J Thromb Haemost 2006; 4:
90–7.
128 Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF,
Lopez-Rodriguez A, Steingrub JS, Garber GE, Helterbrand JD, Ely
EW, Fisher CJ Jr; Recombinant Human Activated Protein C
Worldwide Evaluation in Severe Sepsis (PROWESS) Study Group.
Efficacy and safety of recombinant human activated protein C for
severe sepsis. N Engl J Med 2001; 344: 699–709.
129 Aoki N, Matsuda T, Saito H, Takatsuki K, Okajima K, Takahashi
H, Takamatsu J, Asakura H, Ogawa N. A comparative double-blind
randomized trial of activated protein C and unfractionated heparin in
the treatment of disseminated intravascular coagulation. Int J
Hematol 2002; 75: 540–7.
130 Saito H, Maruyama I, Shimazaki S, Yamamoto Y, Aikawa N, Ohno
R, Hirayama A, Matsuda T, Asakura H, Nakashima M, Aoki N.
Efficacy and safety of recombinant human soluble thrombomodulin
(ART-123) in disseminated intravascular coagulation: results of a
phase III, randomized, double-blind clinical trial. J Thromb Haemost
2007; 5: 31–41.
131 Abraham E, Reinhart K, Opal S, Demeyer I, Doig C, Rodriguez AL,
Beale R, Svoboda P, Laterre PF, Simon S, Light B, Spapen H, Stone
J, Seibert A, Peckelsen C, De Deyne C, Postier R, Pettilla¨ V, Artioas
A, Percell SR, et al.; OPTIMIST Trial Study Group. Efficacy and
safety of tifacogin (recombinant tissue factor pathway inhibitor) in
severe sepsis: a rendomaized controlled trial. JAMA 2003; 290: 238–
47.
132 Nemerson Y, Turitto VT. The effect of flow on hemostasis and
thrombosis. Thromb Haemost 1991; 66: 272–6.
133 Falati S, Gross P, Merrill-Skoloff G, Furie BC, Furie B. Real-time in
vivo imaging of platelets, tissue factor and fibrin during arterial
thrombus formation in the mouse. Nat Med 2002; 8: 1175–81.
134 Nishimura S, Manabe I, Nagasaki M, Seo K, Yamashita H, Hosoya
Y, Ohsugi M, Tobe K, Kadowaki T, Nagai R, Sugiura S. In vivo
imaging in mice reveals local cell dynamics and inflammation in obese
adipose tissue. J Clin Invest 2008; 118: 710–21.
135 Kay MA, Landen CN, Rothenberg SR, Taylor A, Leland F, Wiehle
S, Fang B, Bellinger D, Finegold M, Thompson AR. In vivo hepatic
gene therapy: complete albeit transient correction of factor IX defi-
ciency in hemophilia B dogs. Proc Natl Acad Sci USA 1994; 91: 2353–
7.
136 Chuah MK, Schiedner G, Thorrez L, Brown B, Johnston M, Gillijns
V, Hertel S, von Rooijen N, Lillicrap D, Collen D, VandenDriessche
T, Kochanek S. Therapeutic factor VIII levels and negligible
toxicity in mouse and dog models of hemophilia A following gene
therapy with high-capacity adenoviral vectors. Blood 2003; 101: 1734–
43.
137 Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ,
Ozelo MC, Hoots K, Blatt P, Konkle B, Dake M, Kaye R, Razavi M,
Zajko A, Zehnder J, Rustagi PK, Nakai H, Chew A, Leonard D,
Wright JF, et al. Successful transduction of liver in hemophilia by
AAV-Factor IX and limitations imposed by the host immune re-
sponse. Nat Med 2006; 12: 342–7.
138 Gailani D, Renne´ T. Intrinsic pathway of coagulation and arterial
thrombosis. Arterioscler Thromb Vasc Biol 2007; 27: 2507–13.
Past, present and future of coagulation research 363
Ó 2011 International Society on Thrombosis and Haemostasis