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D-dimer and von Willebrand factor (vWF) are associated with atherosclerosis. We recently reported that in a post-World War II birth cohort, Japanese men in Japan had lower levels of atherosclerosis than white men in the United States (US). We examined whether the differences in D-dimer and vWF levels are associated with differences in atherosclerosis between the two populations.
Population-based samples of 99 Japanese and 100 white American men aged 40–49 were examined for coronary artery calcification (CAC), carotid intima-media thickness (IMT), D-dimer, vWF, and other factors using a standardized protocol. When compared to white American men, Japanese had similar levels of D-dimer (0.22 ± 0.28 vs. 0.19 ± 0.24 µg/L, respectively, p=0.39) but significantly higher levels of vWF (124.1 ± 36.6 vs. 91.3 ± 48.8 %, respectively, p<0.01). Japanese as compared to white American men had significantly lower prevalence of CAC (13.1 vs. 28.0 %, P<0.01, respectively) and significantly lower IMT (0.61 ± 0.07 vs. 0.66 ± 0.08 mm, p<0.01, respectively). Japanese men had a significant positive association of D-dimar with the prevalence of CAC and a negative association of vWF with IMT, whereas whites American men did not have any significant associations.
In men aged 40–49, Japanese as compared to white Americans had similar levels of D-dimer and higher levels of vWF although Japanese had significantly a lower prevalence of CAC and IMT. These hemostatic factors are unlikely to explain the difference in atherosclerosis in these populations.
Coronary heart disease (CHD) mortality and incidence in Japan have been very low in comparison to other developed countries, which gas beeb attributed to very low levels of serum total cholesterol in Japan in the 1960’s1. After World War II (WWII), the Japanese adopted a morere westernized life styles including substantial increase in dietary intake of saturated fat. As a result, the levels of serum total cholesterol are now similar to those found in the United States (US) 1. However incidence of CHD in Japan remains low and is about a tenth of that in the US.2 Even in men in the post WWII birth cohort who adopted a westernized lifestyle from childhood through young adulthood, CHD mortality in Japan is much lower than in the US 1, 3. In fact, CHD mortality in Japan, including this birth cohort has been decreasing since the 1970’s 4.
We have recently reported that in a post WWII birth cohort, Japanese men in Japan as compared to white American men had less atherosclerosis5 evaluated as coronary artery calcification (CAC) and carotid intima-media thickness (IMT) 6–7. We also reported that Japanese American men in a post WWII birth cohort have similar or higher levels of atherosclerosis as compared to white American men.8 Other migrant studies of the Japanese to the US demonstrated substantial increase in CHD and subclinical atherosclerosis 1, 9. Taken together, the low levels of atherosclerosis in Japanese men in Japan are unlikely due primarily to genetic factors.
Hemostatic factors are associated with atherosclerosis and CHD 10–22. Recent studies showed that D-dimer, a marker of fibrin turnover, and von Willebrand factor (vWF), an endothelial marker and an important co-factor in platelet adhesion, are positively associated with atherosclerosis17–18 and CHD 10–16, 19–23. However the association of these factors with CAC or IMT was inconclusive 24–27. Among other hemostatic factors, we previously reported that the difference in fibrinogen or plasminogen-activator inhibitor-1 is not associated with the lower levels of atherosclerosis in the Japanese in Japan 5, 28.
No previous study has reported the difference in the levels of D-dimer or vWF between the Japanese and whites in a post WWII birth cohort, although a few studies have reported the difference in older age groups or among other ethnic groups 29–32. No previous study has compared the association of D-dimer or vWF with atherosclerosis between Japanese and white populatoins.
The aims of this study were to examine whether D-dimer and vWF levels are different between the Japanese and whites in a post WWII birth cohort, and whether that difference is associated with the difference in atherosclerosis between the two populations. We examined these aims in population-based samples of 99 Japanese and 100 white American men in a post WWII birth cohort.
The research design and methods of the Electron-Beam Tomography, Risk Factor Assessment Among Japanese and U.S. Men in the Post-World War II Birth Cohort (ERA JUMP) Study, a population-based, cross-sectional study of men aged 40–49, were previously described in detail 5. From the ERA JUMP Study, 99 Japanese and 100 whites American men were randomly selected for the current study. Informed consent was obtained from all participants. The study was approved by the Institutional Review Boards of Shiga University of Medical Science, Otsu, Japan and the University of Pittsburgh, Pittsburgh, US.
Body-mass index (BMI), waist circumference and blood pressure (BP) were measured as previously described 5. Serum levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), triglycerides, low-density lipoprotein cholesterol (LDL-C), fasting glucose, fasting insulin and C-reactive protein (CRP) were determined as previously described 5, 8.
Both D-dimer and vWF levels were measured by the STAR automated coagulation analyzer (Diagnostica Stago Inc, Parsippany, NJ), using an immuno-turbidimetric assay. Coefficient variations for D-dimer and vWF were 5% and 4%, respectively. The normal range was 0.06 – 0.77 µg/ml for D-dimer and 51% – 158% for vWF.
Self-administered questionnaires were used to obtain current smoking status and habitual alcohol drinking (Yes = 1, No = 0).
The scanning procedures are described elsewhere in detail 5, 8. Briefly, scanning was performed using a standardized protocol with a GE-Imatron C150 EBT scanner (GE Medical Systems, South San Francisco, CA, US). A total of 30 to 40 contiguous 3-mm-thick transverse images were obtained from the level of the aortic root to the apex of the heart. Images were recorded during a maximal breath hold using ECG-guided triggering of 100-m-second exposures during the same phase of the cardiac cycle. CAC was considered to be present with three contiguous pixels (area=1 mm2) ≥130 Hounsfield Unit. One trained reader at the University of Pittsburgh Cardiovascular Institute, read the image, using a software from the AccuImage Diagnostic Corporation (San Francisco, CA, US), which calculates coronary calcium score using the Agatston scoring method 33. The presence of CAC was defined as a coronary calcium score ≥10 5, 8. The reproducibility of the scans had an intraclass correlation of 0.98.
The scanning procedures are described elsewhere in detail 5, 34. Briefly, before the study began, sonographers at both centers received training for carotid scanning provided by the University of Pittsburgh Ultrasound Research Laboratory. A Toshiba 140A scanner equipped with a 7.5 MHz-linear-array imaging probe were used at both centers. The sonographers scanned the right and left common carotid arteries, the carotid bulbs, and the internal carotid arteries. For the common carotid artery segment, both near and far walls were examined 1 cm proximal to the bulb. For the bulb and internal carotid artery areas, only far walls were examined. The scans were recorded on videotape and sent to the Laboratory for scoring. Trained readers digitized the best image for scoring and then measured the average IMT across 1 cm segments of near and far walls of the common carotid artery and the far wall of the carotid bulb and internal carotid arteries on both sides. Measurements from each location were then averaged to produce an overall measurement of IMT. The readers were blinded to participant’s characteristics and study center. Correlation coefficients of IMT between sonographers and between readers were 0.96 and 0.99, respectively 5.
To compare risk factors including BP, serum lipids, fasting glucose and smoking and levels of atherosclerosis, i.e., prevalence of CAC and IMT between the two populations, a t-test, or Chi-Square test was used, as appropriate. To compare D-dimer and vWF between the two populations, multiple regression analysis was used adjusting for selected variables based on the previous studies 22, 35. To examine the association between D-dimer and vWF, Spearman correlation was used. To examine associations of both D-dimer and vWF with risk factors as well as with prevalence of CAC or IMT in each population, subjects were first divided into tertile groups based on the levels of D-dimer or vWF. Then, a general linear model was used to test a linear trend across the tertile groups.
Values are expressed as mean ± standard deviation (SD) or error (SE). Significance was considered to be at a level of p < 0.05. SPSS software (release 16.0, SPSS Inc., Chicago, IL) was used for all statistical analyses.
The Japanese men, as compared to the white American men had a less favorable or similar profile of many traditional risk factors despite a 15% lower BMI in the Japanese (Table 1). These traditional risk factors included BP, LDL-C, triglycerides, fasting glucose, and smoking. Meanwhile the Japanese man as compared to white American men had a more favorable profile of some risk factors including HDL-C, insulin, and CRP.
Levels of D-dimer were not statistically different between the two populations whereas levels of vWF were significantly higher in the Japanese men than in whites American men (Table 1). The significantly higher levels of vWF in the Japanese men remained even after adjusting for age, BMI, systolic BP, total cholesterol, triglycerides, HDL-C, fasting glucose, fasting insulin, CRP and smoking (123.7 ± 5.3 % for the Japanese vs. 91.6 ± 5.3 for whites Americans, p < 0.01, mean ± SE).
D-dimer and vWF were moderately but significantly correlated in both populations. Spearman correlations between D-dimer and vWF were 0.21 for the Japanese men (p < 0.05) and 0.30 for the white American men (p < 0.01). D-dimer or vWF did not have significant associations with risk factors (data not shown). Only exception was a significant positive association of D-dimer with age in the American white men.
D-dimer had a significant positive association with the prevalence of CAC in the Japanese men (p for trend = 0.04) and a marginally significant positive association with IMT in the white American men (p for trend = 0.06) (Table 2A). The significant association in the Japanese men was attenuated and became non-significant after adjusting for age, BMI, smoking, systolic BP, HDL-C, LDL-C, triglycerides and fasting glucose (p for trend = 0.19) (Table 2A). D-dimer had no significant association with IMT in the Japanese men or with the prevalence of CAC in the whites American men.
vWF had a marginally significant inverse association with IMT in the Japanese men (p for trend = 0.05) (Table 2B). The association of vWF with IMT did not change much after adjusting for age, BMI, smoking, systolic BP, HDL-C, LDL-C, triglycerides and fasting glucose (p for trend = 0.06) (Table 2B). vWF had no significant association with the prevalence of CAC in either the Japanese men or whites American men although vWF appeared to have a positive association with prevalence of CAC in both populations (Table 2B).
Our study shows that in men aged 40–49, Japanese in Japan as compared to white Americans had similar levels of D-dimer and higher levels of vWF. Additionally our study suggests that the difference in D-dimer or vWF was not associated with lower levels of atherosclerosis in Japanese men in Japan as assessed by CAC and IMT. To our knowledge, this is the first study that compared D-dimer levels between Japanese and white American men, although several studies compared D-dimer levels in other ethnic groups.31–32, 36.
The finding that the Japanese had higher levels of vWF than white Americans was somewhat unexpected, since several epidemiological studies have reported that high levels of vWF have been predictive of an adverse prognosis in CHD 16–17, 20–22. Furthermore, the finding is inconsistent with the result from one previous study reporting that the levels of vWF are similar between the Japanese and whites 30. The discrepancy with the result from the previous study may be due to differences in the dietary intake of saturated fat which has a positive association with vWF 37, between the studied Japanese populations. The dietary intake of saturated fat in our study population is likely to be much higher than that in the previous study because our study examined those born after WWII who were exposed to a westernized lifestyle from childhood whereas the previous study examined mostly subjects born before WWII. It is also possible that the difference in the levels of vWF between the Japanese and whites observed in our study is due to genetics since the heritability for vWF is reported to be 32 to 72% 38–39.
D-dimer had a significant positive association with CAC in the Japanese but not in white Americans and had no significant association with IMT in either the Japanese or white Americans. Very few studies have reported the association of D-dimer with CAC. The Atherosclerosis Risk in Community Study reported no significant association of D-dimer with CAC in a predominantly white population.24 The significant positive association of D-dimer with CAC in the Japanese must be confirmed with other studies. Our result showing no significant association of D-dimer with IMT is consistent with results from most, but not all, previous studies 18 25–26.
D-dimer had no significant associations with risk factors. This finding is generally consistent with the results from previous studies. Most previous epidemiological studies found no association of D-dimer with BP, cholesterol, diabetes, or BMI 12, 19, 35, although several studies observed a weak association with CRP 14 or HDL-C/total cholesterol ratio 40.
vWF had no significant association with CAC and had a negative or no significant association with IMT. Generally the results do not contradict those from previous studies 17, 24–27. As for the association of vWF with CAC, our result showing no significant association is consistent with those from previous studies 24–25. As for the association of vWF with IMT, the Coronary Artery Risk Development in Young Adults Study found a significant negative association of vWF wth IMT in white American men, but not in white American women or black American men or women.25 Additionally, one previous study in Japan showed no significant association of vWF with IMT 27. One exception is a study in Spain which showed a significant positive association of vWF with IMT, even after adjusting for potential cofounders 17.
We didn’t find any association of vWF with risk factors in either population, except the negative association with age in white American men. Several studies have shown significant associations of vWF with risk factors but the results are inconsistent; some showed positive associations with CRP 21, diabetes status 29, HOMA-IR 29, 41, and BMI 29–30, whereas others showed no association with lipids 21, 30, fasting glucose 21, BMI 21, and smoking 21, 29–30.
It is hypothesized that D-dimer and vWF are less involved in early plaque development and atherogenesis 24–26, and are more related to CHD events and thrombogenesis10–16, 19–22. It is also hypothesized that D-dimer predicts future CHD risk independent of traditional risk factors 10–15, 18–19, 23. Our results showing a weak association of both D-dimer and vWF with two measures of atherosclerosis: CAC and IMT, may be consistent with the first hypothesis. Our results neither support nor oppose the second hypothesis.
Since we have reported that in men in a post WWII birth cohort, Japanese men in Japan have lower levels of atherosclerosis than American white men,5 we have investigated many factors that might be responsible for the lower levels in Japanese men. Traditional risk factors including BP, smoking, LDL-C, HDL-C, and diabetes as well as CRP and fibrinogen did not explain the difference in atherosclerosis between Japanese men and white American men5. The difference in the distribution of lipoprotein particles did not explain the difference34. Adiponectin, which has anti-atherogenic, anti-inflammatory, and anti-diabetic properties, is unexpectedly lower in Japanese man than white American men42 and is unlikely to explain the difference. Lipoprotein-associated phospholipase A2, which is considered as a pro-inflammatory mediator involved in the vascular inflammation, did not explain the difference43. PAI-1, an independent predictor of cardiovascular disease, is unexpectedly higher in Japanese men than white American men28 and is unlikely to explain the difference. We have recently reported that the difference in serum levels of n-3 fatty acids may be responsible for the difference in atherosclerosis between Japanese men and white American men8. However the study is cross sectional and we cannot establish the causality. The ongoing follow-up study would provide more information on the association of n-3 with atherosclerosis. The current study adds an important piece of information showing that differences in D-dimer or vWF between Japanese men and white American men are unlikely to explain the difference in atherosclerosis between the two groups.
Several limitations of the study warrant consideration. The sample size was relatively small. However, it appears to be unlikely that the difference in the levels of D-dimer or vWF between Japanese men and white American men in this post WWII birth cohort would account for the difference in the levels of atherosclerosis between the two groups. We analyzed the data using a cutoff point of coronary calcium score of 0 because this is reported to be clinical significant44. However, we did analyze the data using a cutoff point of coronary calcium score of 0 and the results were (data not shown). We only examined men aged 40–49; thus the results are not generalizable to women or other age groups. Finally, we acknowledge that we cannot determine causal relationships due to the cross-sectional nature of the study.
In conclusion, our study shows that in men aged 40–49, Japanese in Japan as compared to white Americans had similar levels of D-dimer and higher levels of vWF. Japanese men had significant positive association of D-dimer with prevalence of CAC, and marginally significant association of vWF with IMT. Other than these associations, D-dimer or vWF did not have significant associations with prevalence of CAC or IMT either in Japanese or white American men. Our results suggest that the difference in D-dimer or vWF is not associated with lower levels of atherosclerosis in Japanese men in Japan as assessed by CAC and IMT.
We thank you for Dr. Nancy Glynn, Research Assistant Professor of Epidemiology, Graduate School of Public Health, University of Pittsburgh for her English editing.
This research was supported by grants R01 HL68200 and HL071561 from the National Institutes of Health, B 16790335 and A 13307016 from the Japanese Ministry of Education, Culture, Sports, Science and Technology.
Names of institutions
University of Pittsburgh, Pittsburgh, PA, USA and Shiga University of Medical Science, Otsu, Japan
Conflict of interest