Hemostasis in the 21st century

The Netherlands Journal of Medicine 1999;55:280–286

Hemostasis in the 21st century Marcel Levi 1 Department of Vascular Medicine and Internal Medicine F-4, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands

Introduction In the last decades of the 20th century, our insight into the mechanism of blood coagulation has dramatically increased. The discovery of numerous factors and pathways, either contributing to blood clot formation and degradation or playing a regulatory role in hemostasis, has emphasised the dual function of the coagulation system. On the one hand the hemostatic system ensures the rapid formation of a hemostatic plug, in the case of vessel wall disruption, to prevent ongoing blood loss, while on the other hand this same system is essential in maintaining blood fluidity within the vessels to guarantee proper circulation and organ perfusion. The function of primary hemostasis (the initial defence mechanism upon injury, leading to a platelet plug) and subsequent blood coagulation (resulting in a fibrin clot), as well as the fibrinolytic system (which ultimately removes the clot to restore normal tissue circulation and function) are in its main features well understood [1–3]. However, many important issues, such as the precise regulation and interaction of these mechanisms in vivo or the ultimate impact of this knowledge on the management of bleeding or thrombosis, have yet to be defined. In view of recent achievements there is sufficient reason to assume that, in the first decades of the 21st century, major 1

M.L. is an Investigator of the Royal Netherlands Academy of Science.

progress in this area is to be expected. In this paper, a number of imminent important developments in hemostasis are discussed in greater detail.

Refinement of the coagulation scheme The last 10 years have provided definitive proof that the ‘classic’ division of coagulation in an intrinsic and an extrinsic pathway has become untenable and the pivotal role of tissue factor in the initiation of coagulation in vivo has been established (Fig. 1) [2,4]. In fact, the pathways involved in the activation of coagulation in vivo have been clearly defined and the impact of physiological anticoagulants, such as antithrombin III and (activated) protein C, on each of these pathways has been clarified [5]. Newly identified factors have been discovered that govern the regulation between the activation of coagulation and the fibrinolytic system, such as the carboxypeptidase thrombin-activatable fibrinolytic inhibitor (TAFI) [6]. Nevertheless, virtually all our knowledge on the mechanisms involved in coagulation and physiological anticoagulation comes from observations done in blood or plasma, derived from the circulation. In contrast, coagulation is usually a localised process, which takes place at the site of the injured or, for example, atherosclerotic vessel wall. We do not know whether the mechanisms that occur in circulating blood are identical to the mechanism of local, endothelial cell-associated

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Fig. 1. Schematic representation of the coagulation cascade at the end of the 20th century. The activation of coagulation is initiated by tissue factor and factor VII(a), which subsequently activate factor X and factor II. Thrombin generation is amplified by tissue factor / factor VIIa-mediated activation of factor IX and thrombin-mediated activation of factor XI, both leading to more factor Xa activation and subsequent prothrombin to thrombin conversion. Regulation (circles) takes place by three different physiological anticoagulant systems: antithrombin III (inhibiting factor Xa and factor IIa), the protein C system (degrading co-factors VIII and V) and tissue factor pathway inhibitor (TFPI, the main inhibitor of tissue factor activity).

blood coagulation. Only recently, techniques have become available to study the activation of coagulation at the surface of endothelial cells ex vivo [7]. Further refinement of these methods and application of these techniques in various disease states will help us to further refine our knowledge on the mechanisms of coagulation and thrombus formation in vivo. Another unsettled issue is with regard to the control of the level of circulating coagulation factors. The plasma concentration of virtually each coagulation factor has been directly and independently related to an increased risk of thrombosis. At present, however, we do not know which factors regulate this plasma concentration. A relatively common polymorphism in the 39-untranslated region of coagulation factor II (the prothrombin 20210 G → A mutation) results in a slightly higher plasma concentration of this factor in affected individuals, which has been clearly associated with the occurrence of venous and possibly arterial thrombosis [8]. The underlying molecular mechanism of this mutation has yet to be elucidated. It may be expected that enhanced insight

into the molecular control of the circulating level of coagulation factors and the detection of polymorphisms that may affect this control will lead to a better understanding of determinants that contribute to the activation of coagulation and the development of thrombosis. What will the clotting scheme look like at the end of the 21st century? Extrapolating the latest insights into the mechanism of blood coagulation, the scheme will probably be much simpler than what we have ever thought. The complex pathways that have been in the textbooks for the last 25 years can now already be considered as artefacts of biochemical experiments in an inappropriate environment (i.e., glass). At the beginning of our understanding of coagulation in vivo, all signs indicate that in reality more lucid and straightforward pathways are in place.

A new role of platelets and coagulation beyond hemostasis Platelets and plasma coagulation proteins represent

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the most important components of the coagulation system. However, recent studies have demonstrated that both platelets and plasma coagulation factors contribute importantly to processes beyond hemostasis and thrombosis. Platelets have been found to release, upon their activation, numerous growth factors and cytokines that may play a role in inflammation, atherogenesis and cell migration and proliferation [9]. Thrombin (and recently factor X) have been identified as potent agonists for smooth muscle cell proliferation [10]. Studies of targeted disruption of coagulation factor genes have indicated a pivotal role of coagulation proteins, such as tissue factor, fibrinogen and prothrombin, in embryonal vasculogenesis and postnatal angiogenesis [11,12]. The contact system of coagulation (comprising the factor XII–kallikrein system) plays a role in regulation of hemodynamics and tissue perfusion, rather than being important for blood coagulation [13,14]. However, the precise role of the blood components that were initially thought to play a role in the hemostatic system exclusively, will have to be determined more precisely in the years to come. That knowledge will also provide more insight into the effect of pharmacological interventions directed against platelets or coagulation factors on processes other than coagulation.

A cure for inherited coagulation disorders Although for most congenital coagulation disorders proper management strategies are available, these diseases still represent a substantial burden for affected individuals [15]. Besides the ever present risk (and fear) of potentially life-threatening bleeding, available treatment may be associated with considerable drawbacks, such as immunological complications or the risk of transmission of bloodborne diseases. Following the isolation and characterisation of the cDNAs for several clotting factors, and experiments showing expression of these genes in tissue culture, the production of recombinant coagulation proteins, in particular factor VIII and factor IX, has become feasible [16–18]. The production of both recombinant factor VIII and factor IX may be considered as highly advanced technological

achievements. The factor VIII gene spans 180 000 base pairs and thereby encompasses about 0.1% of the human X-chromosome, and is the first gene of this size that was successfully used for recombinant protein engineering. Recombinant factor IX proved to be a challenge because of the indispensable posttranslational protein modifications, including vitamin K-dependent g-carboxylation and pro-peptide cleavage, but these hurdles have also been overcome. The production of recombinant factor VIII and IX for the first time provided the means for the treatment of patients with severe hemophilia A and B, respectively, without the use of allogeneic blood products. In the 1990s the first hemophiliacs have been treated with recombinant coagulation factors, and it may be foreseen that within 10 years all patients will be using these products, thereby rendering plasma-derived products obsolete [19]. The use of recombinant coagulation proteins, however, may be associated with an enhanced risk of the formation of antibodies towards coagulation factors, although at present the real magnitude of this problem and the underlying mechanism remain unclear [20]. Hence, even more advanced treatment strategies for patients with congenital coagulation defects are required. A logical next step would be treatment by gene transfer, directed at the introduction of functional clotting factor genes into autologous cells of patients to provide a continuous supply of coagulation factors. Indeed, at present much attention is directed to the development of gene therapy for hemophilia [21,22]. Important, currently occurring drawbacks comprise difficulties in efficient gene transfer to somatic cells, achievement of sufficient protein production after integration of the transferred DNA in the host genome, and potential immunogenicity of the viral vectors, which may limit the duration of the effect. However, it may be foreseen that all these pitfalls will be solved eventually. A relatively low protein production will not be a major obstacle for hemophilia treatment, since even the expression of low levels of coagulation factors will already transform a severe hemophiliac into a patient with a mild bleeding tendency only. Many research teams are presently working on better viral and non-viral methods for gene transfer, and the use of modified viruses (such as gutless adenoviruses) or alternative vectors (such

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as lentiviruses or retroviruses) have been shown to result in more efficient gene transfer and less immune response [23,24]. Taking all these ongoing and future developments together, the 21st century is likely to be the era of a better — and probably definitive — treatment for patients with congenital coagulation disorders.

The end of ‘idiopathic’ thrombosis Until not very long ago, for the majority of patients with venous thromboembolism, no clear explanation as to the cause of the thrombosis could be established. Large groups of patients suffered from thrombosis in the absence of precipitating factors such as immobilisation or malignancy. For these patients the term ‘idiopathic thrombosis’ was frequently used. Up to 1990, in about 5–8% of these patients a defect in the coagulation system (such as a deficiency of antithrombin III or protein C) was identified, which could explain the occurrence of thrombosis [25]. The last decade of the 20th century has shown major leaps in this area. The detection of resistance to activated protein C (and the underlying

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defect, i.e., a point mutation in factor V or ‘factor V Leiden’) as a risk factor for thrombosis suddenly provided an explanation for the occurrence of thrombosis in another 20% of patients [26]. The subsequent discovery of other important risk factors for hypercoagulability and thrombosis, such as the earlier mentioned prothrombin 20210 mutation [8], hyperhomocysteinemia [27], and high levels of factor VIII [28], has now provided a cause for thrombosis in the majority of cases that were considered ‘idiopathic’ only 10 years ago [29]. In addition, many ‘candidate’ risk factors for thrombosis are being considered at present. Extrapolating the exponential increase in our knowledge on risk factors for venous thromboembolism over the last decades, it may be predicted that in the early 21st century the cause of thrombosis may be explained in virtually all patients (Fig. 2). Inversely, the early detection of risk factors for thrombosis may facilitate individualised schemes to prevent the disease. Ongoing well-designed epidemiological studies will prove to be very helpful in predicting the risk of thrombosis of patients with any of the clotting system disorders, which will result in tailored and appropriate primary or secondary prophylaxis.

Fig. 2. Percentage of patients with venous thromboembolism in which a cause for the thrombosis may be defined. The figure illustrates the increment in knowledge on factors that may cause thrombosis. Extrapolating the line into the 21st century leads to the prediction that in the coming years, for virtually all episodes of thromboembolism, a precipitating cause may be identified. The Netherlands Journal of Medicine 1999;55:280 – 286

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Novel anticoagulant and prohemostatic agents Improved insight into the mechanisms that play a role in the activation and regulation of coagulation may generate novel therapeutic strategies to optimise treatment of patients with thrombosis or with severe bleeding. In fact, at present a host of new pharmacological agents, based on the newly developed understanding of the coagulation system, is being designed, or even already available for testing in experimental and clinical studies. In view of the central role of tissue factor in the initiation of blood coagulation and the abundant presence of tissue factor at the site of the (ruptured) atherosclerotic plaque, tissue factor is one of the most promising targets for novel anticoagulants. A promising recombinant anti-tissue factor agent, based on an anticoagulant protein of nematodes, is currently being investigated in clinical studies [30]. In addition, a number of other tissue factor-directed agents will be developed and tested in the coming years. Further developments will soon occur in the group of direct thrombin inhibitors. Thrombin occupies a pivotal place in the activation of coagulation and is an excellent target for antithrombotic therapy. Direct thrombin inhibitors, such as hirudin or hirudin-derived compounds, do not require the presence of antithrombin III for thrombin inhibition, are able to block surface-bound thrombin and were shown to be highly effective as anticoagulants [31,32]. Experimental and clinical studies have suggested an increased benefit of a more sustained potent inhibition of thrombin, for example in patients with acute coronary syndromes [33]. A major drawback so far is the exclusive parenteral mode of administration of these agents. In the first years of the 21st century we will see the introduction of several orally available direct thrombin inhibitors, which will potentially replace conventional long-term anticoagulants, such as coumarins, in patients who need (secondary) prophylaxis for atherothrombotic disease or prevention of arterial embolism as a complication of atrial fibrillation. One of the major challenges for the future is the therapeutic width of the novel anticoagulant agents. So far, any new anticoagulant agent with a more effective antithrombotic action has been associated

with an enhanced risk of bleeding as an adverse consequence. The future might prove that it is simply not possible to effectively block coagulation without enhancing the bleeding risk; however, one needs to realise that all anticoagulants so far are based on inhibition of fluid-phase coagulation. A novel approach, aimed at inhibitors of surface-associated or local coagulation activation might be proved to possess a more beneficial antithrombotic to bleeding ratio. The pharmacokinetic profile of the novel agents will also be an important feature here. Most currently available anticoagulants have an unpredictable bioavailability, resulting in a highly variable inter- and intra-individual effect and necessitating frequent laboratory control. A more stable and predictable effect will result in a larger proportion of patients being in the therapeutic target range, most likely with a more favourable clinical outcome, and obviating the need for repeated laboratory tests in addition. The replacement of unfractionated heparin by low molecular weight heparin in patients with venous and arterial thrombosis has shown the benefit of this approach [34]. Lastly, but importantly, in the next century we will have to make a start in the attempt to more specifically tailor our pharmacotherapy to the individual patient. Major progress in this area may be achieved by taking into account the genetic properties of our patients at the molecular level. A good or inadequate response upon medication might well depend on variations in genetic background. Frequent or infrequent polymorphisms have already been shown to be related to a lack of response to drugs, such as cholesterol-lowering agents [35]. In view of the complex interplay between coagulation factors and coagulation inhibitors, it is certainly not imaginable that a relative resistance to anticoagulation or an increased risk of bleeding while taking anticoagulant agents may be predicted by predefined genetic polymorphisms.

Conclusion Major breakthroughs in the last decades of the 20th century have caused a tremendous increase in our insights into the mode of action of blood

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coagulation and physiological anticoagulant mechanisms. Many issues, however, have remained unsolved, but it may be assumed that our further understanding of coagulation will swiftly progress in the 21st century. Our major challenge for the next era will be the transformation of this newly accumulated knowledge into clinically applicable improvement in the management of patients with bleeding and thrombosis.

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