Historical perspective and future direction of coagulation research

Authors


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

Abstract

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.

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 investigators. 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 important insight into the process of blood clotting. In 1905 Morawitz [1] proposed the so called ‘the classic theory of blood coagulation’ 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.

Figure 1.

 Advances of technology used in coagulation research. Introduction of various technologies was illustrated over time.

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 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].

Figure 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.

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 anticoagulants) 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 disorders, 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 Quick’s one-stage prothrombin time (PT) [25] and the partial thromboplastin 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 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 haemophilia, into the coagulation scheme of Morawitz, because the prothrombin time of haemophilic plasma was normal. Furthermore, 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 understanding 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]. Furthermore, 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, 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 ‘AHF-like’ 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 ‘von Willebrand factor’ 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 reactive 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-thrombomodulin 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 coagulation factor that was cloned and sequenced was factor IX in 1982 [47,48], followed by other factors [49–54]. Davie’s 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 modifications 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/haemBdatabase.html, http://www.vwf.group.shef.ac.uk/) [55,56]. A recent study found a specific mutation in the F9 gene in the recovered DNA extracted from bone fragments of the Russian Tsar’s family, thereby identifying the cause of the Royal Disease of Queen Victoria’s descendants as haemophilia 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 molecular 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 neurologic 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 pathogenesis 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 unexpectedly 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 intermediate 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 understanding of the blood coagulation system in vivo. The late 1990s 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 thrombosis 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 of haemophilia A became possible with the prediction rate of 70–90% 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 within the F8 gene [90] or direct mutation detection [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. The choice of a proper polymorphism is important, as the incidence of common polymorphisms of some genes may be variable among different ethnic groups [93]. Similarly, DNA diagnosis of hereditary deficiency of antithrombin, protein C and 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

Development and clinical application of plasma concentrates (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 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 ‘bypass’ the site of action of factor VIII inhibitor [108,109]. There are, however, some concerns about thrombotic 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 anticoagulant 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 identification 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 γ-carboxyglutamyl (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.

Figure 3.

 Sites of actions of established and novel anticoagulants in the coagulation cascade. The targets of anticoagulants are illustrated in the coagulation 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.

The leading question in the development of anticoagulant drugs has been ‘Is it possible to make a potent anticoagulant without a bleeding risk?’ We assume that this aim is almost impossible to achieve, as the mechanisms underlying intravascular 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).

Table 1.   Characteristics of new oral anticoagulants
 DabigatranRivaroxabanApixabanEdoxaban
  1. *P-gp, P-glycoprotein 1; CYP3A4, cytochrome P450 3A4; NVAF, non-valvular arterial fibrillation.

TargetFllaFXaFXaFXa
ProdrugYesNoNoNo
DosingTwice dailyOnce dailyTwice dailyOnce daily
Coagulation monitoringNoNoNoNo
Bioavailability (%)6805050
Half-life (h)12–175–9129–11
Renal excretion (%)80652535
AntidoteNoneNoneNoneNone
Drug interactionsP-gp* inhibitorsPotent inhibitors of CYP3A4 or P-gpPotent inhibitors of CYP3A4Potent inhibitors of CYP3A4 or P-gp
Clinical statusApproved in Canada and Europe for VTE prophylaxis after major orthopedic surgery Approved in USA and Japan for stroke prevention in NVAFApproved in Canada and Europe for VTE prophylaxis after major orthopedic surgeryNoneApproved in Japan for VTE prophylaxis after major orthopedic surgery

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 thrombocytopenia (HIT). Hirudin is also approved in HIT. Dabigatran, 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 synthesised 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 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 symptoms 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 important 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 techniques 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 application 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 replacement therapy is already available and further attempts to develop recombinant factors with improved properties are under way.

Recent advances in the technology of regenerative medicine 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 maintenance 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 Haemostasis (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.

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.

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