a University Department of Medicine, Royal Infirmary, Glasgow, UK
b Clinical Trial Service Unit and Epidemiological Studies Unit, Nuffield Department of Medicine, University of Oxford, Oxford, UK
c Department of Primary Care and Population Sciences, Royal Free and University College London Medical School, London, UK
d Department of Public Health Sciences, St George's Hospital Medical School, London, UK
* Correspondence to: Professor G. D. O. Lowe, Department of Medicine, Royal Infirmary, 10 Alexandra Parade, Glasgow, G31 2ER, UK. Tel: +44 141 211 5412; Fax: +44 141 211 0414
E-mail address: gdl1j{at}clinmed.gla.ac.uk
Received 14 May 2003; revised 28 October 2003; accepted 6 November 2003
Abstract
Aims To determine whether circulating tissue plasminogen activator (t-PA) antigen concentrations are prospectively related to risk of coronary heart disease (CHD) in the general population
Methods and results We measured baseline concentrations of t-PA antigen in the stored serum samples of 606 CHD cases and 1227 controls nested in a prospective cohort of 5661 men monitored for 16 years, and conducted a meta-analysis of previous relevant studies to place our findings in context. Tissue plasminogen activator antigen values were strongly correlated with several vascular risk factors, including serum lipids, body mass index, alcohol consumption, and markers of systemic inflammation. In a comparison of men in the top third compared with those in the bottom third of baseline t-PA antigen values, the odds ratio for CHD was 2.20 (95% confidence interval (CI) 1.702.85) after adjustment for age and town only, but this fell to 1.48 (1.092.01) after further adjustment. Analysis of t-PA as a continuous variable gave similar results. Similarly, when published information on all seven available prospective cohort studies in general populations (2119 cases and 8832 controls in total) was synthesized, the combined odds ratio was 2.18 (1.772.69) after adjustment for age and sex only, and this fell to 1.47 (1.191.81) after further adjustment.
Conclusion Although there is a statistically significant association between circulating concentrations of t-PA antigen and subsequent CHD, additional studies are needed to determine to what extent this is independent from more established risk factors.
Key Words: Coronary heart disease Epidemiology Fibrinolysis
1. Introduction
Tissue plasminogen activator (t-PA) is a glycoprotein produced mainly by vascular endothelial cells.1,2It activates clot dissolution in the presence of fibrin by conversion of plasminogen to plasmin, thereby cleaving cross-linked fibrin to D-dimer and other degradation products,3and it may also be involved in coronary plaque rupture.2As free active t-PA is difficult to measure in plasma (unless blood is collected into anticoagulants specific for this purpose),1most clinical studies have measured circulating t-PA antigen values.1,2The biological relevance of t-PA antigen is, however, less understood. As a marker of the up-regulation of endogenous fibrinolysis, it might be expected to be associated with a lower incidence of vascular disease. But, as it is mainly a marker of complex formation between t-PA and its major plasma inhibitor, plasminogen activator inhibitor-1 (PAI-1), rather than a measure of free t-PA, it might be expected to be associated with a higher incidence of vascular disease.1,2Tissue plasminogen activator and PAI-1 are also associated with lifestyle variables4and with the inflammatory response, markers of which are related to coronary disease.5
Several epidemiological studies have reported on the relations between circulating t-PA antigen values and coronary heart disease (CHD) in general populations611and in cohorts with existing vascular disease.1221Most studies have involved relatively few CHD cases, and they have yielded apparently conflicting results.621To help clarify the epidemiological evidence, we report the largest and most prolonged community based prospective study of circulating t-PA antigen values and CHD thus far, as well as a meta-analysis of available prospective studies to place our findings in context. Given the proposed association between circulating concentrations of t-PA antigen and PAI-1 complexes, we have also conducted a subsidiary meta-analysis of available prospective studies of PAI-1 and CHD in general populations to help interpret our new data.6,10,11,22,23
2. Methods
2.1. Participants
In 19781980, 7735 males aged 4059 years were randomly selected from general practice registers in each of 24 British towns, and invited to take part in the British Regional Heart Study (response rate 78%). Nurses administered questionnaires, made physical measurements, recorded an ECG, and, in 5661 men in 18 of the towns, collected non-fasting venous blood samples, from which serum was stored at 20°C for subsequent analysis.24Additional questionnaires on car ownership and childhood social circumstances (father's social class and childhood household amenities) were mailed 5 years (98% response among survivors) and 12 years (90% response among survivors) after entry, respectively. All men have been monitored subsequently for all-cause mortality and for cardiovascular morbidity, with a follow up loss of <1% to date.25A prospective, nested, case control study, matched for age and town, was established within the cohort. Eligible cases were 279 men who died from CHD and 364 men who had non-fatal myocardial infarction before 1996.4Fatal cases were ascertained through National Health Service Central Registers on the basis of a death certificate with ICD-9 codes 410414. Non-fatal myocardial infarction was based on reports from general practitioners, supplemented with hospital reports confirming the diagnosis in accordance with World Health Organisation criteria;26a validation study has beenreported.27Cases were frequency matched with 1278 controls, on town of residence and age in 5-year bands, of GP record reviews. These were randomly selected from among men surviving to the end of the study period free from incident CHD. Due to limited sample availability, t-PA antigen measurements were available for only 606 of these cases and for only 1227 of these controls.
2.2. Laboratory and statistical analyses
Laboratory workers, blinded to the case-control status of participants, measured serum concentrations of t-PA antigen with an enzyme immunoassay (Biopool AB, Umea, Sweden) previously used in several other prospective cohort studies.4,8,10,12To validate use of serum, we assayed t-PA antigen in paired plasma and serum samples from 56 healthy individuals and observed a correlation coefficient of 0.95 between the values, as well as very similar mean and SD values (Fig. 1). Because of fluctuations of t-PA antigen concentrations within individuals over time, case-control comparisons of measured baseline values tend tounderestimate any association with CHD risk.2729Measurements of t-PA antigen were made in pairs of samples collected at an interval of 5 years apart in 892 controls in a separate study,8yielding a self-correlation coefficient of 0.52 (Lowe et al.unpublished data from the Edinburgh Artery Study). This was used to estimate the magnitude of regression dilution and to correct for it (see Results).2830C-reactive protein,31serum amyloid A,31albumin,32white cell count,33Chlamydia pneumoniae IgG and IgA titres,34Helicobacter pylori seropositivity,35fibrin D-dimer,36von Willebrand factor37and homocysteine38were measured as previously described.
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3. Results
3.1. Present study
There were highly significant differences between cases and controls with respect to various known vascular risk factors and t-PA antigen (Table 1). Table 2shows that baseline t-PA antigen values in the control population were highly significantly associated with several classical risk factors, including age (2P<0.0001), alcohol consumption (2P<0.00001), body mass index (2P<0.00001), blood pressure (2P<0.0001), total cholesterol (2P<0.00001) and triglyceride (2P<0.00001). There were strong adjusted associations of t-PA antigen values with forced expiratory volume in 1s (2P<0.00001), haematocrit (2P<0.00001), leucocyte count (2P<0.001), and serum concentrations of von Willebrand factor (2P<0.00001), C-reactive protein (2P<0.00001), insulin (2P<0.0001) and urea (2P<0.00001). No strong associations were observed of t-PA antigen values with serum concentrations of homocysteine or amyloid A protein, markers of persistent infection, or indicators of socioeconomic status (data not shown).
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4. Discussion
Previous studies have generally involved too few CHD cases to determine reliably whether there is a statistically significant association between circulating concentrations of t-PA antigen and subsequent CHD, independent of known cardiovascular risk factors. Whereas the previous largest published prospective study included 326 patients with CHD and 720 controls,6the present report involves almost twice as much new data, involving 606 CHD cases and 1223 controls. It also includes a meta-analysis of available prospective studies, increasing the numbers available for analysis to 2119 CHD cases and to 8832 controls. A combined analysis of these studies, based on published odds ratios that wereadjusted for some risk factors, suggests that CHD risk is about 50% greater in those in the top third compared with those in the bottom third of baseline t-PA antigen values. The relationship of t-PA antigen to CHD, however, still remains uncertain. A tendency to more extreme odds ratios reported in the smaller studies in Fig. 1suggests the likelihood of some exaggeration in the overall estimate due to publication bias.41Moreover, because incomplete adjustment for some risk factors reduced the odds ratio substantially, it is not known how much, if any, of the residual association between t-PA antigen and CHD would persist with more complete adjustment for these (and other) factors. On the other hand, we have demonstrated that the predictive ability of t-PA antigen for CHD has been under-estimated in previous studies due to lack of correction for its within-individual variation over time.
In the present study, a comparison of those in the top third with those in the bottom third of baseline t-PA antigen values yielded an odds ratio for CHD of 2.20 (1.702.85; 21=36) after adjustment for age and town only, and of 1.48 (1.092.01;
21=6) after further adjustment for classical CHD risk factors. A combined analysis of all six available prospective studies in approximately general populations yielded an odds ratio of 2.18 (1.772.69;
21=55) based on published associations that were adjusted for age and sex only; and an odds ratio of 1.47 (1.191.81;
21=13: Fig. 3) based on published associations that were adjusted for age, sex, and baseline values of some risk factors. This pattern of attenuation in the odds ratio for CHD with increasing degree of adjustment was also seen in the six prospective studies of patients with previous vascular disease. The fact that adjustments based just on baseline measurements of these risk factors reduced the chi-squared values so substantially suggests that exact adjustment for these (and other) confounders would produce even greater reductions. The estimates of effect in patients with previous vascular disease, although rather weaker than those in patients without previous vascular disease, are not markedly different.
Epidemiological studies of t-PA antigen consistently show strong correlations with PAI-1 activity or antigen.42This association may reflect simultaneous release from endothelial cells, delayed clearance of t-PA-PAI-1 complexes, acute-phase reactions, or mutual correlations with measures of insulin resistance.43Our subsidiary meta-analysis of available prospective studies of CHD and PAI-1 was largely inconclusive, yielding a null odds ratio with a wide confidence interval (0.98, 0.531.81). Larger observational studies involving measurement of both t-PA antigen and of PAI-1 in the same participants (enabling assessment of the relevance of each factor to the other and to CHD) and involving serial measurements of t-PA antigen and of several potential confounding factors (enabling correction for fluctuations of values of risk factors within individuals over time)29,30should help to elucidate the strength of the association seen between t-PA antigen and CHD.
Potential limitations of the present study include measurement of t-PA antigen in serum instead of plasma, and prolonged storage at 20°C (1116 years). However, our validation study showed high correlation (r=0.95) between serum and plasma t-PA antigen levels (with very similar mean and SD values), as in a previous report.44We recently performed a study of repeat t-PA antigen assays after 9 years storage at 50°C in 255 samples. There was no significant degradation in t-PA values (mean and SD) during this time (Rumley et al., unpublished). Moreover, values for serum t-PA antigen levels in CHD cases and controls in the present study were very similar to those reported for plasma t-PA antigen (usually stored for a shorter period of time) in previous prospective studies,621and showed the expected correlations with some classical vascular risk factors.4
What is the potential biological significance of the positive association between circulating t-PA antigen and risk of CHD? The possible influence of PAI-1 on t-PA antigen has already been noted. Tissue plasminogen activator is released from vascular endothelium, hence increased circulating levels may be a marker of endothelial disturbance: in the present study, circulating t-PA antigen correlated strongly with circulating levels of von Willebrand factor (vWF), another endothelial release product,37as well as with risk markers associated with endothelial dysfunction (Table 2). Tissue plasminogen activator (and PAI-1) levels also increase as part of the inflammatory response:43in the present study, circulating t-PA antigen correlated with several inflammation-related measures (e.g., C-reactive protein, leucocyte count). Hence it is possible that the association of t-PA with CHD may partly reflect their mutual associations with the inflammatory response. While increased circulating free t-PA might increase fibrin lysis, no significant association of t-PA antigen was observed with fibrin D-dimer levels in the present study. Finally, it has been proposed that t-PA may play a role in coronary plaque rupture.2In a prospective study of patients with stable angina, both t-PA antigen and leucocyte elastase (another protease which may play a role in plaque rupture) were predictive of myocardial infarction (which is usually preceded by plaque rupture).45However, at present the pathophysiological relevance of circulating levels of t-PA to plaque rupture remains uncertain.
5. Conclusions
There is a statistically significant association between circulating concentrations of t-PA antigen and subsequent CHD, but additional studies are needed to determine to what extent this association is independent from more established risk factors, and to determine the biological significance of t-PA antigen.
Acknowledgments
The views expressed in this paper are those of the authors and not necessarily the funding agencies. Professor A. G. Shaper established the British Regional Heart Study, which is a British Heart Foundation Research Group and also receives support form the Department of Health. Paul Appleby plotted the figures. K. Craig, F. Key, and L. Oxford provided t-PA, von Willebrand factor and fibrin D-dimer assays; Professor M. B. Pepys and J. R. Gallimore provided C-reactive protein and serum amyloid A assays; H. Refsum and P. Ueland provided homocysteine assays; M. Thomas, Yuk-ki Wong and Professor M. Ward provided C pneumoniae serology; J. Atherton and Professor C. Hawkey provided H pylori serology; and J. John provided valuable assistance. J.D. was supported by a programme grant from the British Heart Foundation and by the Raymond and Beverly Sackler Award in the Medical Sciences. A.R. and G.D.O.L. are supported by project and programme grants from the British Heart Foundation.
References