Haemostatic function, arterial disease and the prevention of arterial thrombosis

BaillieÁre's Clinical Haematology Vol. 12, No. 3, pp. 577±599, 1999

14 Haemostatic function, arterial disease and the prevention of arterial thrombosis P. K. MacCallum*

MD, MRCP, MRCPath

Senior Lecturer in Haematology MRC Epidemiology and Medical Care Unit, and Department of Haematology, St Bartholomew's and The Royal London School of Medicine and Dentistry, London, UK

T. W. Meade

CBE, DM, FRCP, FRS

Director MRC Epidemiology and Medical Care Unit, St. Bartholemew's and The Royal London School of Medicine and Dentistry, London, UK

Recent years have seen the expansion of information linking raised plasma levels of individual clotting factors and evidence of disturbances of ®brinolytic activity with the risk of thrombotic manifestations of arterial disease, both in community-based, apparently healthy populations and in patients with known atherosclerosis. Some of these prothrombotic changes in the haemostatic system may result partly from underlying chronic in¯ammation or acute infection and may, in turn, contribute substantially to the thrombotic risk which accompanies these underlying processes. The importance of the coagulation system in the pathogenesis of arterial thrombosis is further illustrated by the bene®t in the Thrombosis Prevention Trial of lowintensity, dose-adjusted warfarin in the primary prevention of ischaemic heart disease. Clinical trials of beza®brate, which is being used for its ®brinogen-lowering as well as lipid-modifying properties, are in progress. Key words: ischaemic heart disease; myocardial infarction; risk factors; thrombosis; ®brinogen; ®brinolysis; clotting factors.

It is now generally accepted that the majority of acute coronary syndromes (sudden coronary death, acute myocardial infarction and unstable angina) are caused by thrombosis secondary to atheromatous plaque disruption.1,2 About three-quarters of strokes are also thrombotic in origin. There is increasing evidence that levels of individual components of the haemostatic system are associated with the risk of these events. Besides the clinical manifestations of thrombosis, the contribution of platelets and of the coagulation system to the progression of atheroma itself is increasingly recognized with regard to ischaemic heart disease (IHD) and lower extremity arterial *Address for correspondence: Dr P. K. MacCallum, MRC Epidemiology and Medical Care Unit, Wolfson Institute of Preventive Medicine, St Bartholomew's and The Royal London School of Medicine and Dentistry, Charterhouse Square, London EC1M 6BQ, UK. 1521±6926/99/030577+23 $12.00/00

c 1999 Harcourt Publishers Ltd. *

578 P. K. MacCallum and T. W. Meade

disease (LEAD). There are obvious and potentially substantial implications of this growing body of knowledge for identifying those whose risk of clinically manifest arterial disease may be due to an increased thrombotic tendency and for the way in which the risk of arterial disease is investigated and managed. The data are broadly consistent with the emerging understanding of atherosclerosis as a chronic in¯ammatory process3, which itself may contribute to systemic prothrombotic changes, thereby increasing the likelihood of future thrombotic events. On top of this chronic process, acute infections may add to risk in the shorter term4, possibly in part as a result of transient prothrombotic disturbances. In keeping with the role of in¯ammation, prospective data are emerging on other new systemic predictors of risk, such as C-reactive protein (CRP) and soluble intercellular adhesion molecules.5±7 Much work is clearly needed to distinguish markers of biological importance to clinical events or disease progression from those which merely re¯ect the presence of underlying in¯ammation, although the latter may contribute to changes in the former. The results of primary prevention trials that modify platelet function or reduce activity of the coagulation system8,9 demonstrate the independent contribution that the haemostatic system makes to arterial thrombosis. This further supports the view that many of the pathogenic factors involved in atherothrombosis, such as in¯ammation, endothelial dysfunction and the cluster of features which make up the insulin resistance syndrome, may in¯uence components of the haemostatic system which, in turn, may contribute to thrombotic risk. There is no doubt as to the central role of platelets in thrombosis and IHD. So far, however, there is no generally accepted and validated index of platelet function that has been correlated with the onset of clinically manifest arterial disease, although spontaneous platelet aggregation10 and large platelet volume11 are associated with increased rates of the recurrence of IHD in those who have already experienced events. Platelet receptor polymorphisms are being actively investigated for associations with IHD in di€erent populations and may provide further insights into the prothrombotic contribution of platelets. However, studies of the coagulation system have so far been considerably more rewarding, and it is worth bearing in mind that components of this system in¯uence platelet behaviour. Thus, thrombin is a potent platelet aggregating agent and ®brinogen enhances the rate of ADP-induced platelet aggregation.12,13 So the coagulation system a€ects platelet function as well as being responsible for the more familiar conversion of ®brinogen to ®brin. This chapter summarizes prospective studies of individual components of the haemostatic system in IHD, draws attention to their relations with stroke and LEAD (particularly in the case of ®brinogen) and outlines recent trials in the primary and secondary prevention of arterial disease with antithrombotic therapy. Because of the possible e€ects of clinical events on clotting factor levels, prevalence or cross-sectional studies comparing those who have or have not previously had episodes are referred to only where prospective data are not available.

CLOTTING FACTORS AND CLINICALLY MANIFEST DISEASE Fibrinogen The Northwick Park Heart Study (NPHS) was the ®rst to report the association between high ®brinogen levels and IHD14±16, as shown in Figure 1. NPHS also showed that the association persists in the longer term, the mean follow-up being 16 years,

Haemostatic function 579

Figure 1. Standardized regression e€ects (SRE), 95% con®dence intervals and P values for relations of variables with ischaemic heart disease incidence in the Northwick Park Heart Study.16 SRE is the di€erence in IHD risk associated with a di€erence of 1 standard deviation in the variable, i.e. high ®brinogen and cholesterol levels and low ®brinolytic activity.

although the strength of the association may be greater for events in the short term.14,16 Subsequent studies have consistently con®rmed the initial report, and two recent meta-analyses of prospective studies have been published.17,18 In 18 studies included in the ®rst of these, in which there were 4018 cases of IHD, those in the top tertile of ®brinogen had a risk ratio of 1.8 (95% con®dence interval 1.6±2.0) compared to the bottom tertile.17 The risk ratio was similar in the 12 studies of ®rst events and six studies of individuals with previously diagnosed vascular disease (Figure 2). The cause and e€ect nature of the association of ®brinogen and IHD is supported by the strength and consistency of the association and the plausibility of the mechanisms through which the association could be mediated (e.g. increased ®brin formation, platelet aggregation, plasma viscosity, contribution to atherosclerosis). The second metaanalysis essentially came to a similar conclusion.18 A recent nested case±control analysis from the Physicians' Health Study and a cohort study in elderly males further support the strength and independence of the association.19,20 Combinations of high ®brinogen and classical cardiovascular risk factors may be particularly important. For example, three of the prospective studies21±23 suggest that risk may be particularly high in those with the combination of raised ®brinogen and raised blood pressure levels. In the Prospective Cardiovascular Munster (PROCAM) study24, those with both low-density lipoprotein (LDL) cholesterol and ®brinogen levels in the high thirds of the distribution had ®ve times the incidence of IHD compared with those whose levels of both variables were in the low thirds of their distributions. The ®brinogen level also

580 P. K. MacCallum and T. W. Meade

Figure 2. Prospective studies of ®brinogen and coronary heart disease. Risk ratios compare top and bottom thirds of baseline measurements. Shaded squares indicate the risk ratio in each study, with the square size proportional to the number of cases and the horizontal lines representing the 99% con®dence intervals (CI). The combined risk ratio and its 95% CI are indicated by unshaded diamonds for subtotals and by shaded diamonds for grand totals. NR indicates not reported; ‡ ˆ adjustment for age and sex only; ‡‡ ˆ for these plus smoking; ‡‡‡ ˆ for these plus some other standard vascular risk factors; ‡‡‡‡ ˆ for these plus markers of social class; and ‡‡‡‡‡ ˆ for these plus information on chronic disease at baseline. c 1999 Reproduced from Danesh et al (1998, Journal of the American Medical Association 279: 1477±1482, * American Medical Association) with permission.

contributed substantially to the risk of future events in those with elevated serum total cholesterol in the European Concerted Action on Thrombosis (ECAT) study of patients with angina pectoris.5 However, not all studies have con®rmed such interactions.25 There are fewer published data for women than for men but both the Framingham study26 and the Scottish Heart Health Study25 suggest that ®brinogen is associated with arterial disease in women as well as in men. The Framingham data on relationships of ®brinogen with the onset of IHD, stroke and LEAD are summarized in Table 1. High levels of ®brinogen are also associated with the recurrence or progression of other arterial disease, for example after stroke27 and in patients with intermittent claudication28±30 as shown in Figure 3. Fowkes et al31 found ®brinogen to be the strongest independent predictor of death from IHD in a 1-year follow-up of 617 patients with intermittent claudication. High levels of ®brinogen are also associated with the incidence of graft occlusion following bypass surgery in patients with LEAD32, the relation of ®brinogen with graft occlusion being stronger than the in¯uence of smoking. Some studies have reported that ®brinogen is associated with an increased risk of restenosis following coronary angioplasty and angioplasty of the lower limb33,34, whereas others have not.35

Haemostatic function 581 Table 1. Age-adjusted 18-year incidence of clinical events per 1000 in Framingham study participants aged 47±79 years according to entry ®brinogen levels.

Fibrinogen (mg/dl) 126±264 265±310 311±696

Total cardiovascular disease

Ischaemic heart disease

Stroke

Peripheral arterial disease

Men

Women

Men

Women

Men

Women

Men

Women

411 446 611

245 281 514

273 304 430

112 147 296

101 127 187

113 75 224

69 94 113

41 38 68

Reproduced from Kannel (1992, in Ernst E et al (eds) Fibrinogen: a `New' Cardiovascular Risk Factor, pp 101±109; Vienna: Blackwell-MZV) with permission.

Factor VII activity Prospective data on Factor VII activity, VIIc, and IHD are now available from several studies. In the ®rst Northwick Park Heart Study14±16, based on 183 ®rst IHD events, there was a clear association between high VIIc levels and events, particularly those which occurred within the ®rst 5 years and were fatal rather than non-fatal. The PROCAM study reported signi®cantly higher baseline Factor VII levels in the 130 men experiencing IHD events during an 8-year follow-up period compared with those who remained event-free (P ˆ 0.023).36 The di€erence in VIIc in those with events compared with those remaining event-free was somewhat more pronounced in those experiencing fatal rather than non-fatal events, and this similarity to NPHS is of interest. The association in PROCAM of VIIc with IHD was no longer present after accounting for serum cholesterol and triglyceride, i.e. the association was not independent, but this

Figure 3. Probability of death over 6 years in 333 patients with stable intermittent claudication according to presence or absence of past history of myocardial infarction (MI). Numbers of patients with initial ®brinogen levels between values shown are given in brackets.30

582 P. K. MacCallum and T. W. Meade

does not exclude an important role for VIIc in providing a biological link between lipids and thrombosis. In contrast to these two studies, the Atherosclerosis Risk in Communities (ARIC) study37, the Edinburgh Artery study38 and the Cardiovascular Health Study20 showed no association of Factor VII with coronary events, although in the latter study VIIc was associated with incident angina in men. There are di€erences in the VIIc assay methodologies in the studies mentioned, the NPHS assay being more sensitive to the activated form of Factor VII than those used in PROCAM or ARIC.39 The Edinburgh Artery study used a chromogenic assay.38 However, it is unlikely that this is the sole explanation for the di€erent ®ndings in the studies because an association between VIIc and IHD is not found in the second Northwick Park Heart Study, which used methodology similar to that used in the ®rst Northwick Park study (Miller GJ, personal communication). Case±control and crosssectional data are also con¯icting, associations with prevalent cardiovascular disease being seen in some studies40,41 but not others.42,43 In one case±control study, Factor VII antigen was elevated in young males with previous myocardial infarction whereas activated Factor VII (VIIa) was not44, while in another recent study higher levels of VIIc were found in survivors of myocardial infarction than in controls.45 Clearly, further work is needed to clarify the relationship between Factor VII and IHD, which may be a€ected by the background prevalence of atherosclerosis within the population studied as well as the reduction which has been seen over the years in case-fatality rates. Factor VIII and von Willebrand Factor There are now a number of prospective studies evaluating the association between Factor VIII activity (VIIIc) and/or von Willebrand Factor (vWF) and risk of IHD, both in the general population and in patients with known cardiovascular disease. Sixteen-year follow-up of NPHS, by which time 178 ®rst major episodes of IHD had occurred in 1393 men, showed that baseline VIIIc was associated with IHD incidence.46 As for Factor VIIc, the data suggested a stronger association for fatal than for non-fatal events, although, overall, the association was not as strong as for ®brinogen. The association of VIIIc with IHD was independent of blood group, which is relevant given the considerable di€erences in VIIIc between blood groups and the possibility (observed in NPHS) that blood group also in¯uences the risk of IHD. On adjusting VIIIc for age and blood group, the standardized regression e€ect (i.e. the change in risk associated with a 1 standard deviation [s.d.] rise in the variable) for its association with fatal IHD in NPHS was 1.28 (P ˆ 0.02), for non-fatal events it was 1.13 (P ˆ 0.28) and for all IHD events it was 1.18 (P ˆ 0.05). The ARIC study follow-up at 5 years also showed VIIIc to be associated with IHD events, although this was signi®cant only in females.37 The signi®cance was lost after adjustment for other risk factors, consistent with other risk factors either confounding or partly operating through their e€ects on haemostatic variables such as VIIIc. In keeping with a role for VIIIc are reports of lower than expected IHD incidence in haemophiliac patients47,48 as illustrated in Table 2. Some caution is necessary in interpreting the ®ndings because the high mortality of haemophilic patients from other causes introduces the concept of competing risks as a possible reason for their lower IHD mortality. However, the very considerable reduction in IHD in the Dutch study, coupled with the NPHS and ARIC ®ndings, does indicate a possible contribution of Factor VIII to IHD. In view of autopsy studies showing that haemophilia does not prevent the development of atheroma, it may be an antithrombotic e€ect of low VIIIc levels resulting in reduced ®brin formation that explains the apparent protection of haemophiliac patients against IHD.47 Moreover, a

Haemostatic function 583 Table 2. Ratios of observed to expected deaths in Dutch haemophilic patients. Neoplasm Lung cancer Accidents Renal failure Stroke Ischaemic heart disease

2.5 8.6 2.6 30.0 5.0 0.2

Reproduced from Rosendaal et al (1989, British Journal of Haematology 71: 71±76) with permission.

further recent analysis from The Netherlands suggests that carriers of haemophilia also have a reduced standardized mortality rate49 in keeping with protection against cardiovascular disease by low levels of VIIIc (or IXc). The ®nding in the Leiden Thrombophilia Study of an association of elevated levels of VIIIc with risk of a ®rst venous thrombosis50 further supports a prothrombotic role for Factor VIII because vessel wall disease is not a consideration and, therefore, this association indicates a contribution from the circulating blood. Factor VIII circulates in a complex with von Willebrand Factor (vWF). The two proteins serve di€erent haemostatic functions and have di€erent sites of production but statistically they are closely associated and it is likely that independent contributions of VIIIc and vWF to IHD events would be dicult to demonstrate. It is not therefore surprising that prospective studies of vWF also show associations with IHD. In NPHS, as for VIIIc, there was an association of high vWF levels with increased risk, the strength of the association being similar to that based on VIIIc.46 There is therefore no reason to believe that vWF is a better marker of vessel wall damage than VIIIc, at any rate in their long-term relations with IHD in previously una€ected men. A high VIIIc level may be a marker of endothelial cell dysfunction and vascular damage, although once increased as a result it may both aggravate underlying vessel wall disease and the risk of IHD by enhancing procoagulant activity. In the Edinburgh Artery study in which 268 new vascular events occurred over 5 years in a population cohort of 1592 men and women aged 55 to 74 years, vWF was associated with risk of both myocardial infarction and stroke, although this association was not seen after adjustment for cardiovascular risk factors and baseline disease.38 The ARIC study also reported that vWF was associated with increased risk of IHD in both men and women, although, again, the association was not seen after adjustment for other risk factors.37 In a prospective study from Sweden using a nested case±control design with 78 cases of a ®rst myocardial infarction and 156 matched controls, increasing baseline levels of vWF were associated with risk of myocardial infarction.51 vWF was an independent predictor of subsequent acute coronary risk with a relative risk of 1.24 for each s.d. increase in vWF (P ˆ 0.05) in the ECAT study in which 106 de®nite coronary events occurred in 2960 patients with known angina pectoris followed up for 2 years.5 In a study of survivors of myocardial infarction by Jansson et al52, a high concentration of vWF was associated with an increased risk of cardiovascular death after adjusting for age and hypertension. There are fewer data for cerebrovascular disease, but in a study of 208 cases of stroke categorized by CT scan, vWF levels were associated with risk of death over the ensuing 6 months even after adjustment for stroke type.53

584 P. K. MacCallum and T. W. Meade

Fibrinolytic activity A number of prospective studies, both of ®rst events and in patients with known IHD, have now been reported which lead to the conclusion that disturbances of the ®brinolytic system are associated with IHD. Di€erent studies have used di€erent laboratory methodologies, including global tests of ®brinolytic activity (FA), assays of speci®c components of FA and measurement of the degradation products of ®brinolysis, particularly D-dimer. The Northwick Park Heart Study reported that low FA was associated with increased risk of IHD both in males, particularly under the age of 55 years, and in females.16,54 NPHS used the dilute blood clot lysis time (DCLT) as a `global' measure of ®brinolytic activity. It has been postulated that FA as measured by the DCLT in NPHS may be no more than an indirect measure of the plasma ®brinogen concentration, a high ®brinogen concentration leading to a larger clot which takes longer to be lysed. However, in NPHS the e€ects of low FA and ®brinogen were independent and the strong association between low FA and IHD in younger men persisted after taking account of ®brinogen. Moreover, a recent community-based study showed that the main determinants of the DCLT were plasminogen activator inhibitor type 1 (PAI-1) activity and, in males, tissue-type plasminogen activator (t-PA) activity, whereas ®brinogen did not make an independent contribution.55 This con®rms earlier work showing that ®brinogen did not a€ect DCLT.56 In contrast to NPHS, the Goteborg study in men born in 1913 and aged 54 years at entry to the study reported that ®brinolytic activity in the euglobulin fraction measured by a ®brin plate method was not related to the incidence of IHD.21 Other prospective studies of community-based participants have reported associations of individual components of ®brinolysis with arterial disease. For example, the Physicians' Health Study found associations of t-PA antigen with both IHD and stroke.57,58 In the Edinburgh Artery study in 1592 middle-aged males and females in whom there were 268 new vascular events over 5 years follow-up, t-PA antigen was associated with risk of both IHD and stroke.38 In the prospective population-based study of Thogersen et al51 referred to earlier, elevated levels of PAI-1 and t-PA antigen were associated with risk of development of a ®rst myocardial infarction in both males and females. In the Caerphilly study in 1998 middle-aged males in whom there were 129 major IHD events at mean follow-up of 61 months, t-PA antigen, but not PAI-1 antigen, was associated with risk of IHD.59 Increased levels of D-dimer, indicating increased ®brin turnover, have been found to be predictive of future cardiovascular events in healthy males in the Physicians' Health Study and the Caerphilly study.59,60 In the Cardiovascular Health Study, in which 146 of 5201 males and females 565 years developed myocardial infarction, angina or coronary death during 2.4 years follow-up, elevated D-dimer levels were associated with increased risk of myocardial infarction or coronary death but not angina.61 This risk was independent of ®brinogen and C-reactive protein. The D-dimer ®ndings were supported by very similar results for plasmin-antiplasmin complex (PAP), a measure of recent plasmin production formed by the inactivation of plasmin by a2-antiplasmin and indicating increased ®brinolysis. In contrast, in this elderly population, there was no association of PAI-1 antigen with IHD events.61 Associations of ®brinolytic changes with further events have been described in patients with known IHD. In a study of 49 survivors of acute myocardial infarction, Factor XII-dependent euglobulin lysis was depressed in the 10 who experienced recurrence.62 Hamsten et al63 reported that high concentrations of PAI-1 were

Haemostatic function 585

associated with an increased risk of recurrence in 109 young survivors of myocardial infarction. The PLAT study in patients with atherosclerosis also showed that high PAI-1 activity predicted future IHD events.64 Jansson et al65 reported high levels of t-PA antigen in the 47 out of 213 patients with severe angina pectoris who later experienced myocardial infarction, stroke or cardiovascular death. In a 10-year follow-up study of 123 patients with acute myocardial infarction, during which time there were 57 deaths, 54 from cardiovascular disease, t-PA antigen was associated with cardiovascular mortality independent of age and hypertension.52 In the ECAT study referred to above, in patients with angina pectoris, coronary events were associated with higher baseline levels of both t-PA antigen and PAI-1 antigen and activity.66 After adjustment for non-haemostatic risk factors, t-PA antigen remained independently predictive of IHD events.5 D-dimer has also been reported to be predictive of future cardiovascular events in patients with peripheral arterial disease and atherosclerosis.31,64 In a recent study of 1045 survivors of an index myocardial infarction in whom there were 81 further coronary events over 26 months of follow-up, D-dimer was an independent and uniquely strong predictor of recurrent coronary events.67 On balance these studies demonstrate clear, and in some cases independent, associations of indicators of FA with IHD events which continue to raise a number of interesting points. One is the obvious contrast between studies suggesting that low FA is associated with an increased risk of IHD and those suggesting the reverse. It is in general results based on direct measures of ®brinolytic inhibitors such as PAI-1 or on methods that re¯ect inhibition (including the NPHS method) as well as activation that suggest an association between low FA and IHD. High t-PA antigen levels on the other hand probably indicate high levels of t-PA/PAI-1 complex68 in which the PAI-1 component is of chief functional signi®cance. Thus, t-PA antigen is correlated inversely with t-PA activity and positively with PAI-1.55,69 It is therefore likely that the association of raised t-PA antigen with IHD reported by several of the studies outlined above at least partly re¯ects high PAI-1 levels. However, the situation is complicated by apparent di€erences in the aetiological roles of PAI-1 and t-PA antigen. Analysis of data from the ECAT trial in patients with angina pectoris suggests that the associations of PAI-1 activity and antigen with IHD events are not seen after adjustment for features of insulin resistance (body mass index, triglyceride, HDL cholesterol, blood pressure and diabetes), and therefore that the prognostic role of PAI-1 is related principally to insulin resistance, at least in this patient group.66 In contrast, t-PA antigen was a€ected to a similar degree by adjustment for variables which re¯ected insulin resistance, in¯ammation (®brinogen, CRP) and endothelial cell damage (vWF), suggesting that it is more widely in¯uenced than PAI-1 by a variety of pathophysiological pathways.66 The positive association with IHD of D-dimer and PAP, indicating increased ®brinolytic activity, seen in some studies where no association was found for PAI-159,61 suggests that di€erent assays may measure di€erent aspects of ®brinolysis whose importance may vary in di€erent populations with di€erent degrees of prevalent atherosclerosis. Of possible relevance is a tendency in the early crosssectional studies and in NPHS for the relation between low FA and IHD to be evident mainly in younger men, an observation that might explain the absence of any association in other studies whose participants were older. Low FA is not, of course, the only characteristic to show a stronger association with IHD in younger than in older men. Both cigarette smoking and cholesterol levels also show this contrast.70,71 In addition to an association with thrombosis, it has been suggested that elevated PAI-1 in evolving atheroma may predispose to plaque rupture through inhibition of vascular smooth muscle cell migration and proliferation.72 It is also important to bear in mind

586 P. K. MacCallum and T. W. Meade

the considerable within-person variability in FA and its components. Statistically independent associations of these variables with IHD may not always be demonstrable, particularly in the multiple regression techniques commonly used nowadays as methods of ®rst rather than last analysis and which may well obscure the e€ects of some variables on others in an aetiologically important pathway. What are clearly needed are further detailed prospective studies in di€erent populations in which both global tests and assays of individual components of ®brinolysis are performed. Other haemostatic proteins There are of course other potential candidates within the haemostatic system which may be associated with IHD but for which the available data are less extensive. For example, thrombomodulin is an endothelial receptor which binds thrombin thereby enabling it to activate Protein C. Although an integral membrane protein, circulating thrombomodulin can be detected in plasma and raised levels are found in disorders which a€ect the microvascular bed.73 The ARIC study has recently reported an inverse association of thrombomodulin with incident IHD, with an odds ratio of 0.29 (P ˆ 0.005) for the highest compared to the lowest quintile.74 The relationship of thrombomodulin with IHD was in¯uenced by VIIIc with a greater reduction in risk at higher VIIIc levels. In contrast, a small prospective study in 54 survivors of myocardial infarction showed that soluble thrombomodulin levels were positively rather than inversely associated with risk of a recurrent cardiovascular event during 49 months of follow-up.75 A key goal of investigation in the next few years will be to integrate the di€erent components of haemostasis into an overall system which links conventional and novel risk factors with background atherosclerosis and with clinical outcome. This may be complicated by the varying functions di€erent haemostatic proteins play depending on their particular location and, as recently summarized by Rosenberg and Aird76, by di€erences in the nature of the haemostatic balance in di€erent vascular beds. THROMBOGENIC MECHANISMS The hypothesis underlying much of the contemporary work on coagulation and IHD is that high clotting factor levels would increase the incidence of IHD by predisposing to thrombosis. However, even if (for example) the undoubted association of high ®brinogen levels with IHD risk may now be useful in a purely predictive sense, association does not of itself prove causation, and other sorts of evidence have to be taken into account. Good evidence is often provided by randomized controlled trials, providing as they do unbiased assessments of the consequences of modifying pathways that may be involved in the onset of IHD, particularly when the main outcome is the onset of clinical disease (rather than intermediate or surrogate measures such as changes in platelet sensitivity or in clotting factor levels, relevant though these outcomes also are). Indeed, the value of trials as a means of understanding pathogenesis is still too often overlooked, largely because the main interest understandably centres on their clinical implications. However, attributing any bene®ts of antithrombotic regimens to the rationale for their use in a trial is not always straightforward because many agents have more than one e€ect. An example is provided by some of the ®brates, which not only lower plasma ®brinogen levels but also exert what would be considered bene®cial

Haemostatic function 587

in¯uences on lipid levels, i.e. lowering total cholesterol, LDL cholesterol and triglyceride levels while raising HDL cholesterol levels. In both of the Medical Research Council's hypertension trials77,78 it appeared that thiazide diuretics conferred bene®ts over and above those attributable to their blood pressure lowering e€ect, although the additional pathway or pathways involved were unidenti®ed. The possibility that an agent operates through more than one pathway is of limited clinical interest but is clearly of the greatest relevance if a trial's results are being used for an understanding of pathogenesis. Against this general background, the results of trials of anticoagulants, of aspirin and of ®brinogen-lowering agents form an important part of the evidence for or against a role for high clotting factor levels or platelet sensitivity in the onset of IHD. Oral anticoagulants The confusion that followed the early trials of oral anticoagulants following myocardial infarction has been replaced by widespread recognition of their undoubted value in this setting.79±81 More recently the demonstration in the Thrombosis Prevention Trial (TPT), outlined more fully below, that lower than conventional intensities of oral anticoagulation are e€ective in the primary prevention of IHD, particularly fatal IHD, is powerful evidence that levels of vitamin K-dependent clotting factors, and therefore the ability to generate thrombin, in¯uence the risk of clinically important thrombogenesis. Aspirin The evidence that aspirin reduces the incidence of major vascular events in those with previous or developing clinical episodes of arterial disease is now overwhelming.8 The clear bene®t of other antiplatelet agents, such as clopidogrel82, in the prevention of thrombotic events further supports the platelet contribution to arterial thrombosis. With regard to primary prevention, in a more recent analysis from the Physicians' Health Study the bene®t of aspirin was greater in those with higher baseline CRP levels (though still in the normal range), which is of interest bearing in mind the antiin¯ammatory activity of aspirin6, although this is regarded as an e€ect usually seen only at higher doses than the alternate day regimen of 325 mg. A puzzle, however, remains as to the rather uncertain value of aspirin in primary prevention (i.e. the prevention of initial rather than recurrent episodes) by contrast with its unequivocal value in secondary prevention, particularly with regard to fatal events. Fibrinogen-lowering agents The strong and independent relation of high ®brinogen levels with the onset and progression of clinical disease at each of the three major arterial sites ± coronary, cerebral and peripheral ± is now being followed up in several trials with clinical (as distinct from biochemical) end-points. Beza®brate reduces plasma ®brinogen levels by about 15% and also has what would be regarded as bene®cial e€ects on lipid levels. Three randomized controlled trials using beza®brate have been carried out or are in progress. The Beza®brate Coronary Atherosclerosis Intervention Trial (BECAIT) in dyslipidaemic young male survivors of myocardial infarction showed that beza®brate reduced further coronary events, slowed progression of focal coronary atherosclerosis, lowered plasma ®brinogen and improved dyslipidaemia.83 The Beza®brate Infarction

588 P. K. MacCallum and T. W. Meade

Prevention (BIP) trial using beza®brate for the secondary prevention of myocardial infarction is completed and the ®nal results are awaited.84 The third trial, the Lower Extremity Arterial Disease Event Reduction (LEADER) trial, for the prevention of major arterial events in patients with lower extremity arterial disease, is in progress. Gem®brozil, the clinical value of which was demonstrated in the Helsinki Heart Project85, leads to raised rather than reduced ®brinogen levels, an e€ect that is clearly more than o€set by its lipid-modifying properties and by the lower plasma level of thrombin it also induces.86 The need to distinguish between the ®brinogen-lowering and lipid-modifying e€ects of beza®brate, for example, has already been noted. If the former explanation for any clinical bene®ts is eventually demonstrated, this will provide good evidence that high ®brinogen levels are of pathogenetic signi®cance. In many ways the situation is similar to that of 20 years ago for cholesterol where the observational studies had to be followed by clinical trials to demonstrate conclusively a cause-and-e€ect relationship. EPIDEMIOLOGY OF CLOTTING FACTORS The causality, or otherwise, of high clotting factor levels in IHD ± or of low ®brinolytic activity ± can also be assessed by the extent to which these levels are associated with characteristics known to in¯uence the risk of IHD. With a high degree of consistency, this approach does suggest that variations in haemostatic variables are involved in the development of IHD. Earlier evidence has been extensively reviewed.87 The recent ®brinogen meta-analysis of Maresca et al18 referred to earlier takes account also of cross-sectional data. Reference has already been made to the importance of in¯ammation in atherothrombosis. A role for speci®c chronic infections in this process, particularly with Chlamydia pneumoniae, Helicobacter pylori or cytomegalovirus, has also been suggested, although the epidemiological associations which have been observed may simply re¯ect confounding by other risk factors rather than a true aetiological role.88 This issue is still not resolved and, for example, it has been suggested that virulent strains of Helicobacter pylori (possessing the cytotoxin-associated gene-A) may be of importance in the risk of IHD89, perhaps because of a greater host in¯ammatory response.90 If these chronic infections do prove to be relevant, the mechanism may in part be through the haemostatic system and perhaps ®brinogen in particular.91 It is interesting to note that in a recent small randomized clinical trial in IHD patients seropositive for Helicobacter pylori or Chlamydia pneumoniae, ®brinogen levels were signi®cantly reduced by a short course of antibiotic therapy.92 Clinical trials of appropriate antibiotic therapy are ongoing. In the ROXIS trial, for example, the macrolide roxithromycin, which has activity against Chlamydia pneumoniae and may also have anti-in¯ammatory properties, reduced the risk of further coronary events for at least 6 months after initial treatment in patients with unstable angina.93 Recent acute infections, too, may be associated with risk of acute myocardial infarction and stroke.4,94 Patients with chronic obstructive pulmonary disease (COPD) or symptoms of chronic bronchitis have an increased risk of IHD which may be mediated through higher levels of ®brinogen.95±97 Figure 4 shows how ®brinogen levels rise acutely following infective exacerbations of COPD, thereby acutely increasing the risk of thrombotic events.98 Infection leads to changes in many acute and chronic phase proteins, only one of which is an increase in ®brinogen, so it does not necessarily follow that infections predispose to IHD or stroke through ®brinogen

Haemostatic function 589

Figure 4. Changes in plasma levels of ®brinogen and interleukin 6 (IL-6) with exacerbation and during convalescence in patients with chronic obstructive pulmonary disease (Wedzicha et al, submitted for publication).

levels. However, if raised ®brinogen levels do partly explain the increase in risk associated with these conditions, they illustrate the importance of not dismissing elevations that may be due to relatively non-speci®c stimuli as biologically insigni®cant. There is no doubt as to the markedly increased mortality from IHD and stroke during the winter months, and there is a signi®cant seasonal variation in plasma ®brinogen concentrations99±101, levels being highest during the coldest months when mortality from both myocardial infarction and stroke are also at their highest levels. It may be that the high ®brinogen, and thus mortality, levels during the colder months are partly explained by respiratory and other infections, which are, of course, also commonest during colder months. This hypothesis receives further support from a study in older patients from whom serial samples were taken during the year along with information on upper respiratory infections.102 Fibrinogen levels were highest when the prevalence of upper respiratory infections reached its peak, as was the case for other risk factors including cholesterol, blood pressure and VIIc levels (Table 3). While still not conclusive, these ®ndings do suggest that at least part of the excess winter mortality from IHD and stroke may be attributable to high ®brinogen levels associated with infections. The association of diabetes mellitus and, more broadly, of the so-called insulin resistance syndrome with atherosclerotic disease is well recognized and levels of ®brinogen, VIIIc, vWF and PAI-1 are raised in such individuals.103 The observation that Table 3. Clotting factors and symptoms in 96 men and women aged 65±74 years, according to season. Annual mean Fibrinogen (g/l) VIIc (%) Neutrophils (108/l) Cough (% reporting) Coryza (% reporting)

2.83 117.3 4.11 29 13

Seasonal di€erencea Peak month 0.13 4.2 0.35 18 19

Feb Jan Jan Feb Feb

Body mass index, blood pressure and cholesterol levels are also highest Jan±Feb. between highest and lowest mean values by season. Reproduced from Woodhouse et al (1994, Lancet 343: 435±439) with permission.

aDi€erence

P value 0.003 0.02 0.002 0.0005 50.0001

590 P. K. MacCallum and T. W. Meade

there is a signi®cant increase in plasma VIIa, VIIc and VII antigen with increasing urinary albumin excretion in non-insulin dependent diabetic patients104 suggests that the degree of vessel wall damage may in¯uence the onset of IHD through the production of VIIa. Recent cross-sectional data from the PRIME study illustrate the greater strength of the association of insulin resistance with PAI-1 than with ®brinogen.105 For example, characteristics of insulin resistance explained 23% of the variance in PAI-1 compared to 1.5% of the variance in ®brinogen. It has recently been determined that adipose tissue is an important contributor to PAI-1 levels in obese individuals106 and weight loss in obese subjects is associated with reduction in PAI-1.107 Factor XIIa, one of the components of the contact system, is also emerging as a possible risk marker for IHD. Levels have been reported to cluster with features of insulin resistance and to be higher in survivors of acute myocardial infarction than in controls.108 In middle-aged men XIIa is associated with the major conventional risk factors for IHD109 and results of prospective studies are awaited. There are indications from a number of studies that haemostatic variables are associated with subclinical arterial disease as assessed by carotid intima-media thickness (IMT). In the Edinburgh Artery study in which IMT was measured in 1106 men and women aged 60±80 years, IMT in men was associated with ®brinogen and blood and plasma viscosity, although not with t-PA, D-dimer or vWF.110 In the ARIC study111 and the Cardiovascular Health Study112 ®brinogen was also associated in IMT, and in the case of the Cardiovascular Health Study, an association was additionally found with VIIIc. In contrast, much weaker associations have been found in other studies.113

MANAGEMENT AND PREVENTION Antithrombotic medication now forms an important part of the management and prevention of IHD, stroke and other vascular conditions. Pathological evidence leaves little doubt that thrombosis is the ®nal structural event leading to myocardial infarction or sudden coronary death, and this is represented in Figure 5. If this is a fair representation of the chain of events, reducing thrombogenic potential may be a particularly e€ective way of preventing the onset or recurrence of clinical events, whichever the particular risk factors are that contribute to these events in di€erent people. Preventing thrombosis and modifying individual risk factors are not, of course, mutually exclusive (provided due attention is paid to any adverse drug interactions).

Figure 5. Primary prevention of ischaemic heart disease: epidemiological and pathological implications. BP ˆ blood pressure.

Haemostatic function 591

Secondary prevention Antithrombotic treatment in the early stages of myocardial infarction or in angina merges into the longer-term, secondary prevention of further episodes. There is no doubt as to the value of aspirin in the early stages of myocardial infarction, the risk of a further major vascular event (myocardial infarction, stroke or vascular death) being reduced by some 25%.8 Since many patients have more than one clinical manifestation of arterial disease and are at risk of further episodes at sites other than those initially a€ected, it is not only reasonable but necessary to consider the e€ects of antithrombotic agents (especially aspirin) in terms of the bene®ts for further episodes at all major sites and in all clinical groupings. There is also no doubt as to the even greater value of aspirin combined with thrombolytic therapy114 ± in other words, both antiplatelet and ®brin-modifying treatment, considered again later on. The question of the most appropriate dose of aspirin is still not ®nally resolved. Recent evidence again suggests that there is no advantage to doses more than 300 mg daily115 and, within the lower dose range, 75 mg daily may be as e€ective as higher doses.116 However, to establish high blood levels during the early stages of myocardial infarction it is prudent to start with 160±300 mg and then either to maintain these dose levels or to use 75±100 mg daily for longer-term management. Aspirin may reduce non-fatal recurrent episodes of myocardial infarction to a somewhat greater extent than further episodes that are fatal. Because of the predominance of death from vascular causes in those with prior manifestations of arterial disease, total mortality rates are also signi®cantly reduced by about 16% in those taking aspirin.8 The proportional bene®ts of aspirin are similar in older and younger patients, in men and women, in normotensive and hypertensive patients, and in non-diabetic and diabetic patients. Absolute reductions, however, are greater in the higher-risk groups (e.g. patients who are older, hypertensive or diabetic) because of their higher event rates. There is at present little or no formal evidence on how long aspirin should be taken after an initial event, but since those who have already experienced episodes of arterial disease are likely to remain at high risk inde®nitely, antithrombotic treatment should probably also be continued long term. This decision needs to take account of possible side-e€ects, particularly bleeding. There is little doubt that the incidence of gastrointestinal bleeding and of peptic ulceration, as well as of other less serious digestive tract symptoms, is increased in those taking aspirin. This risk is doserelated.117 There is still an appreciable mortality from major gastrointestinal haemorrhage. The possibility also has to be considered that aspirin may slightly increase the risk of cerebral haemorrhage, as has been con®rmed in a recent meta-analysis.118 These hazards by no means outweigh the bene®ts of aspirin in reducing the incidence of major vascular events in high-risk groups, but they do need to be borne in mind. Despite the clear value of aspirin in secondary prevention, the bene®ts of oral anticoagulation in secondary prevention79±81 should not be overlooked, for two main reasons. First, it is possible that conventional intensities of anticoagulation confer slightly greater protection against recurrence than aspirin. Despite the disadvantages of anticoagulation, this extra bene®t (if real) may still be worthwhile for a common condition with a high risk of recurrence, i.e. in absolute terms. Second, the value of oral anticoagulants in modifying ®brin production may add to the value of aspirin in reducing platelet aggregability. The value of combined antithrombotic treatment is well illustrated in the results of the ISIS-2 trial114, in which ®brin modi®cation ± in this instance with streptokinase ± added to the value of aspirin so that event rates in those

592 P. K. MacCallum and T. W. Meade

on the combination were about half those on either active treatment alone (in whom, in turn, rates were signi®cantly lower than those on double placebo treatment). Several early trials using conventional levels of anticoagulation and also conventional aspirin doses and without the bene®t of recent developments in anticoagulant control (reviewed in Meade et al119), had already suggested the potential value of combined treatment, but at the cost of a high rate of bleeding complications. A Canadian trial found that 100 mg aspirin daily reduced thromboembolism and vascular death in patients on conventional doses of warfarin undergoing heart valve surgery, the risk of serious bleeding being only slightly and not signi®cantly greater in those on combined treatment compared with those on warfarin alone.120 Combined aspirin and conventional intensity anticoagulation (heparin followed by warfarin) was also shown to be bene®cial in the setting of acute coronary syndromes.121 In contrast, three recent trials122±124 have cast doubt on the potential bene®t of combined antithrombotic therapy. However, these trials used ®xed or capped low-dose warfarin, and the ®ndings almost certainly re¯ect the need to administer warfarin in a dose-adjusted manner rather than in a ®xed or capped manner. Thus, in the CARS trial122 in patients with a previous MI, warfarin was administered in a ®xed dose of 1 mg or 3 mg daily combined with aspirin 80 mg daily and did not reduce the risk of a major cardiovascular event compared to aspirin 160 mg alone. The mean achieved International Normalized Ratio (INR) on 3 mg was 1.5 at week 1, falling to 1.27 at week 4 and 1.19 at 6 months. In the Post Coronary Artery Bypass Graft Trial123 the dose of warfarin was capped at 4 mg and the mean trial INR was 1.4. This failed to prevent angiographic progression of atherosclerosis in grafts, and no di€erences were apparent in clinical outcome. In phase 1 of the OASIS pilot study124 in patients with unstable angina or non-Q-wave infarction, a 3 mg daily ®xed dose of warfarin in combination with aspirin failed to prevent further ischaemic events compared to standard therapy. Since 1994, the clear success of statin therapy in the secondary prevention of IHD125 has had a major impact on prescribing habits. While statins substantially reduce cholesterol, particularly LDL-cholesterol, there is continuing debate as to their mechanism of prevention of IHD, for example, in terms of cholesterol reduction, plaque stabilization or antithrombotic changes.126 In apparent contrast, hormone replacement therapy (HRT), which observational data suggest is likely to reduce the risk of a ®rst myocardial infarction by 30% or so, has been reported in a recent randomized controlled trial to be of no bene®t in secondary prevention of IHD.127 Indeed there may have been an early increase in recurrent coronary events followed by apparent protection after several years in women receiving HRT compared to placebo, although there is considerable uncertainty about this point because the analysis was not pre-speci®ed and is very data-dependent. HRT also increased the risk of venous thromboembolism, again predominantly at an early stage127, presumably through prothrombotic changes. Thus, the bene®cial e€ects of HRT on many metabolic and haemostatic variables128±131 may have been partly negated by prothrombotic changes, at least in women with established coronary disease. Primary prevention By contrast with the unequivocal value of aspirin in secondary prevention, its value in primary prevention, i.e. in much lower risk circumstances, is unclear, and the Antiplatelet Trialists' Collaboration8 concluded that there was at present no reliable evidence on the balance of risks and bene®ts of antiplatelet therapy in this context. Currently available evidence now comes from four trials, in American and British

Haemostatic function 593

doctors and, more recently, in high-risk middle-aged men in the Thrombosis Prevention Trial (TPT) and in hypertensive patients in the Hypertension Optimal Treatment (HOT) trial.132 In the American doctors, IHD incidence rates were particularly low, almost certainly re¯ecting the selected and health-conscious nature of the trial population. The signi®cant reduction in non-fatal myocardial infarction in the American trial133 contrasts with the absence of any obvious e€ect in the British trial.134 In an overview of both trials135, there is a suggestive although not statistically signi®cant excess of strokes in those taking aspirin, for which an increase in cerebral haemorrhage may be a partial explanation. However, in the HOT trial in patients with hypertension, aspirin 75 mg daily reduced the risk of myocardial infarction compared to placebo without an increase in stroke or fatal bleeds.132 The TPT investigated the balance between bene®ts and hazards of combined antiplatelet and anticoagulant treatment.9 TPT was a randomized trial conducted in 5499 men at high risk of IHD who had not yet experienced clinical manifestations, i.e. with primary prevention in a higher-risk group than those in the American and British doctors' trials. Low-dose aspirin (75 mg daily) and low-intensity oral anticoagulation with warfarin adjusted to a target INR of 1.5 were administered in a factorial design. There were thus four treatment groups: active aspirin and active warfarin, placebo aspirin and active warfarin, active aspirin and placebo warfarin and double placebo treatment. Aspirin was taken in a controlled-release formulation intended to inhibit thromboxane production while sparing prostacyclin. The main intention to treat ®ndings were that aspirin and warfarin both reduced the risk of a ®rst myocardial infarction by about 20%, although aspirin prevented mainly non-fatal and warfarin fatal events. Warfarin signi®cantly reduced all-cause mortality by 17%. The combination of warfarin and aspirin was particularly e€ective, reducing the risk of a ®rst myocardial infarction by 34%. Individually, each active agent signi®cantly increased the risk of minor but not major bleeding. Combined antithrombotic therapy increased the risk of haemorrhagic stroke only in those who were hypertensive. In particular, and in contrast to the Physicians' Health Study, aspirin did not increase the risk of stroke, although it is unclear whether this re¯ects the lower dose of aspirin used in TPT or di€erences in the populations studied. Taking account of these ®ndings, the most recent (5th) ACCP Consensus Conference recommends aspirin for the primary prevention of IHD in highrisk men and combined aspirin and low-intensity, dose-adjusted warfarin in those at very high risk.136

REFERENCES

* * * *

1. Burke AP, Farb A, Malcolm GT et al. Coronary risk factors and plaque morphology in men with coronary disease who died suddenly. New England Journal of Medicine 1997; 336: 1276±1281. 2. Theroux P & Fuster V. Acute coronary syndromes. Unstable angina and non-Q-wave myocardial infarction. Circulation 1998; 97: 1195±1206. 3. Ross R. Atherosclerosis ± an in¯ammatory disease. New England Journal of Medicine 1999; 340: 115±126. 4. Meier CR, Jick SS, Derby LE et al. Acute respiratory-tract infections and risk of ®rst-time acute myocardial infarction. Lancet 1998; 351: 1467±1471. 5. Thompson SG, Kienast J, Pyke SDM et al for the European Concerted Action on Thrombosis Angina Pectoris Study Group. Hemostatic factors and the risk of myocardial infarction or sudden death in patients with angina pectoris. New England Journal of Medicine 1995; 332: 635±641. 6. Ridker PM, Cushman M, Stampfer MJ et al. In¯ammation, aspirin, and the risk of cardiovascular disease in apparently healthy men. New England Journal of Medicine 1997; 336: 973±979.

594 P. K. MacCallum and T. W. Meade 7. Ridker PM, Hennekens CH, Roitman-Johnson B et al. Plasma concentration of soluble intercellular adhesion molecule 1 and risks of future myocardial infarction in apparently healthy men. Lancet 1998; 351: 88±92. 8. Antiplatelet Trialists' Collaboration. Collaborative overview of randomised trials of antiplatelet therapy ± I: prevention of death, myocardial infarction, and stroke by prolonged antiplatelet therapy in various categories of patients. British Medical Journal 1994; 308: 81±106. * 9. Medical Research Council's General Practice Research Framework. Thrombosis prevention trial: randomised trial of low-intensity oral anticoagulation with warfarin and low-dose aspirin in the primary prevention of ischaemic heart disease in men at increased risk. Lancet 1998; 351: 233±241. 10. Trip MD, Cats VM, van Capelle FJL & Vreeken J. Platelet hyperreactivity and prognosis and survivors of myocardial infarction. New England Journal of Medicine 1990; 322: 1549±1554. 11. Martin JF, Bath PM & Burr ML. In¯uence of platelet size on outcome after myocardial infarction. Lancet 1991; 338: 1409±1411. 12. Meade TW, Vickers MV, Thompson SG et al. Epidemiological characteristics of platelet aggregability. British Medical Journal 1985; 290: 428±432. 13. Meade TW, Vickers MV, Thompson SG & Seghatchian MJ. The e€ect of physiological levels of ®brinogen on platelet aggregation. Thrombosis Research 1985; 38: 527±534. 14. Meade TW, North WRS, Chakrabarti R et al. Haemostatic function and cardiovascular death: early results of a prospective study. Lancet 1980; i: 1050±1054. 15. Meade TW, Mellows S, Brozovic M et al. Haemostatic function and ischaemic heart disease: principal results of the Northwick Park Heart Study. Lancet 1986; ii: 533±537. 16. Meade TW, Ruddock V, Stirling Y & Miller GJ. Fibrinolytic activity, clotting factors, and long-term incidence of ischaemic heart disease in the Northwick Park Heart Study. Lancet 1993; 342: 1076±1079. * 17. Danesh J, Collins R, Appleby P & Peto R. Association of ®brinogen, C-reactive protein, albumin, or leukocyte count with coronary heart disease. Meta-analysis of prospective studies. Journal of the American Medical Association 1998; 279: 1477±1482. 18. Maresca G, Di Blasio A, Marchioli R & Di Minno G. Measuring plasma ®brinogen to predict stroke and myocardial infarction. An update. Arteriosclerosis, Thrombosis and Vascular Biology 1999; 19: 1368±1377. 19. Ma J, Hennekens CH, Ridker PM & Stampfer MJ. A prospective study of ®brinogen and risk of myocardial infarction in the Physicians' Health Study. Journal of the American College of Cardiology 1999; 33: 1347±1352. * 20. Tracy RP, Arnold AM, Ettinger W et al. The relationship of ®brinogen and factors VII and VIII to incident cardiovascular disease and death in the elderly. Results from the Cardiovascular Health Study. Arteriosclerosis, Thrombosis and Vascular Biology 1999; 19: 1776±1783. 21. Wilhelmsen L, Svardsudd K, Korsan-Bengtsen K et al. Fibrinogen as a risk factor for stroke and myocardial infarction. New England Journal of Medicine 1984; 311: 501±505. 22. Stone MC & Thorp JM. Plasma ®brinogen ± a major coronary risk factor. Journal of the Royal College of General Practitioners 1985; 35: 565±569. 23. Kannel WB. Hypertension and other risk factors in coronary heart disease. American Heart Journal 1987; 114: 918±925. 24. Heinrich J, Balleisen L, Schulte H et al. Fibrinogen and factor VII in the prediction of coronary risk. Results from the PROCAM study in healthy men. Arteriosclerosis and Thrombosis 1994; 14: 54±59. 25. Woodward M, Lowe GDO, Rumley A & Tunstall-Pedoe H. Fibrinogen as a risk factor for coronary heart disease and mortality in middle-aged men and women. European Heart Journal 1998; 19: 55±62. 26. Kannel WB. Fibrinogen: a major cardiovascular risk factor. In Ernst E, Koenig W, Lowe GDO & Meade TW (eds) Fibrinogen: a `New' Cardiovascular Risk Factor, pp 101±109. Vienna: Blackwell-MZV, 1992. 27. Resch KL, Ernst E, Matrai A & Paulsen HF. Fibrinogen and viscosity as risk factors for subsequent cardiovascular events in stroke survivors. Annals of Internal Medicine 1992; 117: 371±375. 28. Dormandy JA, Hoare E, Colley J et al. Clinical, haemodynamic, rheological, and biochemical ®ndings in 126 patients with intermittent claudication. British Medical Journal 1973; 4: 576±581. 29. Dormandy JA, Hoare E, Khattab AH et al. Prognostic signi®cance of rheological and biochemical ®ndings in patients with intermittent claudication. British Medical Journal 1973; 4: 581±583. 30. Banerjee AK, Pearson J, Gilliland EL et al. A six year prospective study of ®brinogen and other risk factors associated with mortality in stable claudicants. Thrombosis and Haemostasis 1992; 68: 261±263. 31. Fowkes FGR, Lowe GDO, Housley E et al. Cross-linked ®brin degradation products, progression of peripheral arterial disease, and risk of coronary heart disease. Lancet 1993; 342: 84±86. 32. Wiseman S, Kenchington G, Dain R et al. In¯uence of smoking and plasma factors on patency of femoropopliteal vein grafts. British Medical Journal 1989; 299: 643±646. 33. Montalescot G, Ankri A, Vicaut E et al. Fibrinogen after coronary angioplasty as a risk factor for restenosis. Circulation 1995; 92: 31±38.

Haemostatic function 595 34. Tschopl M, Tsakiris DA, Marbet GA et al. Role of hemostatic risk factors for restenosis in peripheral arterial occlusive disease after transluminal angioplasty. Arteriosclerosis, Thrombosis and Vascular Biology 1997; 17: 3208±3214. 35. Price JF, Mamode N, Smith FB et al. Haemostatic and rheological factors as predictors of restenosis following percutaneous transluminal angioplasty. European Journal of Vascular and Endovascular Surgery 1997; 14: 392±398. 36. Junker R, Heinrich J, Schulte H et al. Coagulation factor VII and the risk of coronary heart disease in healthy men. Arteriosclerosis, Thrombosis and Vascular Biology 1997; 17: 1539±1544. 37. Folsom AR, Wu KW, Rosamond WD et al. Prospective study of hemostatic factors and incidence of coronary heart disease. The Atherosclerosis Risk in Communities (ARIC) study. Circulation 1997; 96: 1102±1108. 38. Smith FB, Lee AJ, Fowkes FGR et al. Hemostatic factors as predictors of ischemic heart disease and stroke in the Edinburgh Artery study. Arteriosclerosis, Thrombosis and Vascular Biology 1997; 17: 3321±3325. 39. Miller GJ, Stirling Y, Esnouf MP et al. Factor VII-de®cient substrate plasmas depleted of protein C raise the sensitivity of the factor VII bio-assay to activated factor VII: an international study. Thrombosis and Haemostasis 1994; 71: 38±48. 40. Broadhurst P, Kelleher C, Hughes L et al. Fibrinogen, factor VII clotting activity and coronary artery disease severity. Atherosclerosis 1990; 85: 169±173. 41. Kario K, Miyata T, Sakata T et al. Fluorogenic assay of activated factor VII. Plasma factor VIIa levels in relation to arterial cardiovascular diseases in Japanese. Arteriosclerosis and Thrombosis 1994; 14: 265±274. 42. Cushman M, Yanez D, Psaty BM et al. Association of ®brinogen and coagulation factors VII and VIII with cardiovascular risk factors in the elderly. The Cardiovascular Health Study. American Journal of Epidemiology 1996; 143: 665±676. 43. Heywood DM, Ossei-Gerning N & Grant PJ. Association of factor VII:C levels with environmental and genetic factors in patients with ischaemic heart disease and coronary atheroma characterised by angiography. Thrombosis and Haemostasis 1996; 76: 161±165. 44. Moor E, Silveira A, van't Hooft F et al. Coagulation factor VII mass and activity in young men with myocardial infarction at a young age. Role of plasma lipoproteins and factor VII genotype. Arteriosclerosis, Thrombosis and Vascular Biology 1995; 15: 655±664. 45. Redondo M, Watzke HH, Stucki B et al. Coagulation factors II, V, VII, and X, Prothrombin gene 20210G ! A transition, and factor V Leiden in coronary artery disease. High factor V clotting activity is an independent risk factor for myocardial infarction. Arteriosclerosis, Thrombosis and Vascular Biology 1999; 19: 1020±1025. 46. Meade TW, Cooper JC, Stirling Y et al. Factor VIII, ABO blood group and the incidence of ischaemic heart disease. British Journal of Haematology 1994; 88: 601±607. 47. Rosendaal FR, Varekamp I, Smit C et al. Mortality and causes of death in Dutch haemophiliacs, 1973±86. British Journal of Haematology 1989; 71: 71±76. 48. Rosendaal FR, Briet E, Stibb J et al. Haemophilia protects against ischaemic heart disease: a study of risk factors. British Journal of Haematology 1990; 75: 525±530. 49. Sramek A, Kriek M & Rosendaal FR. A reduced mortality in carriers of hemophilia. Thrombosis and Haemostasis 1999; (supplement): 486. 50. Koster T, Blann AD, Briet E et al. Role of clotting factor VIII in e€ect of von Willebrand factor on occurrence of deep-vein thrombosis. Lancet 1995; 345: 152±155. 51. Thogersen AM, Jansson J-H, Boman K et al. High plasminogen activator inhibitor and tissue plasminogen activator levels in plasma precede a ®rst acute myocardial infarction in both men and women. Circulation 1998; 98: 2241±2247. 52. Jansson JH, Nilsson TK & Johnson O. von Willebrand factor, tissue plasminogen activator, and dehydroepiandrosterone sulphate predict cardiovascular death in a 10 year follow up of survivors of acute myocardial infarction. Heart 1998; 80: 334±337. 53. Catto AJ, Carter AM, Barrett JH et al. Von Willebrand factor and factor VIII:C in acute cerebrovascular disease. Relationship to stroke subtype and mortality. Thrombosis and Haemostasis 1997; 77: 1104±1108. 54. Meade TW, Cooper JA, Chakrabarti R et al. Fibrinolytic activity and clotting factors in ischaemic heart disease in women. British Medical Journal 1996; 312: 1581. 55. MacCallum PK, Cooper JA, Howarth DJ et al. Sex di€erences in the determinants of ®brinolytic activity. Thrombosis and Haemostasis 1998; 79: 587±590. 56. Chakrabarti R, North WRS & Meade TW. The e€ect of plasma ®brinogen levels on measures of ®brinolytic activity. Thrombosis and Haemostasis 1978; 39: 450±454. 57. Ridker PM, Vaughan DE, Stampfer MJ et al. Endogenous tissue-type plasminogen activator and risk of myocardial infarction. Lancet 1993; 341: 1165±1168.

596 P. K. MacCallum and T. W. Meade 58. Ridker PM, Hennekens CH, Stampfer MJ et al. Prospective study of endogenous tissue plasminogen activator and risk of stroke. Lancet 1994; 343: 940±943. * 59. Lowe GDO, Yarnell JWG, Sweetnam PM et al. Fibrin D-dimer, tissue plasminogen activator, plasminogen activator inhibitor, and the risk of major ischaemic heart disease in the Caerphilly Study. Thrombosis and Haemostasis 1998; 79: 129±133. 60. Ridker PM, Hennekens CH, Cerskus A & Stampfer MJ. Plasma concentration of cross-linked ®brin degradation product (D-dimer) and the risk of myocardial infarction among apparently healthy men. Circulation 1994; 90: 2236±2240. * 61. Cushman M, Lemaitre RN, Kuller LH et al. Fibrinolytic activation markers predict myocardial infarction in the elderly. The Cardiovascular Health Study. Arteriosclerosis, Thrombosis and Vascular Biology 1999; 19: 493±498. 62. Pedersen OD, Munkvad S, Gram J et al. Depression of factor XII-dependent ®brinolytic activity in survivors of acute myocardial infarction at risk of reinfarction. European Heart Journal 1993; 14: 785±789. 63. Hamsten A, de Faire U, Wallius G et al. Plasminogen activator inhibitor in plasma: risk factor for recurrent myocardial infarction. Lancet 1987; ii: 3±9. 64. Cortellaro M, Cofrancesco E, Boschetti C et al for the PLAT Group. Increased ®brin turnover and high PAI-1 activity as predictors of ischemic events in atherosclerotic patients: a case±control study. Arteriosclerosis and Thrombosis 1993; 13: 1412±1417. 65. Jansson JH, Nilsson TK & Olofsson BO. Tissue plasminogen activator and other risk factors as predictors of cardiovascular event in patients with severe angina pectoris. European Heart Journal 1991; 12: 157±161. 66. Juhan-Vague I, Pyke SDM, Alessi MC et al on behalf of the ECAT Study Group. Fibrinolytic factors and the risk of myocardial infarction or sudden death in patients with angina pectoris. Circulation 1996; 94: 2057±2063. 67. Moss AJ, Goldstein RE, Marder VJ et al. Thrombogenic factors and recurrent coronary events. Circulation 1999; 99: 2517±2522. 68. Chandler WL, Alessi MC, Aillaud MF et al. Clearance of tissue plasminogen activator (TPA) and TPA/ plasminogen activator inhibitor type 1 (PAI-1) complex. Circulation 1997; 96: 761±768. 69. Haverkate F, Thompson SG & Duckert F. Haemostasis factors in angina pectoris: relation to gender, age and acute-phase reaction. Results of the ECAT Angina Pectoris Study Group. Thrombosis and Haemostasis 1995; 73: 561±567. 70. Doll R & Peto R. Mortality in relation to smoking: 20 years' observations on male British doctors. British Medical Journal 1976; 2: 1525±1536. 71. Rose G & Shipley M. Plasma cholesterol concentration and death from coronary heart disease: 10 years results of the Whitehall study. British Medical Journal 1986; 293: 306±307. 72. Sobel BE. Increased plasminogen activator inhibitor-1 and vasculopathy. A reconcilable paradox. Circulation 1999; 99: 2496±2498. 73. Nawroth PP & Haring H-U. Thrombomodulin and coronary heart disease. Lancet 1999; 353: 1722±1723. 74. Salomaa V, Matei C, Aleksic N et al. Soluble thrombomodulin as a predictor of incident coronary heart disease and symptomless carotid artery atherosclerosis in the Atherosclerosis Risk in Communities (ARIC) study: a case±control study. Lancet 1999; 353: 1729±1734. 75. Blann AD, Admiral J & McCollum CN. Prognostic value of increased soluble thrombomodulin and increased soluble E-selectin in ischaemic heart disease. European Journal of Haematology 1997; 59: 115±120. 76. Rosenberg RD & Aird WC. Vascular-bed ± speci®c hemostasis and hypercoagulable states. New England Journal of Medicine 1999; 340: 1555±1564. 77. Medical Research Council Working Party. MRC trial of mild hypertension: principal results. British Medical Journal 1985; 291: 97±104. 78. Medical Research Council Working Party. Medical Research Council trial of treatment of hypertension in older adults: principal results. British Medical Journal 1992; 304: 405±412. 79. Sixty-Plus Reinfarction Study Research Group. A double-blind trial to assess long-term oral anticoagulant therapy in elderly patients after myocardial infarction. Lancet 1980; ii: 989±994. 80. Smith P, Arnesen H & Holme I. The e€ect of warfarin on mortality and reinfarction after myocardial infarction. New England Journal of Medicine 1990; 323: 147±152. 81. Anticoagulants in the Secondary Prevention of Events in Coronary Thrombosis (ASPECT) Research Group. E€ect of long-term oral anticoagulant treatment on mortality and cardiovascular morbidity after myocardial infarction. Lancet 1994; 343: 499±503. 82. CAPRIE Steering Committee. A randomised, blinded, trial of clopidogrel versus aspirin in patients at risk of ischaemic events (CAPRIE). Lancet 1996; 348: 1329±1339. 83. Ericsson CG, Hamsten A, Nilsson J et al. Angiographic assessment of e€ects of beza®brate on progression of coronary artery disease in young male postinfarction patients. Lancet 1996; 347: 849±853.

Haemostatic function 597 84. Behar S for the BIP Study Group. Lowering ®brinogen levels: clinical update. Blood Coagulation and Fibrinolysis 1999; 10 (supplement 1): S41±S43. 85. Frick MH, Elo O, Haapa K et al. Helsinki Heart Study: primary-prevention trial with gem®brozil in middle-aged men with dyslipidemia. Safety of treatment, changes in risk factors, and incidence of coronary heart disease. New England Journal of Medicine 1987; 317: 1237±1245. 86. Wilkes HC, Meade TW, Barzegar S et al. Gem®brozil reduces plasma prothrombin fragment F1 ‡ 2 concentration, a marker of coagulability, in patients with coronary heart disease. Thrombosis and Haemostasis 1992; 67: 503±506. 87. Meade TW. The epidemiology of atheroma, thrombosis and ischaemic heart disease. In Bloom AL, Forbes CD, Thomas DP & Tuddenham EGD (eds) Haemostasis and Thrombosis, 3rd edn, pp 1199±1227. Edinburgh: Churchill Livingstone, 1994. 88. Danesh J, Collins R & Peto R. Chronic infections and coronary heart disease: is there a link? Lancet 1997; 350: 430±436. 89. Pasceri V, Cammarota G, Patti G et al. Association of virulent Helicobacter pylori strains with ischemic heart disease. Circulation 1998; 97: 1675±1679. 90. Peek RM Jr, Miller GG, Tham KT et al. Heightened in¯ammatory response and cytokine expression in vivo to cagA‡ Helicobacter pylori strains. Laboratory Investigation 1995; 73: 760±770. 91. Patel P, Mendall MA, Carrington D et al. Association of Helicobacter pylori and Chlamydia pneumoniae infections with coronary heart disease and cardiovascular risk factors. British Medical Journal 1995; 311: 711±714. 92. Torgano G, Cosentini R, Mandelli C et al. Treatment of Helicobacter pylori and Chlamydia pneumoniae infections decreases ®brinogen plasma level in patients with ischemic heart disease. Circulation 1999; 99: 1555±1559. 93. Gur®nkel E, Bozovich G, Beck E et al for the ROXIS Study Group. Treatment with the antibiotic roxithromycin in patients with acute non-Q-wave coronary syndromes. European Heart Journal 1999; 20: 121±127. 94. Syrjanen J, Valtonen VV, Ilvnainen M et al. Preceding infection as an important risk factor for ischaemic brain infarction in young and middle aged patients. British Medical Journal 1988; 296: 1156±1160. 95. Jousilahti P, Vartiainen E, Tuomilehto J & Puska P. Symptoms of chronic bronchitis and the risk of coronary disease. Lancet 1996; 348: 567±572. 96. Jousilahti P, Salomaa V, Rasi V & Vahtera E. Symptoms of chronic bronchitis, haemostatic factors, and coronary heart disease risk. Atherosclerosis 1999; 142: 403±407. 97. Haider AW, Larson MG, O'Donnell CJ et al. The associations of chronic cough with the risk of myocardial infarction: The Framingham Heart Study. American Journal of Medicine 1999; 106: 279±284. 98. Seemungal TAR, MacCallum PK, Wedzicha JA et al. Change in plasma ®brinogen following acute exacerbation of chronic obstructive pulmonary disease. British Journal of Haematology 1999; 105 (supplement 1): 44. 99. Stout RW & Crawford V. Seasonal variations in ®brinogen concentrations amoung elderly people. Lancet 1991; 338: 9±13. 100. Frohlich M, Sund M, Russ S et al. Seasonal variations of rheological and hemostatic parameters and acute phase reactants in young, healthy subjects. Arteriosclerosis, Thrombosis and Vascular Biology 1997; 17: 2692±2697. 101. van der Bom JG, de Maat MP, Bots ML et al. Seasonal variation in ®brinogen in the Rotterdam Study. Thrombosis and Haemostasis 1997; 78: 1059±1062. 102. Woodhouse PR, Shaw K-T, Plummer M et al. Seasonal variation of plasma ®brinogen and factor VII activity in an elderly population and the relationship of ®brinogen to winter infections. Lancet 1994; 343: 435±439. 103. Juhan-Vague I, Thompson SG & Jespersen J on behalf of the ECAT Angina Pectoris Study Group. Involvement of the hemostatic system in the insulin resistance syndrome. A study of 1500 patients with angina pectoris. Arteriosclerosis and Thrombosis 1993; 13: 1865±1873. 104. Kario K, Sakata T, Matsuo T & Miyata T. Factor VII in non-insulin-dependent diabetic patients with microalbuminuria. Lancet 1993; 342: 1552. 105. Scarabin PY, Aillaud MF, Amouyel P et al. Association of ®brinogen, factor VII, and PAI-1 with baseline ®ndings among 10 500 male participants in a prospective study of myocardial infarction. The Prime Study. Thrombosis and Haemostasis 1998; 80: 749±756. 106. Alessi MC, Peiretti F, Morange P et al. Production of plasminogen activator inhibitor 1 by human adipose tissue. Possible link between visceral fat accumulation and vascular disease. Diabetes 1997; 46: 860±867. 107. Folsom AR, Quamheih HT, Wing RR et al. Impact of weight loss on plasminogen activator inhibitor (PAI-1), factor VII, and other hemostatic factors in moderately overweight adults. Arteriosclerosis and Thrombosis 1993; 13: 162±169.

598 P. K. MacCallum and T. W. Meade 108. Kohler HP, Carter AM, Stickland MH & Grant PJ. Levels of activated FXII in survivors of myocardial infarction ± association with circulating risk factors and extent of coronary artery disease. Thrombosis and Haemostasis 1998; 79: 14±18. 109. Miller GJ, Esnouf MP, Burgess AI et al. Risk of coronary heart disease and activation of factor XII in middle-aged men. Arteriosclerosis, Thrombosis and Vascular Biology 1997; 17: 2103±2106. 110. Lee AJ, Mowbray PI, Lowe GDO et al. Blood viscosity and elevated carotid intima-media thickness in men and women. The Edinburgh Artery Study. Circulation 1998; 97: 1467±1473. 111. Folsom AR, Wu KK, Shahar E et al for the ARIC Study Investigation. Association of haemostatic variables with prevalent cardiovascular disease and asymptomatic carotid artery atherosclerosis. Arteriosclerosis and Thrombosis 1993; 13: 1829±1836. 112. Tracy RP, Bovill EG, Yanez D et al for the Cardiovascular Health Study. Fibrinogen and factor VIII, but not factor VII, are associated with measures of subclinical cardiovascular disease in the elderly: results from the Cardiovascular Health Study. Arteriosclerosis, Thrombosis and Vascular Biology 1995; 15: 1269±1279. 113. Salonen R & Salonen JT. Determinants of carotid intima-media thickness: a population-based ultrasonography study in eastern Finnish men. Journal of Internal Medicine 1991; 229: 225±231. 114. ISIS-2 Collaborative Group. Randomised trial of intravenous streptokinase, oral aspirin, both, or neither among 17 187 cases of suspected acute myocardial infarction: ISIS-2. Lancet 1988; ii: 349±360. 115. Taylor DW, Barnett HJM, Haynes RB et al for the ASA and Carotid Endarterectomy (ACE) Trial Collaborators. Low-dose and high-dose acetylsalicylic acid for patients undergoing carotid endarterectomy: a randomised controlled trial. Lancet 1999; 353: 2179±2184. 116. Patrono C, Coller B, Dalen JE et al. Platelet-active drugs. The relationships among dose, e€ectiveness, and side e€ects. Chest 1998; 114: 470S±488S. 117. Roderick PJ, Wilkes HC & Meade TW. The gastrointestinal toxicity of aspirin: an overview of randomised controlled trials. British Journal of Clinical Pharmacology 1993; 35: 219±226. 118. He J, Whelton PK, Vu B & Klag MJ. Aspirin and risk of hemorrhagic stroke. A meta-analysis of randomized controlled trials. Journal of the American Medical Association 1998; 280: 1930±1935. 119. Meade TW, Roderick PJ, Brennan PJ et al. Extra-cranial bleeding and other symptoms due to low dose aspirin and low intensity oral anticoagulation. Thrombosis and Haemostasis 1992; 68: 1±6. 120. Turpie AGG, Gent M, Laupacis A et al. A comparison of aspirin with placebo in patients treated with warfarin after heart-valve replacement. New England Journal of Medicine 1993; 329: 524±529. 121. Cohen M, Adams PC, Parry G et al. Combination antithrombotic therapy in unstable rest angina and non-Q-wave infarction in nonprior aspirin users: primary end points, analysis from the ATACS trial. Circulation 1994; 89: 81±88. 122. Coumadin Aspirin Reinfarction Study (CARS) Investigators. Randomised double-blind trial of ®xed lowdose warfarin with aspirin after myocardial infarction. Lancet 1997; 350: 389±396. 123. Post Coronary Artery Bypass Graft Trial Investigators. The e€ect of aggressive lowering of low-density lipoprotein cholesterol levels and low-dose anticoagulation on obstructive changes in saphenous-vein coronary-artery bypass grafts. New England Journal of Medicine 1997; 336: 153±162. 124. Anand SS, Yusuf S, Pogue J et al for the OASIS Pilot Study Investigators. Long-term oral anticoagulant therapy in patients with unstable angina or suspected non-Q-wave myocardial infarction. Organization to assess strategies for ischemic syndromes (OASIS) pilot study results. Circulation 1998; 98: 1064±1070. 125. Scandinavian Simvastatin Survival Study Group. Randomised trial of cholesterol lowering in 4444 patients with coronary heart disease: the Scandinavian Simvastatin Survival Study (4S). Lancet 1994; 344: 1383±1389. 126. Rosenson RS & Tangney CC. Antiatherothrombotic properties of statins: implications for cardiovascular event reduction. Journal of the American Medical Association 1998; 279: 1643±1650. *127. Hulley S, Grady D, Bush T et al. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. Journal of the American Medical Association 1998; 280: 605±613. 128. Nabulsi AA, Folsom AR, White A et al. Association of hormone-replacement therapy with various cardiovascular risk factors in postmenopausal women. New England Journal of Medicine 1993; 328: 1069±1075. 129. Writing Group for the PEPI Trial. E€ects of estrogen or estrogen/progestin regimens on heart disease risk factors in postmenopausal women. Journal of the American Medical Association 1995; 273: 199±208. 130. Medical Research Council's General Practice Research Framework. Randomised comparison of oestrogen versus oestrogen plus progestogen hormone replacement therapy in women with hysterectomy. British Medical Journal 1996; 312: 473±478. 131. Koh KK, Mincemoyer R, Bui MN et al. E€ects of hormone-replacement therapy on ®brinolysis in postmenopausal women. New England Journal of Medicine 1997; 336: 683±690.

Haemostatic function 599 132. Hansson L, Zanchetti A, Carruthers SG et al. E€ects of intensive blood-pressure lowering and low-dose aspirin in patients with hypertension: principal results of the Hypertension Optimal Treatment (HOT) randomised trial. Lancet 1998; 351: 1755±1762. 133. Physicians' Health Study Research Group. Final report on the aspirin component of the ongoing Physicians' Health Study. New England Journal of Medicine 1989; 321: 129±135. 134. Peto R, Gray R, Collins R et al. Randomized trial of prophylactic daily aspirin in British male doctors. British Medical Journal 1988; 296: 262±264. 135. Hennekens CH, Buring JE, Sandercock P et al. Aspirin and other antiplatelet agents in the secondary and primary prevention of cardiovascular disease. Circulation 1989; 80: 749±756. 136. Cairns JA, Theroux P, Lewis HD Jr et al. Antithrombotic agents in coronary heart disease. Chest 1998; 114: 611S±633S.