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Prepublished online as a Blood First Edition Paper on October 24, 2002; DOI 10.1182/blood-2002-07-2181.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Julius Center for Health Sciences and Primary
Care, University Medical Center Utrecht; Van Creveldkliniek, University
Medical Center Utrecht; Gaubius Laboratory, TNO-PG, Leiden; and the
Department of Epidemiology and Biostatistics, Erasmus University
Medical Center, Rotterdam, the Netherlands.
Plasminogen activator inhibitor type I (PAI-1) antigen
concentrations follow a circadian oscillation peaking in the morning. Some individuals show no apparent circadian rhythm, while others show
up to a 10-fold variation in PAI-1 over 24 hours. Results from
experimental studies suggest that a polymorphism in the promoter of the
gene for PAI-1 (4G5G) directly influences the circadian expression of the PAI-1 gene. We studied whether the diurnal
variation of PAI-1 antigen differs for the genotypes of the
4G5G polymorphism. A population-based, cross-sectional
study was performed among 263 subjects selected from the Rotterdam
Study, a population-based cohort of 7983 men and women aged 55 years
and older. The 4G allele was associated with a more
pronounced circadian expression of PAI-1 antigen. Morning PAI-1 antigen
concentrations were 79 ng/mL (95% confidence interval [CI],
68-92) in subjects homozygous for 4G, 62 ng/mL (95% CI, 54-72) in heterozygous subjects, and 59 ng/mL (95% CI,
49-71) in subjects homozygous for 5G. While respective PAI-1 antigen concentrations in the afternoon were 40 ng/mL (95% CI,
33-49), 41 ng/mL (95% CI, 37-47), and 40 ng/mL (95% CI, 49-71). These
findings suggest that the morning increase in PAI-1 antigen concentration is more pronounced among subjects homozygous for the
4G allele compared with the morning increase among the
other genotypes. Additionally, these findings show that homozygosity for the 4G allele is associated with increased PAI-1 levels
during the morning only.
(Blood. 2003;101:1841-1844) Although acute cardiovascular events occur
throughout the day, it has been shown that acute myocardial infarction,
sudden cardiac death, silent ischemia, and stroke have a circadian
pattern of occurrence with a peak in the morning.1-4 A
meta-analysis suggested that 9% of all myocardial infarctions are
attributable to the morning excess incidence of cardiac
events.5 The morning excess of cardiac events may result
in part from the circadian variation of fibrinolytic activity.
Plasminogen activator inhibitor type 1 (PAI-1) is the major component
of inhibitors of fibrinolytic activity. PAI-1 shows a clear circadian
oscillation peaking in the morning.6-8
Recently, a novel transcription factor, CLIF (cyclelike factor), has
been identified that regulates the circadian oscillation of PAI-1 gene
expression.9 In the PAI-1 promoter, 2 E-box
elements (CACGTG) are responsible for the activation of
PAI-1 by CLIF; one of the E-boxes is located at Population
Clinical investigation
Laboratory investigations Blood sampling and storage have been described elsewhere.19 Blood samples were collected during the first visit to the research center between 8 AM and 4 PM, using CTAD vacutainers (0.11M citrate, 15 mM theophylline, 3.7 mM adenosine, and 0.198 mM dipyridamole; Diatube H, Becton and Dickinson, Mylan, Cedex, France).20 Serum total and high-density lipoprotein (HDL) cholesterol were determined with an automated enzymatic procedure. PAI-1 antigen concentration was measured in CTAD plasma using the Innotest PAI-1 (Innogenetics, Zwijndrecht, Belgium).21 Genomic DNA was isolated from blood cells and PAI-1 genotype was determined by allele-specific PCR amplification.22Statistical analysis The distribution of the plasma concentration of PAI-1 antigen was positively skewed, therefore statistical analysis was carried out on log-transformed values, but the presented mean values and 95% confidence intervals (CIs) have been transformed back into the original scale. For each genotype, mean PAI-1 antigen concentration for subjects who visited the research center before noon was compared with that in subjects who visited the center after noon. Linear regression analysis with log-transformed PAI-1 concentrations as dependent variable was used to compare PAI-1 levels between subjects visiting the research center before or after noon, adjusted for systolic blood pressure, serum HDL cholesterol, and body mass index. Back transformation of the regression coefficients has been performed by taking the exponent of the coefficient and the exponent of upper and lower values of the confidence interval. The result is a multiplicative factor.This is a relatively small study with relatively little power to test for interaction. We noticed that the morning/afternoon difference of heterozygous subjects was similar to that of subjects homozygous for 5G. We therefore decided to combine these 2 groups. Interaction between the effect of homozygosity for the 4G allele and the diurnal effect were tested with the product of an indicator variable for 4G4G and an indicator variable for visit before noon, together with both indicators as independent variables in a linear regression model.
Characteristics of the study population After exclusion of those with missing data on either the 4G5G polymorphism (n = 22) or PAI-1 antigen concentration (n = 29), or time of blood collection (n = 4), the study population consisted of 263 subjects (132 cases of myocardial infarction and 131 control subjects). We compared the observed distribution of genotypes among the subjects without cardiovascular disease with that expected for a population according to a Hardy Weinberg equilibrium. The frequency of the 4G allele was 53% and that of the 5G allele was 47%, and the distribution of genotypes was in Hardy-Weinberg equilibrium; 39 control subjects (30%) were found to be homozygous for the 4G allele, whereas 62 (47%) control subjects were heterozygous, and 30 (23%) control subjects were homozygous for the 5G allele.Table 1 presents characteristics of the
study population according to time of blood collection.
4G5G genotypes and PAI-1 levels Homozygosity for the 4G allele was associated with increased levels of PAI-1 antigen. In subjects homozygous for the 4G allele PAI-1 antigen concentration was 62 ng/mL (95% CI, 54-70), in those heterozygous it was 50 ng/mL (95% CI, 46-56), and in those homozygous for the 5G allele it was 50 ng/mL (95% CI, 43-58).Diurnal pattern of PAI-1 PAI-1 antigen levels showed a diurnal pattern with higher values in the morning. Figure 1 presents PAI-1 levels according to the time of blood collection. Between 8 and 9 AM, 28 subjects had a median PAI-1 of 63 ng/mL; between 9 and 10 AM, 69 subjects had a median PAI-1 of 64 ng/mL; between 10 and 11 AM, 44 subjects had a median PAI-1 of 65 ng/mL; between 11 AM and noon, 5 subjects had a median PAI-1 of 62 ng/mL; between noon and 1 PM, blood was drawn in one subject with a PAI-1 of 43 ng/mL; between 1 and 2 PM, 58 subjects had a median PAI-1 of 40 ng/mL; between 2 and 3 PM, 44 subjects had a median PAI-1 of 38 ng/mL; between 3 and 4 PM, 14 subjects had a median PAI-1 of 36 ng/mL. The figure illustrates that high peaks of PAI-1 antigen are predominantly found during the morning hours.
Diurnal pattern of PAI and 4G5G genotypes Differences in PAI-1 expression levels according to time of blood collection appeared to depend somewhat on genotype (test for interaction, P = .057), and for this reason separate analyses were conducted according to genotype. In subjects with the 4G4G genotype the morning/afternoon difference was larger than in the other genotypes (Table 2). Results from univariate linear regression analyses suggest that PAI-1 antigen was 1.95 (95% CI, 1.54-2.51) times higher in the morning among subjects homozygous for 4G; for heterozygous subjects it was 1.50 (95% CI, 1.23-1.82) times higher in the morning; and for subjects homozygous for 5G it was 1.47 (95% CI, 1.08-1.97) times higher in the morning. Similar statistically significant differences between morning and afternoon PAI-1 levels were found after adjustment for the confounders (potential confounders are presented in Table 1). After adjustment for systolic blood pressure, HDL cholesterol, and a history of myocardial infarction, PAI-1 antigen was 1.92 (95% CI, 1.61-2.41) times higher in the morning among subjects homozygous for the 4G allele; 1.58 (95% CI, 1.32-1.90) times higher in the morning among heterozygous subjects; and 1.65 (95% CI, 1.26-2.16) times higher in the morning among those homozygous for 5G.
History of myocardial infarction and diurnal PAI-1 There was no statistically significant difference between the morning effect on PAI-1 among subjects with a history of myocardial infarction compared with those without a history of cardiovascular diseases. PAI-1 antigen was 1.54 (95% CI, 1.28-1.85) times higher in the morning among subjects with a history of myocardial infarction; it was 1.75 (95% CI, 1.43-2.13) times higher in the morning among subjects without a history of cardiovascular diseases.
We assessed whether the circadian oscillation pattern of PAI-1 antigen differs for the genotypes for the 4G5G polymorphism in the PAI-1 gene. Homozygosity for the 4G allele was associated with increased PAI-1 levels only during the morning hours. All 3 genotypes showed higher PAI-1 antigen concentrations in the morning, but this morning effect was more pronounced among subjects homozygous for the 4G allele (test for interaction, P= .057). We believe that our findings provide a valid estimate of the effect of the 4G allele of the 4G5G polymorphism on the circadian oscillation pattern of PAI-1 antigen. PAI-1 antigen levels were measured in a selected group of subjects who participated in a large population-based cohort study. Half of the study population had a history of myocardial infarction and the other half did not have a history of cardiovascular diseases. The proportion of patients with a history of myocardial infarction was slightly higher in the morning, which of course explains higher PAI-1 levels in the morning.23 However, the fact that roughly half the studied population had a history of myocardial infarction does not totally account for the observed differences in PAI-1. History of myocardial infarction was adjusted for in the models, and this adjustment did not qualitatively alter the conclusion. Moreover, the morning effect among subjects with myocardial infarction was not more pronounced than among patients without myocardial infarction. Increased PAI-1 levels have been associated with increased blood pressure, increased body mass index, and decreased HDL cholesterol.16,24 Systolic blood pressure and HDL cholesterol were also related to time of blood collection in our study. Therefore, these factors may also be an alternate explanation for our findings. Yet, adjustment for these factors did not notably change our findings. This study is the first to show an association between the 4G5G polymorphism and the circadian pattern of plasma PAI-1 antigen concentrations. Experimental work had already suggested that the 4G5G polymorphism might influence the circadian oscillation of PAI-1 antigen.9 The observation that the 4G5G polymorphism is associated with the circadian pattern of PAI-1 supports the view that the 4G5G polymorphism may affect the circadian expression of PAI-1. Our findings shed an interesting light on the current knowledge about PAI-1 as a risk factor for myocardial infarction. The role of PAI-1 as a risk factor for cardiovascular diseases has been questioned.23 The finding that the 4G allele is associated with increased plasma levels of PAI-1 in the morning confirms that the 4G5G polymorphism in the PAI-1 gene is of functional importance in regulating the expression of the PAI-1 gene. Additionally, homozygosity for the 4G allele is associated with a slightly increased risk for myocardial infarction.12 And, subjects with 1 or 2 4G alleles had a faster progression from first anginal symptoms to acute coronary syndromes than 5G5G subjects.25 The present study suggests that the morning increase in PAI-1 is more pronounced in 4G4G subjects. The morning peak corresponds to the diurnal variation of the time of onset of myocardial infarction. It is tempting to suggest that the 4G4G genotype, maybe through the morning increase in PAI-1, plays a role in the excess number of cases of myocardial infarction in the morning. This would confirm a role of PAI-1 as a risk factor for myocardial infarction. In conclusion, our findings support the view that the 4G5G polymorphism in the promoter of the gene for PAI-1 may affect the circadian expression of the PAI-1 gene.
We are grateful to the participants of the Rotterdam Study. We thank all field workers; laboratory technicians in the Ommoord Research center; and G. J. Derksen and the clinical assay unit of the Gaubius Laboratory of TNO-PG for their enthusiasm and skillful contributions to the data collection.
Submitted July 19, 2002; accepted October 2, 2002.
Prepublished online as Blood First Edition Paper, October 24, 2002; DOI 10.1182/blood-2002-07-2181.
Supported by grants from the Netherlands Heart Foundation (no. 92.398) and the Dutch Thrombosis Foundation (no. 91.004). The Rotterdam Study is supported in part by the NESTOR program for geriatric research (Ministry of Health and Ministry of Education), the Netherlands Heart Foundation, the Netherlands Organisation for Scientific Research (NWO), the Rotterdam Medical Research Foundation (ROMERES), and the Municipality of Rotterdam.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: J. G. van der Bom, Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht, Internal address D01.335, PO Box 85500, 3508 GA Utrecht, the Netherlands; e-mail: j.g.vanderbom{at}jc.azu.nl.
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© 2003 by The American Society of Hematology.
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  J. K. Devin, L. S. Blevins Jr., D. K. Verity, Q. Chen, J. R. Bloodworth Jr., J. Covington, and D. E. Vaughan Markedly Impaired Fibrinolytic Balance Contributes to Cardiovascular Risk in Adults with Growth Hormone Deficiency J. Clin. Endocrinol. Metab., September 1, 2007; 92(9): 3633 - 3639. [Abstract] [Full Text] [PDF]
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 S. Zvonic, A. A. Ptitsyn, S. A. Conrad, L. K. Scott, Z. E. Floyd, G. Kilroy, X. Wu, B. C. Goh, R. L. Mynatt, and J. M. Gimble Characterization of peripheral circadian clocks in adipose tissues. Diabetes, April 1, 2006; 55(4): 962 - 970. [Abstract] [Full Text] [PDF]
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 C Roncal, J Orbe, J.A Rodriguez, M Belzunce, O Beloqui, J Diez, and J.A Paramo Influence of the 4G/5G PAI-1 genotype on angiotensin II-stimulated human endothelial cells and in patients with hypertension Cardiovasc Res, July 1, 2004; 63(1): 176 - 185. [Abstract] [Full Text] [PDF]
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 T. A. Sanders, T. de Grassi, J. Acharya, G. J Miller, and S. E Humphries Postprandial variations in fibrinolytic activity in middle-aged men are modulated by plasminogen activator inhibitor I 4G-675/5G genotype but not by the fat content of a meal Am. J. Clinical Nutrition, April 1, 2004; 79(4): 577 - 581. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
677 to 
the PRIME Study: Prospective Epidemiological Study of Myocardial Infarction.
Thromb Haemost.
1998;80:749-756