Impaired fibrinolysis and increased fibrinogen levels in South Asian subjects

Atherosclerosis 156 (2001) 457– 461 www.elsevier.com/locate/atherosclerosis

Impaired fibrinolysis and increased fibrinogen levels in South Asian subjects Kirti Kain *, Andrew J. Catto, Peter J. Grant Academic Unit of Molecular Vascular Medicine, G-Floor, Martin Wing, Leeds General Infirmary, Leeds LS1 3EX, UK Received 28 April 2000; received in revised form 4 September 2000; accepted 3 October 2000

Abstract The potential role of haemostatic risk markers is largely unexplored in South Asians, who have increased morbidity and mortality from cardiovascular disease and an increased prevalence of insulin resistance. To investigate differences in thrombotic risk markers between South Asian and White populations, 42 Asian and 50 White males and 96 Asian and 80 White females, clinically free from vascular disease, were recruited. Venous blood samples were taken for measures of haemostasis and determination of blood lipids. South Asian females showed lower fasting blood glucose than White females (4.6 vs. 4.8 mmol/l, PB 0.008). In the South Asian population, total cholesterol was lower in females, with a similar trend in males (females 5.0 vs. 5.5 mmol/l, P B0.001; males 5.1 vs. WM 5.5 mmol/l, P= 0.09), but no difference in triglyceride levels. South Asian subjects of both genders had markedly higher levels of fibrinogen (females 3.3 vs. 2.8 mg/dl, P B 0.0005; males 3.0 vs. 2.5 mg/dl P B0.002) and PAI-1 activity (females 14.6 vs. 8.7 ng/ml, PB0.0005, males 21.3 vs. 12.2 ng/ml, ) P B 0.0005). Factor VII:C was lower in both South Asian groups (females 110.9 vs. 122.4%, PB0.005; males 103.3 vs. 125%, P B 0.0005). Factor XII was lower in South Asian females and there were no differences in Factor XII levels in male populations. These results suggest that elevated PAI-1 and fibrinogen in Asians of both genders may contribute to the increased vascular risk experienced in this population; however, the role of dyslipidaemia and Factor VII are not clear in these processes. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: South Asians; Whites; Ethnicity; Cardiovascular risk; Haemostasis

1. Introduction The migrant South Asian population resident in the United Kingdom (UK), Singapore, South Africa or Trinidad show markedly increased morbidity and mortality from ischaemic heart disease compared with indigenous populations [1]. The cause of this increase is not known. It is clear that the conventional risk factors for ischaemic heart disease in White populations do not entirely account for these findings. Smoking, alcohol intake, obesity and cholesterol levels [2,3] are the same or lower in South Asian populations and whilst the age adjusted prevalence of hypertension has been found to be higher in South Asians in one study [4], mean blood pressures are the same or lower in other studies [2,5]. There is some evidence that South Asian populations * Corresponding author. Tel.: + 44-113-3922705; fax: +44-1132423811. E-mail address: [email protected] (K. Kain).

are more sedentary [6], although the potential relationship to insulin resistance and risk clustering needs further evaluation. Type II diabetes mellitus due to insulin resistance is more prevalent in South Asians [7] but its contribution to the increased risk appears inconsistent [8,9]. The development of coronary artery disease and myocardial infarction has both atheromatous and thrombotic components and evidence exists to implicate a number of coagulation and fibrinolytic proteins in this process. Case-control and prospective studies of myocardial infarction in Whites have reported elevated fibrinogen [10], FVII:C [11], plasminogen activator inhibitor-1 (PAI-1)[12,13], tissue plasminogen activator (t-PA) [14] and activated FXII [15] as being related to outcome. Little is known regarding haemostasis in healthy Asian populations. Therefore, the aim of this study was to investigate circulating coagulation and fibrinolytic measures in Asian and White subjects free from a

0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 0 ) 0 0 6 8 4 - 5

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personal history of diabetes mellitus, hypertension or vascular disease.

2. Materials and methods

2.1. Selection of Asian and White subjects Ninety-six healthy South Asian females and 42 South Asian males were compared with 80 healthy White females and 50 White males [16]. The subjects in the two groups were matched for age and had no personal or family history of diabetes, hypertension or clinically detectable vascular disease (cardiovascular, cerebrovascular or peripheral vascular). They were randomly selected from the community of West Yorkshire Health Authority general practice registers. South Asians were described as subjects resident in United Kingdom (UK) who had migrated from India, Pakistan or Bangladesh, or their four grandparents were from one of the three South Asian countries. Subjects with mixed parentage were not included in the study. All subjects gave informed consent according to a protocol approved by United Leeds Teaching Hospitals NHS Trust and Bradford Hospitals NHS Trust.

2.2. Clinical details The height and the weight were recorded without shoes and in light clothing. Body mass index (BMI) was calculated from weight in kilograms divided by square of the height in meters. Blood pressure (BP) was taken after 15-min rest and as the mean of three readings with subjects supine and to the nearest 2 mmHg. Smoking history was classified into subjects who never smoked and smokers if they were currently smoking.

2.3. Laboratory methods A 19-g butterfly needle was used to take free flowing venous blood before 10:30 h following an overnight fast and abstention from smoking. For FXII, fibrinogen and FVII:C, blood was taken into 0.1 M trisodium citrate at room temperature and centrifuged at 2500× g for 15 min within the hour. To prevent cold activation of FXII during processing, plasma aliquots were snap frozen in liquid nitrogen and stored at −40°C. Samples were thawed for 10 min at 37°C before measuring FXIIa using a direct enzyme immunoassay, which detects both a-XIIa and b-XIIa in human plasma (Shield Diagnostics, Dundee, UK) [17]. The inter-assay and intra-assay coefficients of variation (CV) were 9.3 and 9.9%, respectively. Fibrinogen levels were determined by the Clauss method [18] on the KC10 Coagulometer (Amelung), the inter-assay CV being 3.5% and intra-assay CV 2%. Plasma Factor

VII:C levels were determined by a clotting assay on the ACL 3000 plus (Instrumentation Laboratory, Warrington, UK) using human Factor VII deficient plasma and rabbit thromboplastin, which contains tissue factor and other components necessary to activate FVII (Instrumentation Laboratory, Warrington, UK) as reagents. Clotting activity of Factor VII:C was expressed as a percentage of activity given by calibration plasma and the inter-assay and intra-assay CV were 4.3 and 3.2%, respectively. Samples for PAI-1 activity were taken into 0.1 M trisodium citrate, placed immediately in an ice-water mixture and separated within 30 min by centrifugation at 3000× g for 30 min at 4°C. Plasma samples were snap-frozen in liquid nitrogen and stored at − 40°C. A chromogenic assay was used to determine PAI-1 activity (Spectrolyse, Biopool, Umea, Sweden). Inter-assay CV for PAI-1 activity was 9% and intra-assay CV 6.3%. Total triglycerides and total cholesterol were measured on the Hitachi 747 autoanalyser (Boehringer Mannheim, Mannheim, Germany). Plasma glucose was determined by a glucose oxidase method.

2.4. Statistical analysis Circulating concentrations of fibrinogen, PAI-1 and triglycerides were log-transformed to normalise the distribution. All log-transformed results are expressed as geometric mean and anti-logged standard deviation (S.D.). All other results are expressed as mean (95% confidence intervals). Differences in levels between Asian and White subjects were compared for both sexes separately using an unpaired Student t-test. Data on sex and smoking were compared by the  2-test. The Mann– Whitney U-test was used for comparison of BMI and glucose levels. Bivariate correlations between classical risk factors and haemostatic factors were analysed by Spearman’s method. All statistical calculations were performed on the SPSS statistical package (version 8.0) (SPSS Inc. Chicago, IL, USA).

3. Results

3.1. Clinical and biochemical characteristics of Asian and White subjects All subjects recruited into the study populations were free of clinically detectable vascular disease (stroke, ischaemic heart disease or peripheral vascular disease). Subjects with a personal history or biochemical evidence of diabetes mellitus or with hypertension (defined as BP \ 140/90 mmHg, or receiving antihypertensive therapy) were excluded from the study. Eleven percent of South Asian subjects and all the White subjects were born in the UK, the rest of the South Asian subjects

K. Kain et al. / Atherosclerosis 156 (2001) 457–461

were migrants. Of all the South Asians, 52% had migrated from Pakistan, 2% from Bangladesh and 46% from India. Seventeen percent of Indians were Gujratis and the rest were Sikhs. Subjects were matched for age. There was no significant difference in the BMI between the two groups (Table 1). The mean age for both South Asian and White females was 40 and for males in both groups, it was 41. There was a higher prevalence of current smokers in Asian men (31 vs. 4% in Whites P= 0.01), but no Asian females were current smokers (0 vs. 11% in White females P = 0.001). Mean (S.D.) total cholesterol concentrations were significantly lower in Asian compared with White females (5.0 (0.9) vs. 5.5 (1.1) mmol/l P B0.001) and a similar trend was observed in males (5.1(1.2) vs. 5.5 (1.1) mmol/l, P =0.09). In contrast, there were no differences in triglyceride concentrations either by gender or ethnicity. Fasting blood glucose (median, range) was lower in Asian females than their White counterparts (4.6(3.8– 6.0) vs. 4.8(3.9–6.4) mmol/l, P =0.008), but no differences were observed between males in the two ethnic groups.

3.2. Haemostatic markers in Asian and White subjects Of all the haemostatic variables measured, levels of fibrinogen (females 3.32 vs. 2.8 mg/dl, P B 0.0005; males 3.0 vs. 2.5 mg/dl, P B0.002) and PAI-1 activity (females 14.6 vs. 8.7 ng/ml, P B0.0005; males 21.3 vs. 12.2 ng/ml, PB 0.0005) were significantly higher in Asians of both genders compared with their White counterparts. Fibrinogen, PAI-1 activity and FVII:C correlated with age, BMI, cholesterol, triglycerides and fasting glucose in Whites. In Asians only, fibrinogen and PAI-1 activity correlated with BMI, PAI-1 and FXII with fasting glucose, and PAI-1, FVII:C and FXIIa with blood lipids. Univariate analysis of variance with age, gender, ethnicity, BMI, cholesterol and triglycerides, smoking and fasting glucose as covariates and PAI-1 activity and fibrinogen as dependant variables was done. Fibrinogen was associated with age,

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gender, BMI, smoking and ethnicity. PAI-1 activity was associated with BMI triglycerides and ethnicity. Therefore, the difference in fibrinogen and PAI-1 between Whites and South Asians was significantly different even after adjustment for age, gender, BMI, cholesterol, triglycerides and fasting glucose. There was no influence of country of origin on fibrinogen and PAI-1 levels (data not shown). FVII:C activity was lower in both Asian males and females compared with White subjects (females 110.9 vs. 122.4%, PB 0.005; males 103.3 vs. 125%, PB 0.0005) and in a regression model, FVII:C was associated only with age and ethnicity (r=0.137, P= B 0.0005). FXIIa, (females 2.13 vs. 2.8 ng/ml, P= B0.002) was significantly lower in Asian females and there were no differences between the male groups.

4. Discussion This study demonstrates marked ethnic differences in cardiovascular risk markers between matched Asian and White populations of both genders. In this study, healthy Asian subjects demonstrated increased levels of fibrinogen and PAI-1 activity and lower concentrations of cholesterol and factor VII:C, irrespective of gender, when compared with healthy White control subjects. Lower Factor XIIa levels were observed only in Asian females compared with White females. The development of atherothrombotic disorders in Asian populations is of considerable importance because of the very high prevalence of both coronary artery and cerebrovascular disease in this ethnic group [19]. Official figures published in 1991 indicated that the standardised mortality ratio in Asian males (aged 20– 69 years) was 146 for coronary artery disease and 155 for stroke [19]. A number of epidemiological studies have attempted to tease out the differences in classical cardiovascular risk factors between Asian and White populations with conflicting results [8,9]. Diabetes mel-

Table 1 Conventional and haemostatic variables in South Asian and White subjectsa Factors

Asian females

White females

P

Asian males

White males

P

Number Age (years) Body mass index (kg/m2) Present smoker Cholesterol (mmol/l) Triglycerides (mmol/l) Glucose (mmol/l) Fibrinogen (mg/dl) PAI-1 activity (ng/ml) FXIIa (ng/ml) FVII:C (%)

96 40 (23–75) 26 (17–41) 0/96 5.0 (0.9) 1.0 (1.6) 4.6 (3.8–6.0) 3.32 (1.28) 14.6 (2.2) 2.1 (1.6) 110.9 (27.0)

80 40 (22–74) 24 (18–52) 8/72 5.5 (1.1) 1.0 (1.6) 4.8 (3.9–6.4) 2.79 (1.23) 8.8 (2.4) 2.8 (1.2) 122.4 (25.0)

0.782 0.165 0.001 B0.001 0.331 0.008 B0.0005 B0.0005 B0.002 0.005

42 41 (23–75) 25 (18–58) 10/32 5.1 (1.2) 1.5 (1.7) 5.0 (3.7–6.7) 2.98 (1.29) 21.3 (1.9) 2.1 (1.0) 103.3 (22.9)

50 41 (27–70) 25 (19–34) 2/48 5.5 (1.1) 1.3 (1.54) 5.0 (4.2–5.8) 2.53 (1.25) 12.2 (2.2) 2.4 (1.0) 125.0 (29.0)

0.98 0.49 0.01 0.09 0.11 0.64 B0.002 B0.0005 0.10 B0.0005

a

Values are median (range) or geometric mean (S.D.).

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litus is three to five times more common than in White populations [20], and there are some reports of lower HDL cholesterol [2] and elevated triglycerides [21,22] in Asians. However, whilst total cholesterol is an important risk factor in Whites, it has been found to be lower in Asian subjects [3]. In Whites, there is substantial evidence linking PAI-1, Factor VII:C, fibrinogen and Factor XII with cardiovascular disease [10– 12,15]. There have been relatively few studies of the thrombotic component of vascular risk in Asian subjects. Levels of fibrinogen have been previously reported as being low in healthy Indian Asians [2,5] when compared with the indigenous population. Similarly, a study of PAI-1 levels showed no difference between South Asian and White subjects with and without diabetes mellitus [23]. These findings are in marked contrast to those in the present study and could be due to different analytical methods or sample size. However, FVII:C has been found lower in Asians compared with Whites (Miller [2] and Lane [24]) and our result accord with these findings. There have been no studies of Factor XII in Asian subjects although Kohler et al. found that FXIIa correlated with PAI-1, triglycerides and tissue plasminogen antigen in healthy White controls and was elevated in White patients with coronary artery disease [15]. In the present study of well-characterised, clinically healthy subjects, fibrinogen levels and PAI-1 activity were elevated markedly in both male and female Asians compared with Whites. Epidemiological evidence from White subjects extrapolated to the Asian community would suggest that this combination of high fibrinogen and increased PAI-1 activity could have an important role in the increased risk of vascular disease associated with Asian ethnicity. The explanation for these differences between the two ethnic groups is at present speculative. Elevated PAI-1 levels could be attributed to increased visceral fat, as omental adipocytes are a potential source of PAI-1 [25]. In support of this, Asians have increased intra-abdominal fat as indicated by the waist-hip ratio [21]. Unfortunately waist-hip ratio was not available for the White subgroup in our study and the present study did not set out to evaluate insulin resistance per se. However, against this argument, a measure of the BMI that is thought to influence fibrinogen [11] and PAI-1 levels [26] was not significantly different between the two groups. Alternatively, low levels of physical fitness may be contributing to increased PAI-1 levels and fibrinogen levels through a similar mechanism [27]. An important environmental determinant of fibrinogen is smoking [11], although this cannot account for all the observed differences, as there were no smokers amongst Asian females. It is, however, possible that part of the differences in fibrinogen between males might be attributed directly to smoking, as smoking rates were higher in Asian males. However, in

our White male population, we observed lower than expected rates of smoking. Genetic determinants influence fibrinogen levels in Whites [28] and it is possible that they vary in Whites and South Asians. There is some controversy as to the role of elevated levels of Factor VII:C as a risk factor for coronary artery disease. In the Northwick Park Heart Study, a 1 S.D. increase in levels was associated with 62% increase in risk of fatal MI in Whites over a 5-year period [11]. Similar results were obtained in the PROCAM study, although this did not reach significance [29]. In the present study, levels of Factor VII:C were higher in Whites than Asians in both sexes. These findings probably militate against the hypothesis that higher levels are associated with an increased risk of vascular disease, although it is possible that Factor VII has different effects on vascular risk in different populations. FVII:C levels correlate with dietary fat intake and South Asians have been shown to reduce intake of saturated fat, when compared with Whites [3]. It is possible that some of the observed differences could be attributed to dietary influences. Alternatively, levels of Factor VII are strongly related to genotype, and there is an increased frequency of the Gln353 allele in Asians, which is associated with 20% lower Factor VII levels [24]. A similar argument may relate to the generally lower levels of Factor XII in the Asian subjects. Levels of Factor XII are also strongly related to genotype at a 3% non-coding polymorphism although the prevalence of this mutation in the Asian populations is presently unknown [30]. This study has its limitations, principal weaknesses being that the numbers are small, and we did not take into account socio-economic status or dietary and lifestyle factors. There is some evidence of heterogeneity amongst the South Asian population [31]. When the data was analysed according to country of origin, there were no differences in levels of fibrinogen and PAI-1. However, the numbers in each of these subgroups is small, and a larger study would be required to tease out likely changes resulting from regional variation. There is gene effect on the levels of the circulating haemostatic proteins, but the genotypic data is missing from this study. In summary, clinically healthy Asian populations show a different pattern of thrombotic risk markers compared with Whites. This is manifested by increased fibrinogen and PAI-1 activity in Asians irrespective of gender, whilst levels of Factor VII:C and Factor XII are generally lower in this population. The results of this study indicate the potential importance of fibrinogen and PAI-1 in the pathogenesis of vascular disease in Asian subjects. In the future, prospective studies in ethnic populations will help to elucidate further the role of thrombotic risk markers in the increased vascular risk in the westernised Asian community.

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Acknowledgements This study was supported by a grant from the Stroke Association (UK).

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