Journal of Thrombosis and Haemostasis, 12: 587–592
DOI: 10.1111/jth.12545
REVIEW ARTICLE
The Northwick Park Heart Study: evidence from the laboratory H . T E N C A T E * and T . M E A D E † *Laboratory of Clinical Thrombosis and Hemostasis, Department of Internal Medicine, Maastricht University Medical Center and Cardiovascular Research Institute, Maastricht, the Netherlands; and †Department of Non-Communicable Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, UK
To cite this article: ten Cate H, Meade T. The Northwick Park Heart Study: evidence from the laboratory. J Thromb Haemost 2014; 12: 587–92.
Abstract. The Northwick Park Heart Study (NPHS) has shown associations of high plasma fibrinogen and factor VII (FVIIc) levels with the risk of death from coronary heart disease (CHD). The finding for fibrinogen has been confirmed in many other studies. Whereas one further study has found a similar prospective association for FVIIc, several have not. Experimental studies have demonstrated the impact that the coagulation activity of fibrinogen and FVIIc have on the progression and phenotype of atherosclerotic lesions. FVIIc-driven thrombin generation and fibrin formation within the vessel wall are important determinants of both plaque (in)stability and atherothrombosis. In blood, local concentrations of FVIIc and thrombin may be sufficient to allow interactions between these serine proteases and protease-activated receptors, to drive cellular inflammatory reactions that further promote these processes. Local fibrinogen concentrations dictate fibrin clot structure and resistance to fibrinolysis. Within the atherosclerotic plaque, coagulation reactions driven by proinflammatory stimuli may initially support lesion stability (as part of wound healing), but, with advanced inflammation, thrombin and fibrin generation diminish because of proteolytic activity contributing to plaque instability. The NPHS findings have proved controversial, but, in the light of current knowledge, a reappraisal of the importance of FVIIc and fibrinogen in atherosclerosis, atherothrombosis and CHD is justified. Hypercoagulability, reflected in turn by thrombin generation capacity, and local concentrations of coagulation proteins, including FVIIc and fibrinogen, is linked to plaque phenotype, and even minute local concentrations
Correspondence: Hugo ten Cate, Laboratory of Clinical Thrombosis and Hemostasis, Department of Internal Medicine, Cardiovascular Research Institute, Maastricht, the Netherlands. Tel.: +31 43 3884262; fax: +31 43 3884159. E-mail: [email protected] Received 19 September 2013 Manuscript handled by: F. R. Rosendaal Final decision: F. R. Rosendaal, 25 February 2014 © 2014 International Society on Thrombosis and Haemostasis
of fibrinogen and proteases such as FVIIc may affect thrombin generation capacity. Keywords: coagulation factors; coronary heart disease; coronary thrombosis; factor VII; fibrinogen.
Introduction Since 1945, there has been extensive research into the onset, progression and complications of arterial vessel wall disease, on the one hand, and on the clinical and epidemiologic features of coronary heart disease (CHD) on the other. Initially, the main premise for this work comprised the chronic, atherogenic changes in the wall of the coronary arteries associated with major clinical events. It was not until the mid-1970s that the thrombi often found in those dying of CHD were agreed to precede and be the cause of infarction, rather than a consequence of the event [1] Following on from this recognition, further studies showed that thrombosis as an acute process occluding the artery is more often than not the immediate cause of myocardial infarction (MI) [2] and sudden coronary death (SCD) [3]. Initially, work on the thrombotic component of CHD was concerned mainly with the undoubted contribution of platelets, and interest in the blood coagulation system is a more recent and less familiar development. This review considers the evidence that components of the coagulation system are causally involved in the onset of major CHD events, particularly factor VII and fibrinogen, whose involvement has hitherto been especially controversial. Pathology There are two main features determining the onset of clinical CHD. One is the development in the arterial vessel wall of atheroma or atherosclerosis; this is a highly complex process, one element of which is the activity of the blood coagulation system (see later) [4–6]. Leakage or disruption of the atheromatous plaque, with the release of tissue factor (TF) and platelet-activating proteins such
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as collagen, triggers the second component, thrombogenesis. Atherogenesis is a slowly developing, chronic process, a degree of which can be tolerated without clinical symptoms. Thrombosis, or atherothrombosis, is a short-term, acute process caused (as indicated) by leakage or rupture of an atheromatous plaque, and is usually responsible for the final mechanical blockage of the whole or most of the coronary artery lumen, leading to a clinical event. Thus, atheroma is, generally, a necessary but insufficient cause of these events, and thrombosis rarely occurs in the absence of atheroma. The previous paragraph illustrates one difficulty in considering the different processes in a way that is universally understood – the confusing terminology used. ‘Atherosclerotic’ and ‘arteriosclerotic’ have often been used to refer either to the vessel wall pathology only, or to clinical manifestations only, or to the whole range of processes from the early development of vessel wall changes through to death from MI or CHD. Consequently, it is often not clear which developments or events are under consideration. ‘Athero-’ is derived from the Greek for porridge, meal, or groats, which does suggest the frequently soft vessel wall lesion, even if this is not an exact description. We suggest that ‘atherosclerotic’ or atheroma (tous) should be confined to consideration of vessel wall disease, and ‘atherothrombotic’ should refer to the thrombotic process. Other terms, such as MI or SCD, then make it clear when clinical events are being considered. Epidemiologic data In 1972, Meade et al. [7] set up the Northwick Park Heart Study (NPHS), a conventional prospective study in design, but novel in the inclusion of a range of hemostatic variables, including factor (F) V, FVII (referred to as FVIIc), and FVIII, all assessed with biological activity assays [8–10]. 1 Antithrombin III was measured with a chromogenic substrate assay [11], fibrinogen with a clot weight method [12], and fibrinolytic activity with the dilute whole blood clot lysis time [13]. Since the early 1970s, assays for several measures of other coagulation indices, including those indicating the activation status of the coagulation system, have been developed, but the NPHS measures were among those that were available and feasible at the time for large-scale use. The NPHS recruited 3500 people of all ages working in three occupational groups in northwest London. Information at entry about past events of clinically manifest CHD were recorded, e.g. MI or angina pectoris. Details about new events were then collected, all participants having previously been ‘flagged’ in the National Health Service Central Register, thus enabling automatic notification of deaths. Summaries of information 1
The NPHS used clotting assays for these factors, with a subscript ‘c’ in publications to indicate this. FVIIa is used where the activation status of FVII is under consideration.
from general practitioners, hospitals and coroners were obtained and independently assessed ‘blind’ to characteristics such as smoking or blood results at recruitment. Diagnoses obtained in this way occasionally differed from hospital records or death certificates, but were considered to be more accurate in some instances. (For example, an apparent MI occurring as a terminal event immediately before death in a patient with widespread metastatic cancer and given as the certified cause of death would be considered inaccurate, cancer being taken as the actual cause). Early results were published in 1980 [14] and full results in 1986 [7], the latter showing strong independent associations with new CHD events for FVIIc and fibrinogen. Nearly 30 years after recruitment, further results were published [15], again showing independent associations with death from CHD of FVIIc and fibrinogen. These long-term results omitted events occurring within 10 years of recruitment, when the associations for both FVIIc and fibrinogen were particularly strong, so that they reflect continuing and not initial associations. At all stages, FVIIc has been associated only with fatal CHD. Interest in the early NPHS findings opened up a wide program by others of epidemiologic, clinical and basic research focused on the hemostatic system. Nearly all other studies have confirmed the association of fibrinogen with CHD [16]. However, the interpretation of this relationship has been controversial. On the one hand, it is claimed that raised fibrinogen levels reflect only a chronic-phase response to the inflammatory nature of underlying vessel wall pathology, and that there is no causal involvement of fibrinogen in CHD. In support of this view, Mendelian randomization (MR) has been considered to show no association of fibrinogen with CHD [17]. Early reservations about MR [18] have since been resolved in terms of study size and numbers of singlenucleotide polymorphisms in the more recent investigation by Sabater-Lleal et al. [19]. These findings and the results of other MR studies certainly have to be recognized. However, questions still remain about both the quality and the quantity of fibrinogen determined with the measurement methods used. The c chain of fibrinogen has a variable extension at the C-terminus known as c0 / fibrinogen c0 , and its significance for fibrin clot structure is unclear. These and other factors [18] may influence the thrombotic potential of fibrinogen. At the same time, fibrinogen undoubtedly influences a number of processes that have been shown to be associated with CHD. In particular, whole blood and plasma viscosity [20], of which fibrinogen is a major determinant, are associated with clinical events, and high fibrinogen levels also appear to influence the size and stability of thrombi [21] and platelet aggregability [22]. In fact, the two polarized views on fibrinogen (as either reflecting only inflammation of the vessel wall or as indicating causal significance) miss the point that there may be validity in both. Thus, fibrinogen levels may indeed be raised genetically or in response to © 2014 International Society on Thrombosis and Haemostasis
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the inflammatory properties of vessel wall pathology, but, once elevated, they may contribute causally to the onset of clinical events through several pathways. Only one other epidemiologic study has reported an association between high FVIIc levels and CHD [23], while others having failed to demonstrate any relationship [24–27]. However, a variety of FVIIc assays were used in these other studies, and differences between their methods have earlier [28] and more recently [29] been pointed out, with the conclusion that the FVIIc assay used in NPHS was probably more sensitive than others to FVIIa. Fibrinogen is probably not affected to any marked degree by dietary constituents, but its level is clearly increased by smoking. FVIIc is not influenced by smoking, but rises rapidly and substantially in response to an increase in dietary fat [30]. In general, however, the associations of the two clotting factors with other known risk factors for thrombotic events tend to strengthen the case for their causal involvement in CHD [31]. (The NPHS has also reported an association between the FVIII complex and CHD [8]. This report also presented data on ABO blood groups. Similar results on FVIII were obtained by Rumley et al. [32] and Biere-Rafi et al. [33]). Coagulation and thrombosis The contribution of thrombosis to the vessel wall lesion was shown by Duguid many years ago [34], and subsequently by Smith [35] and Falk [36]. Since then, numerous experimental studies have also demonstrated that increased hemostatic activity drives not only thrombus formation, but also plaque development [4]. This effect comprises stimulation of inflammatory responses, cell proliferation and angiogenesis mediated by platelets, leukocytes, and coagulation proteases [37–39]. Platelets, interacting with leukocytes and releasing microparticles, but also soluble mediators such as cytokines and CD40L, act in a proatherogenic manner. Different serine proteases, including FXa and thrombin, act on vessel wall cells through protease-activated receptors (PARs) to stimulate different cellular functions [4]. TF is another important cellular receptor that, upon binding of FVIIc, also stimulates proinflammatory mechanisms, in part via PAR-2, and in part via PAR-independent pathways [40]. The net effect of these mechanisms probably depends on the balance between procoagulant and anticoagulant forces, which may, in turn, result from other vascular risk factors (such as hypertension or metabolic syndromerelated mediators) affecting the vessel wall. A proinflammatory environment leads to ‘perturbation’ of the vascular endothelium, although the response may be quite heterogeneous, depending on the vascular bed [41]. This endothelial response includes many features that stimulate proinflammatory mechanisms, such as increased leukocyte adhesion and permeation. It also involves the downregulation of receptors that are important in anticoagulant defence, i.e. © 2014 International Society on Thrombosis and Haemostasis
thrombomodulin (TM) (which binds thrombin) and endothelial protein C receptor (EPCR) [42]. It has been demonstrated that cell receptor occupancy is one of the critical factors determining the switch in the effects of thrombin from anticoagulant (TM/EPCR-mediated generation of activated protein C) to proinflammatory action, mediated through PAR-1 [43–45]. At the same time, the interplay of environmental inflammatory factors may direct the effects of FVIIc–TF signaling towards proinflammatory actions, depending on the availability of TF-inhibiting forces (TF pathway inhibitor [TFPI]) and cellular receptors that act in concert with FVIIc–TF, including PAR-2 [46]. Hence, the biological actions of serine proteases, including FVIIc and thrombin, depend not only on their systemic plasma concentrations and activity, but perhaps more importantly on whether the vascular milieu facilitates specific interactions with the vessel wall (endothelium). Thus, local concentrations and interactions are critical in determining the local responses of the vascular endothelium, for which the temporary presence of even minute amounts of a free enzyme such as FVIIc, engaging in TF-mediated reactions, may be sufficient. This conclusion is important, because the presence of free enzymes able to interact with cellular receptors would be almost impossible if FVIIc, FXa and thrombin were generated only by a systemic process that is quickly tuned down by a panel of natural inhibitors such as antithrombin and TFPI. Other intriguing molecules in this context are fibrinogen and its derivative fibrin, which constitute the final element in the coagulation cascade and a critical building block of the fibrin clot. Fibrinogen concentrations in plasma are regulated both by genetic and by acute-phase stimulation in the course of inflammation [47]. Although the contribution to clotting depends primarily on the availability of free thrombin, to convert fibrinogen to fibrin monomers, the actual concentration and type of fibrinogen have an impact on the clotting process. Elevated levels of fibrinogen translate into increased thrombosis in animal models [48,49]. As already indicated, the probable mechanisms involve increased viscosity, increased thrombin-mediated fibrin formation (fibrinogen being an important determinant of thrombin generation in plasma), denser clot structure, and resistance to lysis. In addition, the fibrinogen c0 /fibrinogen ratio is a determinant of fibrin clotting properties, and is related to cardiovascular risk factors such as C-reactive protein. As for FVIIc, there is either no or uncertain evidence of haplotype associations with CHD [50]. In addition, fibrinogen, fibrin and fibrin cleavage products have been detected in immunohistochemical studies of human atherosclerotic lesions [35]. This fibrin formation probably results from local thrombin activity [51], and fibrin cleavage from the action of local fibrinolytic proteases (plasmin) and leukocyte-released enzymes such as elastase. Thus, fibrin cleavage products may also result from increased local inflammatory actions. Whereas fibrin
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and the cleavage fragments do not activate PARs, many in vitro studies have shown that fibrin degradation products are important mediators of inflammation, cell migration, and proliferation [52,53]. In humans, however, the contribution of hemostasis to atherosclerosis is still disputed, usually because of the lack of obvious protection against atherosclerosis in congenitally deficient subjects, such as patients with hemophilia [54]. Although the absence of one specific clotting factor may not provide sufficient protection against a complex disorder such as atherosclerosis, the opposite phenotype, hypercoagulability, is linked to atherosclerosis and atherothrombosis. One argument in favor of the latter is that, in a large metaanalysis, FV Leiden and prothrombin 20210 carriage were associated with a modest, but statistically significant, elevations in CHD [55]. Second, several studies have indicated that biomarkers of activated coagulation and fibrinolysis (including D-dimer fragment) are linked to CHD [25,56–59]. A third argument is that both immunohistochemical and functional studies have linked coagulation activity to atherosclerotic plaque instability. Indeed, an increased TF level in plaques correlates with features of instability [60,61]. Increased coagulation protease activity is linked to early atherogenesis, whereas diminishing coagulation activity has been noted in areas of advanced, complex plaque (within the same individuals) [51], suggesting a mechanistic contribution of coagulation proteases in human atherogenesis and atherothrombosis. Finally, a biological link is also suggested by a limited number of studies showing correlations between markers of hypercoagulability and measures of atherosclerosis, i.e. intima– media thickness, or levels of thrombin–antithrombin complexes in plasma linked to coronary calcification [62–64]. The contribution of coagulation activity of the plaque in triggering atherothrombosis is not disputed. Plaque erosion or rupture exposes platelet-activating proteins such as collagen and TF to blood, which is sufficient to start thrombosis in an ordered sequence [65]. Collagen plays a double role, linking platelet activation to FXII activation, which may play a role in stabilization of the arterial thrombus, making it more resistant to clot lysis [66,67]. Thus initiated, coagulation may culminate in an atherothrombotic lesion, which may become a nidus for subsequent clot formation, precipitating symptomatic atherothrombosis, and causing MI or SCD. In the Thrombosis Prevention Trial (TPT) [68], lowintensity warfarin (International Normalized Ratio of 1.5) significantly reduced only fatal CHD events, an effect in line with the observation that FVIIc is associated with fatal but not non-fatal CHD in the NPHS. It may not, of course, have been FVIIc rather than one of the other vitamin K-dependent factors, or a combination, that was responsible for the reduction in events, but it is worth drawing attention to the similarity of the results for FVIIc only for fatal CHD in both the NPHS and the TPT.
In summary, the coagulation system contributes to the pathologic changes leading to MI/CHD, and FVIIc and fibrinogen are involved in these. Conclusion On the basis of experimental and epidemiologic studies, a reappraisal of the importance of the hemostatic system, particularly coagulation, in the processes of atherosclerosis and atherothrombosis is justified. The NPHS and other studies have clearly indicated that high fibrinogen levels are associated with increased risks of CHD, and the same cannot be ruled out for FVIIc. Although part of the association for fibrinogen may indeed reflect the underlying effect of chronic inflammation, causal contributions are also likely. Recent clinical observational data and several experimental studies have clearly indicated that hypercoagulability, which is dependent, among other influences, on the concentrations of FVIIc and fibrinogen (in terms of thrombin generation capacity), is associated with atherosclerosis. In contrast, hypocoagulability protects against atherosclerosis in mice. Importantly, the effects of fibrinogen and serine proteases such as FVIIc may depend significantly on local concentrations and the availability of cellular receptors. Moreover, hypercoagulability is linked to plaque phenotype [51], with effects that support either plaque stability or instability, depending on the plaque stage and the age of the animal, respectively [69,70]. These effects may well be clinically relevant, as three experimental studies have shown that the thrombin inhibitor dabigatran substantially attenuates atherosclerosis in apoE-/- mice [70–72]. Although these observations in mouse models are obviously not directly translatable to humans, even minute effects in humans may turn out to be relevant upon prolonged exposure to anticoagulants. For warfarin, it took some time to realize that prolonged exposure accelerates arterial calcification through inhibition of Gla-dependent proteins in the vasculature [73]. It is therefore not unthinkable that longterm exposure of patients to new oral anticoagulants may also affect atherogenesis and/or plaque phenotype, but it will be some time before the necessary data become available. It is fascinating to observe that the original epidemiologic findings suggesting a causal effect of clotting proteins on CHD now appear to find support in mechanisms linking coagulation activity with the complex pathophysiology of atherosclerosis and atherothrombosis. This creates new avenues for clinical research to unravel the arterial coagulation–inflammation axis and to establish the consequences of long-term anticoagulation in subjects at risk of atherosclerosis. Disclosure of Conflict of Interests The authors state that they have no conflict of interest. © 2014 International Society on Thrombosis and Haemostasis
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DOI: 10.1111/jth.12545
REVIEW ARTICLE
The Northwick Park Heart Study: evidence from the laboratory H . T E N C A T E * and T . M E A D E † *Laboratory of Clinical Thrombosis and Hemostasis, Department of Internal Medicine, Maastricht University Medical Center and Cardiovascular Research Institute, Maastricht, the Netherlands; and †Department of Non-Communicable Disease Epidemiology, London School of Hygiene & Tropical Medicine, London, UK
To cite this article: ten Cate H, Meade T. The Northwick Park Heart Study: evidence from the laboratory. J Thromb Haemost 2014; 12: 587–92.
Abstract. The Northwick Park Heart Study (NPHS) has shown associations of high plasma fibrinogen and factor VII (FVIIc) levels with the risk of death from coronary heart disease (CHD). The finding for fibrinogen has been confirmed in many other studies. Whereas one further study has found a similar prospective association for FVIIc, several have not. Experimental studies have demonstrated the impact that the coagulation activity of fibrinogen and FVIIc have on the progression and phenotype of atherosclerotic lesions. FVIIc-driven thrombin generation and fibrin formation within the vessel wall are important determinants of both plaque (in)stability and atherothrombosis. In blood, local concentrations of FVIIc and thrombin may be sufficient to allow interactions between these serine proteases and protease-activated receptors, to drive cellular inflammatory reactions that further promote these processes. Local fibrinogen concentrations dictate fibrin clot structure and resistance to fibrinolysis. Within the atherosclerotic plaque, coagulation reactions driven by proinflammatory stimuli may initially support lesion stability (as part of wound healing), but, with advanced inflammation, thrombin and fibrin generation diminish because of proteolytic activity contributing to plaque instability. The NPHS findings have proved controversial, but, in the light of current knowledge, a reappraisal of the importance of FVIIc and fibrinogen in atherosclerosis, atherothrombosis and CHD is justified. Hypercoagulability, reflected in turn by thrombin generation capacity, and local concentrations of coagulation proteins, including FVIIc and fibrinogen, is linked to plaque phenotype, and even minute local concentrations
Correspondence: Hugo ten Cate, Laboratory of Clinical Thrombosis and Hemostasis, Department of Internal Medicine, Cardiovascular Research Institute, Maastricht, the Netherlands. Tel.: +31 43 3884262; fax: +31 43 3884159. E-mail: [email protected] Received 19 September 2013 Manuscript handled by: F. R. Rosendaal Final decision: F. R. Rosendaal, 25 February 2014 © 2014 International Society on Thrombosis and Haemostasis
of fibrinogen and proteases such as FVIIc may affect thrombin generation capacity. Keywords: coagulation factors; coronary heart disease; coronary thrombosis; factor VII; fibrinogen.
Introduction Since 1945, there has been extensive research into the onset, progression and complications of arterial vessel wall disease, on the one hand, and on the clinical and epidemiologic features of coronary heart disease (CHD) on the other. Initially, the main premise for this work comprised the chronic, atherogenic changes in the wall of the coronary arteries associated with major clinical events. It was not until the mid-1970s that the thrombi often found in those dying of CHD were agreed to precede and be the cause of infarction, rather than a consequence of the event [1] Following on from this recognition, further studies showed that thrombosis as an acute process occluding the artery is more often than not the immediate cause of myocardial infarction (MI) [2] and sudden coronary death (SCD) [3]. Initially, work on the thrombotic component of CHD was concerned mainly with the undoubted contribution of platelets, and interest in the blood coagulation system is a more recent and less familiar development. This review considers the evidence that components of the coagulation system are causally involved in the onset of major CHD events, particularly factor VII and fibrinogen, whose involvement has hitherto been especially controversial. Pathology There are two main features determining the onset of clinical CHD. One is the development in the arterial vessel wall of atheroma or atherosclerosis; this is a highly complex process, one element of which is the activity of the blood coagulation system (see later) [4–6]. Leakage or disruption of the atheromatous plaque, with the release of tissue factor (TF) and platelet-activating proteins such
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as collagen, triggers the second component, thrombogenesis. Atherogenesis is a slowly developing, chronic process, a degree of which can be tolerated without clinical symptoms. Thrombosis, or atherothrombosis, is a short-term, acute process caused (as indicated) by leakage or rupture of an atheromatous plaque, and is usually responsible for the final mechanical blockage of the whole or most of the coronary artery lumen, leading to a clinical event. Thus, atheroma is, generally, a necessary but insufficient cause of these events, and thrombosis rarely occurs in the absence of atheroma. The previous paragraph illustrates one difficulty in considering the different processes in a way that is universally understood – the confusing terminology used. ‘Atherosclerotic’ and ‘arteriosclerotic’ have often been used to refer either to the vessel wall pathology only, or to clinical manifestations only, or to the whole range of processes from the early development of vessel wall changes through to death from MI or CHD. Consequently, it is often not clear which developments or events are under consideration. ‘Athero-’ is derived from the Greek for porridge, meal, or groats, which does suggest the frequently soft vessel wall lesion, even if this is not an exact description. We suggest that ‘atherosclerotic’ or atheroma (tous) should be confined to consideration of vessel wall disease, and ‘atherothrombotic’ should refer to the thrombotic process. Other terms, such as MI or SCD, then make it clear when clinical events are being considered. Epidemiologic data In 1972, Meade et al. [7] set up the Northwick Park Heart Study (NPHS), a conventional prospective study in design, but novel in the inclusion of a range of hemostatic variables, including factor (F) V, FVII (referred to as FVIIc), and FVIII, all assessed with biological activity assays [8–10]. 1 Antithrombin III was measured with a chromogenic substrate assay [11], fibrinogen with a clot weight method [12], and fibrinolytic activity with the dilute whole blood clot lysis time [13]. Since the early 1970s, assays for several measures of other coagulation indices, including those indicating the activation status of the coagulation system, have been developed, but the NPHS measures were among those that were available and feasible at the time for large-scale use. The NPHS recruited 3500 people of all ages working in three occupational groups in northwest London. Information at entry about past events of clinically manifest CHD were recorded, e.g. MI or angina pectoris. Details about new events were then collected, all participants having previously been ‘flagged’ in the National Health Service Central Register, thus enabling automatic notification of deaths. Summaries of information 1
The NPHS used clotting assays for these factors, with a subscript ‘c’ in publications to indicate this. FVIIa is used where the activation status of FVII is under consideration.
from general practitioners, hospitals and coroners were obtained and independently assessed ‘blind’ to characteristics such as smoking or blood results at recruitment. Diagnoses obtained in this way occasionally differed from hospital records or death certificates, but were considered to be more accurate in some instances. (For example, an apparent MI occurring as a terminal event immediately before death in a patient with widespread metastatic cancer and given as the certified cause of death would be considered inaccurate, cancer being taken as the actual cause). Early results were published in 1980 [14] and full results in 1986 [7], the latter showing strong independent associations with new CHD events for FVIIc and fibrinogen. Nearly 30 years after recruitment, further results were published [15], again showing independent associations with death from CHD of FVIIc and fibrinogen. These long-term results omitted events occurring within 10 years of recruitment, when the associations for both FVIIc and fibrinogen were particularly strong, so that they reflect continuing and not initial associations. At all stages, FVIIc has been associated only with fatal CHD. Interest in the early NPHS findings opened up a wide program by others of epidemiologic, clinical and basic research focused on the hemostatic system. Nearly all other studies have confirmed the association of fibrinogen with CHD [16]. However, the interpretation of this relationship has been controversial. On the one hand, it is claimed that raised fibrinogen levels reflect only a chronic-phase response to the inflammatory nature of underlying vessel wall pathology, and that there is no causal involvement of fibrinogen in CHD. In support of this view, Mendelian randomization (MR) has been considered to show no association of fibrinogen with CHD [17]. Early reservations about MR [18] have since been resolved in terms of study size and numbers of singlenucleotide polymorphisms in the more recent investigation by Sabater-Lleal et al. [19]. These findings and the results of other MR studies certainly have to be recognized. However, questions still remain about both the quality and the quantity of fibrinogen determined with the measurement methods used. The c chain of fibrinogen has a variable extension at the C-terminus known as c0 / fibrinogen c0 , and its significance for fibrin clot structure is unclear. These and other factors [18] may influence the thrombotic potential of fibrinogen. At the same time, fibrinogen undoubtedly influences a number of processes that have been shown to be associated with CHD. In particular, whole blood and plasma viscosity [20], of which fibrinogen is a major determinant, are associated with clinical events, and high fibrinogen levels also appear to influence the size and stability of thrombi [21] and platelet aggregability [22]. In fact, the two polarized views on fibrinogen (as either reflecting only inflammation of the vessel wall or as indicating causal significance) miss the point that there may be validity in both. Thus, fibrinogen levels may indeed be raised genetically or in response to © 2014 International Society on Thrombosis and Haemostasis
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the inflammatory properties of vessel wall pathology, but, once elevated, they may contribute causally to the onset of clinical events through several pathways. Only one other epidemiologic study has reported an association between high FVIIc levels and CHD [23], while others having failed to demonstrate any relationship [24–27]. However, a variety of FVIIc assays were used in these other studies, and differences between their methods have earlier [28] and more recently [29] been pointed out, with the conclusion that the FVIIc assay used in NPHS was probably more sensitive than others to FVIIa. Fibrinogen is probably not affected to any marked degree by dietary constituents, but its level is clearly increased by smoking. FVIIc is not influenced by smoking, but rises rapidly and substantially in response to an increase in dietary fat [30]. In general, however, the associations of the two clotting factors with other known risk factors for thrombotic events tend to strengthen the case for their causal involvement in CHD [31]. (The NPHS has also reported an association between the FVIII complex and CHD [8]. This report also presented data on ABO blood groups. Similar results on FVIII were obtained by Rumley et al. [32] and Biere-Rafi et al. [33]). Coagulation and thrombosis The contribution of thrombosis to the vessel wall lesion was shown by Duguid many years ago [34], and subsequently by Smith [35] and Falk [36]. Since then, numerous experimental studies have also demonstrated that increased hemostatic activity drives not only thrombus formation, but also plaque development [4]. This effect comprises stimulation of inflammatory responses, cell proliferation and angiogenesis mediated by platelets, leukocytes, and coagulation proteases [37–39]. Platelets, interacting with leukocytes and releasing microparticles, but also soluble mediators such as cytokines and CD40L, act in a proatherogenic manner. Different serine proteases, including FXa and thrombin, act on vessel wall cells through protease-activated receptors (PARs) to stimulate different cellular functions [4]. TF is another important cellular receptor that, upon binding of FVIIc, also stimulates proinflammatory mechanisms, in part via PAR-2, and in part via PAR-independent pathways [40]. The net effect of these mechanisms probably depends on the balance between procoagulant and anticoagulant forces, which may, in turn, result from other vascular risk factors (such as hypertension or metabolic syndromerelated mediators) affecting the vessel wall. A proinflammatory environment leads to ‘perturbation’ of the vascular endothelium, although the response may be quite heterogeneous, depending on the vascular bed [41]. This endothelial response includes many features that stimulate proinflammatory mechanisms, such as increased leukocyte adhesion and permeation. It also involves the downregulation of receptors that are important in anticoagulant defence, i.e. © 2014 International Society on Thrombosis and Haemostasis
thrombomodulin (TM) (which binds thrombin) and endothelial protein C receptor (EPCR) [42]. It has been demonstrated that cell receptor occupancy is one of the critical factors determining the switch in the effects of thrombin from anticoagulant (TM/EPCR-mediated generation of activated protein C) to proinflammatory action, mediated through PAR-1 [43–45]. At the same time, the interplay of environmental inflammatory factors may direct the effects of FVIIc–TF signaling towards proinflammatory actions, depending on the availability of TF-inhibiting forces (TF pathway inhibitor [TFPI]) and cellular receptors that act in concert with FVIIc–TF, including PAR-2 [46]. Hence, the biological actions of serine proteases, including FVIIc and thrombin, depend not only on their systemic plasma concentrations and activity, but perhaps more importantly on whether the vascular milieu facilitates specific interactions with the vessel wall (endothelium). Thus, local concentrations and interactions are critical in determining the local responses of the vascular endothelium, for which the temporary presence of even minute amounts of a free enzyme such as FVIIc, engaging in TF-mediated reactions, may be sufficient. This conclusion is important, because the presence of free enzymes able to interact with cellular receptors would be almost impossible if FVIIc, FXa and thrombin were generated only by a systemic process that is quickly tuned down by a panel of natural inhibitors such as antithrombin and TFPI. Other intriguing molecules in this context are fibrinogen and its derivative fibrin, which constitute the final element in the coagulation cascade and a critical building block of the fibrin clot. Fibrinogen concentrations in plasma are regulated both by genetic and by acute-phase stimulation in the course of inflammation [47]. Although the contribution to clotting depends primarily on the availability of free thrombin, to convert fibrinogen to fibrin monomers, the actual concentration and type of fibrinogen have an impact on the clotting process. Elevated levels of fibrinogen translate into increased thrombosis in animal models [48,49]. As already indicated, the probable mechanisms involve increased viscosity, increased thrombin-mediated fibrin formation (fibrinogen being an important determinant of thrombin generation in plasma), denser clot structure, and resistance to lysis. In addition, the fibrinogen c0 /fibrinogen ratio is a determinant of fibrin clotting properties, and is related to cardiovascular risk factors such as C-reactive protein. As for FVIIc, there is either no or uncertain evidence of haplotype associations with CHD [50]. In addition, fibrinogen, fibrin and fibrin cleavage products have been detected in immunohistochemical studies of human atherosclerotic lesions [35]. This fibrin formation probably results from local thrombin activity [51], and fibrin cleavage from the action of local fibrinolytic proteases (plasmin) and leukocyte-released enzymes such as elastase. Thus, fibrin cleavage products may also result from increased local inflammatory actions. Whereas fibrin
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and the cleavage fragments do not activate PARs, many in vitro studies have shown that fibrin degradation products are important mediators of inflammation, cell migration, and proliferation [52,53]. In humans, however, the contribution of hemostasis to atherosclerosis is still disputed, usually because of the lack of obvious protection against atherosclerosis in congenitally deficient subjects, such as patients with hemophilia [54]. Although the absence of one specific clotting factor may not provide sufficient protection against a complex disorder such as atherosclerosis, the opposite phenotype, hypercoagulability, is linked to atherosclerosis and atherothrombosis. One argument in favor of the latter is that, in a large metaanalysis, FV Leiden and prothrombin 20210 carriage were associated with a modest, but statistically significant, elevations in CHD [55]. Second, several studies have indicated that biomarkers of activated coagulation and fibrinolysis (including D-dimer fragment) are linked to CHD [25,56–59]. A third argument is that both immunohistochemical and functional studies have linked coagulation activity to atherosclerotic plaque instability. Indeed, an increased TF level in plaques correlates with features of instability [60,61]. Increased coagulation protease activity is linked to early atherogenesis, whereas diminishing coagulation activity has been noted in areas of advanced, complex plaque (within the same individuals) [51], suggesting a mechanistic contribution of coagulation proteases in human atherogenesis and atherothrombosis. Finally, a biological link is also suggested by a limited number of studies showing correlations between markers of hypercoagulability and measures of atherosclerosis, i.e. intima– media thickness, or levels of thrombin–antithrombin complexes in plasma linked to coronary calcification [62–64]. The contribution of coagulation activity of the plaque in triggering atherothrombosis is not disputed. Plaque erosion or rupture exposes platelet-activating proteins such as collagen and TF to blood, which is sufficient to start thrombosis in an ordered sequence [65]. Collagen plays a double role, linking platelet activation to FXII activation, which may play a role in stabilization of the arterial thrombus, making it more resistant to clot lysis [66,67]. Thus initiated, coagulation may culminate in an atherothrombotic lesion, which may become a nidus for subsequent clot formation, precipitating symptomatic atherothrombosis, and causing MI or SCD. In the Thrombosis Prevention Trial (TPT) [68], lowintensity warfarin (International Normalized Ratio of 1.5) significantly reduced only fatal CHD events, an effect in line with the observation that FVIIc is associated with fatal but not non-fatal CHD in the NPHS. It may not, of course, have been FVIIc rather than one of the other vitamin K-dependent factors, or a combination, that was responsible for the reduction in events, but it is worth drawing attention to the similarity of the results for FVIIc only for fatal CHD in both the NPHS and the TPT.
In summary, the coagulation system contributes to the pathologic changes leading to MI/CHD, and FVIIc and fibrinogen are involved in these. Conclusion On the basis of experimental and epidemiologic studies, a reappraisal of the importance of the hemostatic system, particularly coagulation, in the processes of atherosclerosis and atherothrombosis is justified. The NPHS and other studies have clearly indicated that high fibrinogen levels are associated with increased risks of CHD, and the same cannot be ruled out for FVIIc. Although part of the association for fibrinogen may indeed reflect the underlying effect of chronic inflammation, causal contributions are also likely. Recent clinical observational data and several experimental studies have clearly indicated that hypercoagulability, which is dependent, among other influences, on the concentrations of FVIIc and fibrinogen (in terms of thrombin generation capacity), is associated with atherosclerosis. In contrast, hypocoagulability protects against atherosclerosis in mice. Importantly, the effects of fibrinogen and serine proteases such as FVIIc may depend significantly on local concentrations and the availability of cellular receptors. Moreover, hypercoagulability is linked to plaque phenotype [51], with effects that support either plaque stability or instability, depending on the plaque stage and the age of the animal, respectively [69,70]. These effects may well be clinically relevant, as three experimental studies have shown that the thrombin inhibitor dabigatran substantially attenuates atherosclerosis in apoE-/- mice [70–72]. Although these observations in mouse models are obviously not directly translatable to humans, even minute effects in humans may turn out to be relevant upon prolonged exposure to anticoagulants. For warfarin, it took some time to realize that prolonged exposure accelerates arterial calcification through inhibition of Gla-dependent proteins in the vasculature [73]. It is therefore not unthinkable that longterm exposure of patients to new oral anticoagulants may also affect atherogenesis and/or plaque phenotype, but it will be some time before the necessary data become available. It is fascinating to observe that the original epidemiologic findings suggesting a causal effect of clotting proteins on CHD now appear to find support in mechanisms linking coagulation activity with the complex pathophysiology of atherosclerosis and atherothrombosis. This creates new avenues for clinical research to unravel the arterial coagulation–inflammation axis and to establish the consequences of long-term anticoagulation in subjects at risk of atherosclerosis. Disclosure of Conflict of Interests The authors state that they have no conflict of interest. © 2014 International Society on Thrombosis and Haemostasis
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