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Increased fibrinogen, von Willebrand factor and tissue plasminogen activator levels in insulin resistant South Asian patients with ischaemic stroke
Atherosclerosis 163 (2002) 371 /376 www.elsevier.com/locate/atherosclerosis
Increased fibrinogen, von Willebrand factor and tissue plasminogen activator levels in insulin resistant South Asian patients with ischaemic stroke Kirti Kain a,*, Andrew J. Catto a, John Young b, John Bamford c, John Bavington d, Peter J. Grant a a
Academic Unit of Molecular Vascular Medicine, University of Leeds, G-Floor, Martin Wing, Leeds General Infirmary, Leeds LS1 3EX, UK b Department of Medicine for the Elderly, St. Luke’s Hospital, Bradford, UK c Department of Neurology, St. James’s University Hospital, Leeds, UK d Transcultural Unit, Lynfield Mount Hospital, Bradford, UK Received 14 August 2001; received in revised form 14 December 2001; accepted 18 January 2002
Abstract The aim of this study was to determine differences in atherothrombotic risk factors in South Asian subjects with a history of ischaemic stroke and South Asian subjects free from personal and family history of clinically detectable stroke. Eighty South Asian patients with ischaemic stroke (confirmed on cranial computerised scan) and 80 South Asian controls with similar age and gender distributions were recruited at random. The frequency of hypertension (P / B/0.0001), myocardial infarction (P /0.003) and diabetes mellitus ( B/0.0001) were significantly higher in stroke patients. Stroke patients had lower high-density lipoprotein cholesterol (0.95 vs. 1.1 mmol/l, P / B/0.0001), higher plasma glucose (8.1 vs. 6.6 mmol/l, P /0.01) and trendwise higher HBA1C (6.4 vs. 6.0%, P /0.09). There was no difference in insulin levels but insulin resistance was significantly higher in stroke patients (3.75 vs. 2.66, P /0.01). Stroke patients showed elevated levels of fibrinogen (3.78 vs. 3.41 mg/dl, P /0.02), von Willebrand factor (1.78 vs. 1.50 IU/ml, P /0.006) and tissue plasminogen activator (12.8 vs. 11.3 ng/ml, P /0.04), but the differences did not persist after adjustment for glucose, triglycerides, HDL, WHR, and BMI. Higher levels of fibrinogen, von Willebrand factor and t-PA in South Asian stroke patients disappeared after adjustment for features of insulin resistance syndrome but persisted after adjustment for presence of diabetes, confirming that these changes are essentially dependant on features of insulin resistance syndrome. A prospective study would be required to elucidate the role of thrombotic risk factors in South Asians with ischaemic stroke. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: South Asian; Stroke; Ethnicity; Cardiovascular risks; Haemostasis
1. Introduction Cerebrovascular disease is 1.5 times more common in the South Asian population resident in UK, compared to the indigenous White population [1]. The precise mechanisms, which account for the excess cerebrovascular mortality among South Asians, are poorly understood. However, several studies indicate that plasma insulin level and insulin resistance is higher in South
* Corresponding author. Tel.: 44-113-392-3472; fax: 44-113242-3811. E-mail address: [email protected] (K. Kain).
Asians compared to Whites [2 /4]. Consequently, South Asians tend to have more abnormalities in those risk factors clustering with hyperinsulinaemia and insulin resistance specifically, higher waist /hip ratio, hypertriglyceridaemia, lower high-density cholesterol and an increased prevalence of type 2 diabetes mellitus and hypertension [2,4,5]. These observations might account for some of the excess risk of cerebrovascular disease in South Asians. This is of interest as several, but not all, prospective studies in Whites demonstrate that hyperinsulinaemia is associated with an increased risk of ischaemic stroke [6 /8]. Furthermore, haemostatic factors and fibrinolytic factors in particular, such as plasminogen activator inhibitor-1 and tissue plasmino-
0021-9150/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 2 ) 0 0 0 2 5 - 4
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K. Kain et al. / Atherosclerosis 163 (2002) 371 /376
gen activator, might be also of interest in this context, as these factors have also been associated with insulin resistance [9 /12]. However, there is no data on conventional features of the insulin resistance syndrome, or its haemostatic components in South Asians with ischaemic stroke. Therefore, the aim of this study was to compare features of the insulin resistance and haemostatic risk factors in South Asian patients with history of confirmed ischaemic stroke and South Asian subjects without stroke.
abdominal girth to the nearest 0.5 cm and dividing it by measurement at maximal protrusion of the hips at the level of symphysis pubica also to the nearest 0.5 cm. Smoking history was classified into subjects who never smoked and smokers if they were ex-smokers or current smokers. Subjects with diabetes and hypertension were classified as known if they were already on treatment or their physician had diagnosed them. The majority of the subjects with diabetes had type 2 diabetes. 2.3. Laboratory methods
2. Materials and methods 2.1. Study subjects Eighty South Asian patients with a clinical diagnosis of ischaemic stroke according to WHO criteria were compared with 80 South Asian control subjects randomly recruited from the community, free from a personal or family history of ischaemic stroke. A history of ischaemic stroke was confirmed in South Asian patients from clinical records and the results of a noncontrast computerised cranial tomography scan. To minimise the confounding influence of the acute phase response on coagulation and fibrinolytic factors, stroke patients were recruited at least 2 months after the acute event. Eligible control subjects matched for ethnicity were recruited at random by a postal questionnaire from Family Health Authority General Practitioner’s lists with the aim of having similar age and gender distribution. Both patients and controls were recruited simultaneously over a period of 2 years and from the same geographical location (West Yorkshire, UK). All subjects were of South Asian origin (India, Pakistan or Bangladesh) and their four grandparents were from one of these countries. Subjects with mixed parentage were not recruited. All the subjects received a volunteer information sheet in English and different translations namely Gujrati, Punjabi, Bengali, Hindi and Urdu according to the subject’s ethnic background. Subjects from India, Pakistan and Bangladesh were equally represented in both groups. All subjects gave written informed consent and Leeds and Bradford Ethics Committee UK gave the ethical approval for study. 2.2. Assessment of anthropometric and clinical risk factors Height and weight were recorded without shoes and in light clothing. Body mass index (BMI) was calculated from weight in kilograms divided by height squared in meters. Blood pressure (BP) was taken after 15 min rest and the mean of three readings, with subjects lying down and to the nearest 2 mmHg. Waist /hip ratio (WHR) was calculated by taking the measurement at minimum
A 19 g-butterfly needle was used to draw free flowing venous blood before 10:30 am following an overnight fast and abstention from smoking. For FXII, fibrinogen, von Willebrand factor (vWF) and FVII antigen, 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 8C. Samples were thawed for 10 min at 37 8C before measuring FXIIa using a direct enzyme immunoassay, which detects both a-XIIa and b-XIIa in human plasma (Shield Diagnostics, Dundee, UK) [13]. 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 [14] on the KC10 Coagulometer (Amelung), the inter-assay CV being 3.5% and intra-assay CV 2%. FVII antigen and vWF antigen (DAKOPATTS) were determined by enzyme linked immunoabsorbant assay (ELISA), inter-assay and intra-assay CV being 8.2, 4.3, 4.7 and 2.8% respectively. Samples for plasminogen activator inhibitor-1 (PAI1) antigen and tissue plasminogen activator (t-PA) antigen 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 8C. Plasma samples were snap-frozen in liquid nitrogen and stored at /40 8C. An ELISA assay was used to determine PAI-1 antigen (Biopool cat #214202) and t-PA antigen (Juhan Vague 1992 ECAT assay procedures) [15], and inter-assay and intra-assay CV were 5, 9% for PAI-1 antigen and 10, 8% for t-PA, respectively. The homeostasis model assessment (HOMA) model was used to calculate insulin resistance (IR), which assumes that normal weight subjects aged less than 35 years have a 100% pancreatic islet b-cell function and IR of unity (HOMA score /fasting insulin /fasting plasma glucose divided by 22.5) [16]. Plasma glucose was determined by a glucose oxidase method. Total triglycerides and total cholesterol were measured on the Hitachi 747 autoanalyser (Boehringer Mannheim, Mannheim, Germany). White cell count
K. Kain et al. / Atherosclerosis 163 (2002) 371 /376
(WBC) was determined by modified flourocytometer and plasma viscosity (PV) was measured by Coulter viscometer. Plasma insulin was also determined by ELISA, and the inter-assay and intra-assay CV were 5.6 and 5.3% at 18 mIU/ml, and 9.8 and 3.0% at 84 mIU/ ml for insulin. 2.4. Statistical analysis Skewed atherothrombotic continuous variables were log (natural) transformed if they were non-normally distributed and then compared by unpaired Student’s t test. Categorical data was compared by x 2 test. Partial correlations were used to study age and gender adjusted correlations. Analysis of variance was used to calculate differences between patients and controls after adjustment for covariates. Regression analysis on log-transformed data was carried out to determine independent predictors of fibrinogen, t-PA and vWF. All the analysis were done by using SPSS (version 9) (SPSS Inc. Chicago, USA).
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the two groups. The prevalence of hypertension, diabetes mellitus and myocardial infarction was higher in patients. No difference was observed in the fasting cholesterol and triglycerides between the two groups, HDL-cholesterol was lower in patients. Insulin levels were the same, but insulin resistance (derived from the HOMA estimation) was greater in patients than in controls (Table 1). The WHR was increased in males compared to females in both patients and controls (1.01 vs. 0.92, P / B/0.0001; 0.97 vs. 0.92, P /0.003). BMI, FPG, cholesterol, triglycerides, HDL-cholesterol and insulin were similar in males and females in patients and controls. In the patients, 15% were on lipid lowering agents and 55% on treatment for diabetes mellitus (16% on diet alone, 64% on oral hypoglycaemic agents and 20% on insulin). Only 1% of the control subjects were on lipid lowering agents and 24% on treatment for diabetes mellitus (47% on diet, 48% on oral hypoglycaemic agents and 5% on insulin). The unadjusted levels of fibrinogen, t-PA and vWF were higher in patients compared to controls (Table 1).
3. Results
3.2. Fibrinogen
3.1. Demographic and clinical characteristics of the South Asian patients and South Asian controls
Age- and gender-adjusted correlates are shown in Table 2. Fibrinogen correlated with PV in both groups. The values of fibrinogen were not significantly different in smokers and non-smokers in patients but were higher in smokers in controls (6.07 vs. 3.38 mg/dl, P /0.03). There
Table 1 shows there were no significant differences in age, gender, and anthropometric measurements between
Table 1 Demographic, conventional and haemostatic variables in South Asian stroke patients and South Asian controls Variables
Patients (n 80)
Controls (n 80)
P
Male: female Age (years) Body mass index (kg/m2) Waist /hip ratio Current smokers (%) Hypertension (%) Myocardial infarction (%) Diabetes mellitus (%) WBC ( 109/l) Plasma viscosity (mPas) Fasting plasma glucose (mmol/l) HbA1C (%) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) Triglycerides (mmol/l) Fasting insulin (mU/l) Insulin resistance (HOMA) Fibrinogen (mg/dl) Activated FXIIa (ng/ml) PAI-1 antigen (ng/ml) Factor VII antigen (%) Tissue plasminogen activator (ng/ml) von Willebrand factor (%)
40:40 63 (34) 25 (1.2) 0.96 (0.09) 13 60 22 61 7.37 (1.6) 1.72 (0.11) 8.10 (4.2) 6.43 (1.3) 5.48 (1.1) 0.95 (1.3) 2.00 (1.6) 11.31 (1.9) 3.75 (2.3) 3.78 (1.3) 2.08 (1.7) 12.63 (2.3) 109 (33) 12.84 (4.6) 1.78 (0.7)
38:42 61(35) 26(1.2) 0.94 (0.08) 1 28 6 30 7.00 (1.5) 1.70 (0.11) 6.60 (3.0) 6.00 (1.3) 5.67 (1.0) 1.10 (1.44) 1.81 (1.7) 9.61 (2) 2.66 (2.3) 3.41 (1.3) 2.10 (1.6) 13.5 (2.6) 105 (27) 11.30 (4.4) 1.50 (0.5)
0.9 0.8 0.5 0.09 0.009 B 0.0001 0.003 B 0.0001 0.16 0.27 0.01 0.09 0.26 0.004 0.14 0.14 0.01 0.02 0.92 0.65 0.43 0.04 0.006
Age-median (range), other values are mean (standard deviation).
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Table 2 Age-and -gender adjusted correlation coefficients of haemostatic risk factors in South Asian stroke patients and controls Risk factors
Fibrinogen patients
Fibrinogen controls
t-PA patients
t-PA controls
vWF patients
vWF controls
BMI WHR Triglycerides HDL-cholesterol Fasting plasma glucose Insulin Plasma viscosity WBC Systolic blood pressure Diastolic blood pressure PAI-1 antigen t-PA VWF Factor VII antigen Factor XII Fibrinogen
0.04 0.03 0.22 0.38** 0.20 0.01 0.50** 0.30 0.12 0.09 0.11 0.11 0.06 0.08 0.24*
0.06 0.10 0.19 0.09 0.09 0.15 0.48* 0.28* 0.11 0.18 0.13 0.00 0.33** 0.11 0.01
0.01 0.04 0.43* 0.02 0.22 0.19 0.25 0.20 0.08 0.04 0.32**
0.29* 0.31* 0.32** 0.26* 0.16 0.30* 0.28* 0.14 0.16 0.09 0.44**
0.04 0.49** 0.01 0.14 0.17 0.20 0.03 0.08 0.14 0.17 0.18 0.25*
0.00 0.02 0.15 0.00 0.03 0.11 0.41** 0.32** 0.10 0.15 0.08 0.04
0.00 0.01 0.06
0.11 0.08 0.32**
0.25* 0.07 0.04 0.11
0.04 0.15 0.07 0.00
*Indicates significance (P B 0.05). **Indicates significance (P B 0.005).
were no differences in fibrinogen values between males and females in patients or controls. The values of fibrinogen were similar between subjects with diabetes mellitus and those without, in both patients and controls. On linear regression analysis with age, gender, BMI, WHR, current smoking, HDL-cholesterol, triglycerides, WBC, PV, FPG, insulin, Factor XII in the model there were no independent predictors of fibrinogen in patients. In controls, the predictors were triglycerides, P / 0.013, and PV, P / B/0.0001, regression coefficients (R 2) /0.47. After adjustment for age, gender, ethnicity and systolic blood pressure there were significant differences in fibrinogen between the two groups (patients 3.79 vs. controls 3.41 mg/dl, P /0.02). When plasma glucose, triglycerides, HDL-cholesterol, WHR and BMI were added to the model the differences disappeared (3.70 vs. 3.42 mg/dl, P /0.13). The levels remained significantly different between the two groups after adjustment for age, gender and (a) diabetes mellitus (3.78 vs. 3.40 mg/dl, P /0.02) and (b) hypertension 3.78 vs. 3.42 mg/dl, P /0.03). 3.3. t-PA Unadjusted t-PA levels were significantly elevated in patients, the mean difference being 1.54 ng/ml P / B/ 0.04 (Table 1). Age-and-gender adjusted t-PA antigen correlates are shown in Table 2. There were no differences between t-PA levels between males and females in patients or controls. The values of t-PA were similar between subjects with diabetes mellitus and ones without in both patients and controls. On linear regression analysis with all the factors used in model for fibrinogen and PAI-1 antigen the independent predictors in patients were current smoking, P /
0.01 and PAI-1 antigen P /0.003, R 2 /0.48. In controls, the predictor was PAI-1 antigen P /0.04, R 2 / 0.35. The differences in t-PA levels between patients and controls persisted, after adjustment for age, gender, systolic blood pressure and ethnic origin [11.6 vs. 10.2 ng/ml, P /0.05]. However, with plasma glucose, triglycerides, HDL-cholesterol, WHR and BMI in the model the t-PA levels became similar in the two groups [12.6 vs. 11.4 ng/ml, P /0.15]. The difference in t-PA between two groups persisted after adjustment for age, gender and hypertension (13.0 vs. 11.2 ng/ml, P /0.03), however after adjustment for age, gender and diabetes mellitus it became insignificant (P /0.07).
3.4. vWF vWF correlated after age- and gender-adjustment are given in Table 2. The levels of vWF were similar in males and females in both patients and controls. There were no significant difference in levels between subjects with diabetes mellitus and without in both the groups. The independent predictors of vWF with factors used in model for fibrinogen along with t-PA, were age (P / 0.04) and insulin (P /0.04) (R 2 /0.33) in patients and PV (P /0.001) plus triglycerides (P /0.03) in controls (R 2 /0.35). After adjustment for age, gender, systolic blood pressure and ethnic origin vWF levels were still higher in patients (1.66 vs. 1.45%, P /0.04) and after adjusting for plasma glucose, triglycerides, HDL-cholesterol, WHR and BMI there was a trend (1.66 vs. 1.46%, P /0.06). The levels remained significantly different after adjustment for age, gender and (a) diabetes mellitus (1.79
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vs. 1.50%, P /0.006) and (b) hypertension [1.80 vs. 1.46%, P /0.001].
4. Discussion South Asians exhibit significantly increased mortality from coronary artery disease and cerebrovascular disease [1]. The adverse conventional risk factor profile and features of the insulin resistance syndrome in South Asians do not fully account for the increased mortality [2 /4]. Recently haemostatic factors have been associated with insulin resistance syndrome in Whites and also play a role in determining cerebrovascular disease [9 /12]. There have been relatively few studies of the thrombotic component of vascular risk in South Asian subjects. Results for fibrinogen in South Asians compared to Whites have been contradictory. Higher fibrinogen levels in South Asians have been demonstrated in some studies [17 /19] while McKeigue et al. [4] found no difference between South Asians and Whites. Although increased insulin resistance and elevated levels of tissue plasminogen activator have been found in firstdegree relatives of South Asian patients with ischemic cerebrovascular disease [20], to our knowledge there are no studies on insulin resistance or haemostatic risk factors in South Asians with ischaemic stroke. The established metabolic and environmental risk factors for strokes in Whites are age, hypertension, diabetes mellitus and smoking. Although we observed a very high prevalence of conventional risk factors in both South Asian ischaemic stroke patients and South Asian control groups, it was higher in patients including diabetes mellitus, hypertension and myocardial infarction. Turning to insulin resistance, the South Asian ischaemic stroke population exhibited higher level of insulin resistance, although insulin levels were similar in the two groups because subjects with diabetes mellitus usually become depleted of insulin with time [21]. The relationship between insulin and insulin resistance is complex and 36% of insulin is contributed to by insulin resistance [22]. The finding of this study that plasma insulin levels were similar in stroke patients and control subjects, despite a two times higher prevalence of diabetes in patients than control subjects, suggests that a large proportion of the stroke patients with diabetes must have had an impaired beta-cell function. As increased insulin resistance in White subjects is associated with an adverse cardiovascular risk factor profile, the results from the present study suggest a similar relationship might exist for South Asians [6 /8]. Superficially, our findings of increased levels of fibrinogen, vWF and t-PA could be accounted for by the much higher prevalence of diabetes mellitus in patients. Diabetes mellitus is associated with higher
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levels of coagulation and fibrinolytic proteins, especially the latter [23 /25]. In this study we have demonstrated that differences in t-PA disappeared after adjustment for diabetes mellitus; however, the observed differences in levels of fibrinogen and vWF between the groups persisted even after correction for the confounding influence of diabetes mellitus. Prospective studies in Whites indicate that elevated fibrinogen, von Willebrand factor levels and t-PA are risk factors for ischaemic stroke and our data suggest that these factors may play a role in the South Asian population as well [9,11,26]. We acknowledge that this is a small study and optimally we should recruit two control subjects per patient but given the complexities in recruiting subjects in this particular ethnic group (especially the elderly) we have made sure that the two groups are matched for age and gender. Also, the South Asians (Indians, Pakistanis and Bangladeshis) are a heterogeneous group, they have different diets, life styles and socio-economic status but because of small numbers, we have grouped them together for analysis [27]. Although we have shown values for insulin resistance as calculated by HOMA, 61 and 30% of patients and controls, respectively, were subjects with diabetes mellitus and they probably do not have 100% pancreatic islet beta-cell function, therefore it is not an ideal surrogate [16]. Conventionally, insulin resistance is measured with euglycaemic insulin clamp technique [28]. We also studied PV and white cell count, which were not different in the two groups, suggesting that acute phase response is not contributing to raised fibrinogen and vWF levels. However, we did not determine ABO blood group, although there is evidence to suggest that non-O blood group may raise levels of vWF [29]. Whether the raised vWF in patients is accounted for by increased frequency of non-O blood group cannot be determined. Likewise, genetic influences have been implicated in regulation of coagulation and fibrinolytic proteins and we have not genotyped subjects in this study. We did not measure C-peptide levels without which it is difficult to be sure, that subjects had type 2 diabetes, and this is a potential limitation to the study. In addition to haemostatic factors mentioned in this study, there is emerging evidence to suggest a relation of adhesion molecules and E-selectin to features of insulin resistance syndrome especially adipose tissue [30,31]. We are not aware of any data involving adhesion molecules in South Asians and it would be interesting to study them in this population. In conclusion, there is clustering of features of the insulin resistance syndrome along with increased levels of fibrinogen, t-PA and vWF in South Asian ischaemic stroke patients with a high prevalence of diabetes. However, the association between fibrinogen, von Willebrand factor and t-PA and stroke disappeared after adjustment for features of insulin resistance syndrome
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(glucose, triglycerides, HDL, WHR and BMI) but persisted after adjustment for the presence of diabetes. This finding strengthens the concept that changes observed are essentially dependent on features of insulin resistance syndrome and not just reflections of diabetes. Nevertheless, no inferences on causality or the proportion of stroke risk, which can be accounted for by these factors, can be made due to the cross-sectional nature of this study. A prospective study would be required to elucidate multiple comparisons and adjustment for confounders.
Acknowledgements This study was supported by a grant from the Stroke Association (UK).
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Increased fibrinogen, von Willebrand factor and tissue plasminogen activator levels in insulin resistant South Asian patients with ischaemic stroke Kirti Kain a,*, Andrew J. Catto a, John Young b, John Bamford c, John Bavington d, Peter J. Grant a a
Academic Unit of Molecular Vascular Medicine, University of Leeds, G-Floor, Martin Wing, Leeds General Infirmary, Leeds LS1 3EX, UK b Department of Medicine for the Elderly, St. Luke’s Hospital, Bradford, UK c Department of Neurology, St. James’s University Hospital, Leeds, UK d Transcultural Unit, Lynfield Mount Hospital, Bradford, UK Received 14 August 2001; received in revised form 14 December 2001; accepted 18 January 2002
Abstract The aim of this study was to determine differences in atherothrombotic risk factors in South Asian subjects with a history of ischaemic stroke and South Asian subjects free from personal and family history of clinically detectable stroke. Eighty South Asian patients with ischaemic stroke (confirmed on cranial computerised scan) and 80 South Asian controls with similar age and gender distributions were recruited at random. The frequency of hypertension (P / B/0.0001), myocardial infarction (P /0.003) and diabetes mellitus ( B/0.0001) were significantly higher in stroke patients. Stroke patients had lower high-density lipoprotein cholesterol (0.95 vs. 1.1 mmol/l, P / B/0.0001), higher plasma glucose (8.1 vs. 6.6 mmol/l, P /0.01) and trendwise higher HBA1C (6.4 vs. 6.0%, P /0.09). There was no difference in insulin levels but insulin resistance was significantly higher in stroke patients (3.75 vs. 2.66, P /0.01). Stroke patients showed elevated levels of fibrinogen (3.78 vs. 3.41 mg/dl, P /0.02), von Willebrand factor (1.78 vs. 1.50 IU/ml, P /0.006) and tissue plasminogen activator (12.8 vs. 11.3 ng/ml, P /0.04), but the differences did not persist after adjustment for glucose, triglycerides, HDL, WHR, and BMI. Higher levels of fibrinogen, von Willebrand factor and t-PA in South Asian stroke patients disappeared after adjustment for features of insulin resistance syndrome but persisted after adjustment for presence of diabetes, confirming that these changes are essentially dependant on features of insulin resistance syndrome. A prospective study would be required to elucidate the role of thrombotic risk factors in South Asians with ischaemic stroke. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: South Asian; Stroke; Ethnicity; Cardiovascular risks; Haemostasis
1. Introduction Cerebrovascular disease is 1.5 times more common in the South Asian population resident in UK, compared to the indigenous White population [1]. The precise mechanisms, which account for the excess cerebrovascular mortality among South Asians, are poorly understood. However, several studies indicate that plasma insulin level and insulin resistance is higher in South
* Corresponding author. Tel.: 44-113-392-3472; fax: 44-113242-3811. E-mail address: [email protected] (K. Kain).
Asians compared to Whites [2 /4]. Consequently, South Asians tend to have more abnormalities in those risk factors clustering with hyperinsulinaemia and insulin resistance specifically, higher waist /hip ratio, hypertriglyceridaemia, lower high-density cholesterol and an increased prevalence of type 2 diabetes mellitus and hypertension [2,4,5]. These observations might account for some of the excess risk of cerebrovascular disease in South Asians. This is of interest as several, but not all, prospective studies in Whites demonstrate that hyperinsulinaemia is associated with an increased risk of ischaemic stroke [6 /8]. Furthermore, haemostatic factors and fibrinolytic factors in particular, such as plasminogen activator inhibitor-1 and tissue plasmino-
0021-9150/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 2 ) 0 0 0 2 5 - 4
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gen activator, might be also of interest in this context, as these factors have also been associated with insulin resistance [9 /12]. However, there is no data on conventional features of the insulin resistance syndrome, or its haemostatic components in South Asians with ischaemic stroke. Therefore, the aim of this study was to compare features of the insulin resistance and haemostatic risk factors in South Asian patients with history of confirmed ischaemic stroke and South Asian subjects without stroke.
abdominal girth to the nearest 0.5 cm and dividing it by measurement at maximal protrusion of the hips at the level of symphysis pubica also to the nearest 0.5 cm. Smoking history was classified into subjects who never smoked and smokers if they were ex-smokers or current smokers. Subjects with diabetes and hypertension were classified as known if they were already on treatment or their physician had diagnosed them. The majority of the subjects with diabetes had type 2 diabetes. 2.3. Laboratory methods
2. Materials and methods 2.1. Study subjects Eighty South Asian patients with a clinical diagnosis of ischaemic stroke according to WHO criteria were compared with 80 South Asian control subjects randomly recruited from the community, free from a personal or family history of ischaemic stroke. A history of ischaemic stroke was confirmed in South Asian patients from clinical records and the results of a noncontrast computerised cranial tomography scan. To minimise the confounding influence of the acute phase response on coagulation and fibrinolytic factors, stroke patients were recruited at least 2 months after the acute event. Eligible control subjects matched for ethnicity were recruited at random by a postal questionnaire from Family Health Authority General Practitioner’s lists with the aim of having similar age and gender distribution. Both patients and controls were recruited simultaneously over a period of 2 years and from the same geographical location (West Yorkshire, UK). All subjects were of South Asian origin (India, Pakistan or Bangladesh) and their four grandparents were from one of these countries. Subjects with mixed parentage were not recruited. All the subjects received a volunteer information sheet in English and different translations namely Gujrati, Punjabi, Bengali, Hindi and Urdu according to the subject’s ethnic background. Subjects from India, Pakistan and Bangladesh were equally represented in both groups. All subjects gave written informed consent and Leeds and Bradford Ethics Committee UK gave the ethical approval for study. 2.2. Assessment of anthropometric and clinical risk factors Height and weight were recorded without shoes and in light clothing. Body mass index (BMI) was calculated from weight in kilograms divided by height squared in meters. Blood pressure (BP) was taken after 15 min rest and the mean of three readings, with subjects lying down and to the nearest 2 mmHg. Waist /hip ratio (WHR) was calculated by taking the measurement at minimum
A 19 g-butterfly needle was used to draw free flowing venous blood before 10:30 am following an overnight fast and abstention from smoking. For FXII, fibrinogen, von Willebrand factor (vWF) and FVII antigen, 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 8C. Samples were thawed for 10 min at 37 8C before measuring FXIIa using a direct enzyme immunoassay, which detects both a-XIIa and b-XIIa in human plasma (Shield Diagnostics, Dundee, UK) [13]. 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 [14] on the KC10 Coagulometer (Amelung), the inter-assay CV being 3.5% and intra-assay CV 2%. FVII antigen and vWF antigen (DAKOPATTS) were determined by enzyme linked immunoabsorbant assay (ELISA), inter-assay and intra-assay CV being 8.2, 4.3, 4.7 and 2.8% respectively. Samples for plasminogen activator inhibitor-1 (PAI1) antigen and tissue plasminogen activator (t-PA) antigen 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 8C. Plasma samples were snap-frozen in liquid nitrogen and stored at /40 8C. An ELISA assay was used to determine PAI-1 antigen (Biopool cat #214202) and t-PA antigen (Juhan Vague 1992 ECAT assay procedures) [15], and inter-assay and intra-assay CV were 5, 9% for PAI-1 antigen and 10, 8% for t-PA, respectively. The homeostasis model assessment (HOMA) model was used to calculate insulin resistance (IR), which assumes that normal weight subjects aged less than 35 years have a 100% pancreatic islet b-cell function and IR of unity (HOMA score /fasting insulin /fasting plasma glucose divided by 22.5) [16]. Plasma glucose was determined by a glucose oxidase method. Total triglycerides and total cholesterol were measured on the Hitachi 747 autoanalyser (Boehringer Mannheim, Mannheim, Germany). White cell count
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(WBC) was determined by modified flourocytometer and plasma viscosity (PV) was measured by Coulter viscometer. Plasma insulin was also determined by ELISA, and the inter-assay and intra-assay CV were 5.6 and 5.3% at 18 mIU/ml, and 9.8 and 3.0% at 84 mIU/ ml for insulin. 2.4. Statistical analysis Skewed atherothrombotic continuous variables were log (natural) transformed if they were non-normally distributed and then compared by unpaired Student’s t test. Categorical data was compared by x 2 test. Partial correlations were used to study age and gender adjusted correlations. Analysis of variance was used to calculate differences between patients and controls after adjustment for covariates. Regression analysis on log-transformed data was carried out to determine independent predictors of fibrinogen, t-PA and vWF. All the analysis were done by using SPSS (version 9) (SPSS Inc. Chicago, USA).
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the two groups. The prevalence of hypertension, diabetes mellitus and myocardial infarction was higher in patients. No difference was observed in the fasting cholesterol and triglycerides between the two groups, HDL-cholesterol was lower in patients. Insulin levels were the same, but insulin resistance (derived from the HOMA estimation) was greater in patients than in controls (Table 1). The WHR was increased in males compared to females in both patients and controls (1.01 vs. 0.92, P / B/0.0001; 0.97 vs. 0.92, P /0.003). BMI, FPG, cholesterol, triglycerides, HDL-cholesterol and insulin were similar in males and females in patients and controls. In the patients, 15% were on lipid lowering agents and 55% on treatment for diabetes mellitus (16% on diet alone, 64% on oral hypoglycaemic agents and 20% on insulin). Only 1% of the control subjects were on lipid lowering agents and 24% on treatment for diabetes mellitus (47% on diet, 48% on oral hypoglycaemic agents and 5% on insulin). The unadjusted levels of fibrinogen, t-PA and vWF were higher in patients compared to controls (Table 1).
3. Results
3.2. Fibrinogen
3.1. Demographic and clinical characteristics of the South Asian patients and South Asian controls
Age- and gender-adjusted correlates are shown in Table 2. Fibrinogen correlated with PV in both groups. The values of fibrinogen were not significantly different in smokers and non-smokers in patients but were higher in smokers in controls (6.07 vs. 3.38 mg/dl, P /0.03). There
Table 1 shows there were no significant differences in age, gender, and anthropometric measurements between
Table 1 Demographic, conventional and haemostatic variables in South Asian stroke patients and South Asian controls Variables
Patients (n 80)
Controls (n 80)
P
Male: female Age (years) Body mass index (kg/m2) Waist /hip ratio Current smokers (%) Hypertension (%) Myocardial infarction (%) Diabetes mellitus (%) WBC ( 109/l) Plasma viscosity (mPas) Fasting plasma glucose (mmol/l) HbA1C (%) Total cholesterol (mmol/l) HDL-cholesterol (mmol/l) Triglycerides (mmol/l) Fasting insulin (mU/l) Insulin resistance (HOMA) Fibrinogen (mg/dl) Activated FXIIa (ng/ml) PAI-1 antigen (ng/ml) Factor VII antigen (%) Tissue plasminogen activator (ng/ml) von Willebrand factor (%)
40:40 63 (34) 25 (1.2) 0.96 (0.09) 13 60 22 61 7.37 (1.6) 1.72 (0.11) 8.10 (4.2) 6.43 (1.3) 5.48 (1.1) 0.95 (1.3) 2.00 (1.6) 11.31 (1.9) 3.75 (2.3) 3.78 (1.3) 2.08 (1.7) 12.63 (2.3) 109 (33) 12.84 (4.6) 1.78 (0.7)
38:42 61(35) 26(1.2) 0.94 (0.08) 1 28 6 30 7.00 (1.5) 1.70 (0.11) 6.60 (3.0) 6.00 (1.3) 5.67 (1.0) 1.10 (1.44) 1.81 (1.7) 9.61 (2) 2.66 (2.3) 3.41 (1.3) 2.10 (1.6) 13.5 (2.6) 105 (27) 11.30 (4.4) 1.50 (0.5)
0.9 0.8 0.5 0.09 0.009 B 0.0001 0.003 B 0.0001 0.16 0.27 0.01 0.09 0.26 0.004 0.14 0.14 0.01 0.02 0.92 0.65 0.43 0.04 0.006
Age-median (range), other values are mean (standard deviation).
K. Kain et al. / Atherosclerosis 163 (2002) 371 /376
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Table 2 Age-and -gender adjusted correlation coefficients of haemostatic risk factors in South Asian stroke patients and controls Risk factors
Fibrinogen patients
Fibrinogen controls
t-PA patients
t-PA controls
vWF patients
vWF controls
BMI WHR Triglycerides HDL-cholesterol Fasting plasma glucose Insulin Plasma viscosity WBC Systolic blood pressure Diastolic blood pressure PAI-1 antigen t-PA VWF Factor VII antigen Factor XII Fibrinogen
0.04 0.03 0.22 0.38** 0.20 0.01 0.50** 0.30 0.12 0.09 0.11 0.11 0.06 0.08 0.24*
0.06 0.10 0.19 0.09 0.09 0.15 0.48* 0.28* 0.11 0.18 0.13 0.00 0.33** 0.11 0.01
0.01 0.04 0.43* 0.02 0.22 0.19 0.25 0.20 0.08 0.04 0.32**
0.29* 0.31* 0.32** 0.26* 0.16 0.30* 0.28* 0.14 0.16 0.09 0.44**
0.04 0.49** 0.01 0.14 0.17 0.20 0.03 0.08 0.14 0.17 0.18 0.25*
0.00 0.02 0.15 0.00 0.03 0.11 0.41** 0.32** 0.10 0.15 0.08 0.04
0.00 0.01 0.06
0.11 0.08 0.32**
0.25* 0.07 0.04 0.11
0.04 0.15 0.07 0.00
*Indicates significance (P B 0.05). **Indicates significance (P B 0.005).
were no differences in fibrinogen values between males and females in patients or controls. The values of fibrinogen were similar between subjects with diabetes mellitus and those without, in both patients and controls. On linear regression analysis with age, gender, BMI, WHR, current smoking, HDL-cholesterol, triglycerides, WBC, PV, FPG, insulin, Factor XII in the model there were no independent predictors of fibrinogen in patients. In controls, the predictors were triglycerides, P / 0.013, and PV, P / B/0.0001, regression coefficients (R 2) /0.47. After adjustment for age, gender, ethnicity and systolic blood pressure there were significant differences in fibrinogen between the two groups (patients 3.79 vs. controls 3.41 mg/dl, P /0.02). When plasma glucose, triglycerides, HDL-cholesterol, WHR and BMI were added to the model the differences disappeared (3.70 vs. 3.42 mg/dl, P /0.13). The levels remained significantly different between the two groups after adjustment for age, gender and (a) diabetes mellitus (3.78 vs. 3.40 mg/dl, P /0.02) and (b) hypertension 3.78 vs. 3.42 mg/dl, P /0.03). 3.3. t-PA Unadjusted t-PA levels were significantly elevated in patients, the mean difference being 1.54 ng/ml P / B/ 0.04 (Table 1). Age-and-gender adjusted t-PA antigen correlates are shown in Table 2. There were no differences between t-PA levels between males and females in patients or controls. The values of t-PA were similar between subjects with diabetes mellitus and ones without in both patients and controls. On linear regression analysis with all the factors used in model for fibrinogen and PAI-1 antigen the independent predictors in patients were current smoking, P /
0.01 and PAI-1 antigen P /0.003, R 2 /0.48. In controls, the predictor was PAI-1 antigen P /0.04, R 2 / 0.35. The differences in t-PA levels between patients and controls persisted, after adjustment for age, gender, systolic blood pressure and ethnic origin [11.6 vs. 10.2 ng/ml, P /0.05]. However, with plasma glucose, triglycerides, HDL-cholesterol, WHR and BMI in the model the t-PA levels became similar in the two groups [12.6 vs. 11.4 ng/ml, P /0.15]. The difference in t-PA between two groups persisted after adjustment for age, gender and hypertension (13.0 vs. 11.2 ng/ml, P /0.03), however after adjustment for age, gender and diabetes mellitus it became insignificant (P /0.07).
3.4. vWF vWF correlated after age- and gender-adjustment are given in Table 2. The levels of vWF were similar in males and females in both patients and controls. There were no significant difference in levels between subjects with diabetes mellitus and without in both the groups. The independent predictors of vWF with factors used in model for fibrinogen along with t-PA, were age (P / 0.04) and insulin (P /0.04) (R 2 /0.33) in patients and PV (P /0.001) plus triglycerides (P /0.03) in controls (R 2 /0.35). After adjustment for age, gender, systolic blood pressure and ethnic origin vWF levels were still higher in patients (1.66 vs. 1.45%, P /0.04) and after adjusting for plasma glucose, triglycerides, HDL-cholesterol, WHR and BMI there was a trend (1.66 vs. 1.46%, P /0.06). The levels remained significantly different after adjustment for age, gender and (a) diabetes mellitus (1.79
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vs. 1.50%, P /0.006) and (b) hypertension [1.80 vs. 1.46%, P /0.001].
4. Discussion South Asians exhibit significantly increased mortality from coronary artery disease and cerebrovascular disease [1]. The adverse conventional risk factor profile and features of the insulin resistance syndrome in South Asians do not fully account for the increased mortality [2 /4]. Recently haemostatic factors have been associated with insulin resistance syndrome in Whites and also play a role in determining cerebrovascular disease [9 /12]. There have been relatively few studies of the thrombotic component of vascular risk in South Asian subjects. Results for fibrinogen in South Asians compared to Whites have been contradictory. Higher fibrinogen levels in South Asians have been demonstrated in some studies [17 /19] while McKeigue et al. [4] found no difference between South Asians and Whites. Although increased insulin resistance and elevated levels of tissue plasminogen activator have been found in firstdegree relatives of South Asian patients with ischemic cerebrovascular disease [20], to our knowledge there are no studies on insulin resistance or haemostatic risk factors in South Asians with ischaemic stroke. The established metabolic and environmental risk factors for strokes in Whites are age, hypertension, diabetes mellitus and smoking. Although we observed a very high prevalence of conventional risk factors in both South Asian ischaemic stroke patients and South Asian control groups, it was higher in patients including diabetes mellitus, hypertension and myocardial infarction. Turning to insulin resistance, the South Asian ischaemic stroke population exhibited higher level of insulin resistance, although insulin levels were similar in the two groups because subjects with diabetes mellitus usually become depleted of insulin with time [21]. The relationship between insulin and insulin resistance is complex and 36% of insulin is contributed to by insulin resistance [22]. The finding of this study that plasma insulin levels were similar in stroke patients and control subjects, despite a two times higher prevalence of diabetes in patients than control subjects, suggests that a large proportion of the stroke patients with diabetes must have had an impaired beta-cell function. As increased insulin resistance in White subjects is associated with an adverse cardiovascular risk factor profile, the results from the present study suggest a similar relationship might exist for South Asians [6 /8]. Superficially, our findings of increased levels of fibrinogen, vWF and t-PA could be accounted for by the much higher prevalence of diabetes mellitus in patients. Diabetes mellitus is associated with higher
375
levels of coagulation and fibrinolytic proteins, especially the latter [23 /25]. In this study we have demonstrated that differences in t-PA disappeared after adjustment for diabetes mellitus; however, the observed differences in levels of fibrinogen and vWF between the groups persisted even after correction for the confounding influence of diabetes mellitus. Prospective studies in Whites indicate that elevated fibrinogen, von Willebrand factor levels and t-PA are risk factors for ischaemic stroke and our data suggest that these factors may play a role in the South Asian population as well [9,11,26]. We acknowledge that this is a small study and optimally we should recruit two control subjects per patient but given the complexities in recruiting subjects in this particular ethnic group (especially the elderly) we have made sure that the two groups are matched for age and gender. Also, the South Asians (Indians, Pakistanis and Bangladeshis) are a heterogeneous group, they have different diets, life styles and socio-economic status but because of small numbers, we have grouped them together for analysis [27]. Although we have shown values for insulin resistance as calculated by HOMA, 61 and 30% of patients and controls, respectively, were subjects with diabetes mellitus and they probably do not have 100% pancreatic islet beta-cell function, therefore it is not an ideal surrogate [16]. Conventionally, insulin resistance is measured with euglycaemic insulin clamp technique [28]. We also studied PV and white cell count, which were not different in the two groups, suggesting that acute phase response is not contributing to raised fibrinogen and vWF levels. However, we did not determine ABO blood group, although there is evidence to suggest that non-O blood group may raise levels of vWF [29]. Whether the raised vWF in patients is accounted for by increased frequency of non-O blood group cannot be determined. Likewise, genetic influences have been implicated in regulation of coagulation and fibrinolytic proteins and we have not genotyped subjects in this study. We did not measure C-peptide levels without which it is difficult to be sure, that subjects had type 2 diabetes, and this is a potential limitation to the study. In addition to haemostatic factors mentioned in this study, there is emerging evidence to suggest a relation of adhesion molecules and E-selectin to features of insulin resistance syndrome especially adipose tissue [30,31]. We are not aware of any data involving adhesion molecules in South Asians and it would be interesting to study them in this population. In conclusion, there is clustering of features of the insulin resistance syndrome along with increased levels of fibrinogen, t-PA and vWF in South Asian ischaemic stroke patients with a high prevalence of diabetes. However, the association between fibrinogen, von Willebrand factor and t-PA and stroke disappeared after adjustment for features of insulin resistance syndrome
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(glucose, triglycerides, HDL, WHR and BMI) but persisted after adjustment for the presence of diabetes. This finding strengthens the concept that changes observed are essentially dependent on features of insulin resistance syndrome and not just reflections of diabetes. Nevertheless, no inferences on causality or the proportion of stroke risk, which can be accounted for by these factors, can be made due to the cross-sectional nature of this study. A prospective study would be required to elucidate multiple comparisons and adjustment for confounders.
Acknowledgements This study was supported by a grant from the Stroke Association (UK).
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