Signs of dysregulated fibrinolysis precede the development of type 2 diabetes mellitus in a population-based study

We conducted a nested case-referent (case–control) study within the Västerbotten Intervention Programme (VIP)[26], a cardiovascular disease intervention project (Figure 1). All patients in Västerbotten County in northern Sweden were invited to participate in a health survey at their local primary-care centre when they were 30, 40, 50, or 60 years of age. At the health examination, data about age, sex, and cardiovascular risk factors were collected. Participants were also asked to donate a blood sample for research, which was stored at the Northern Sweden Medical Research Bank. The participation rate was high; the total study population from 1989 to 2000 represented 52% of the eligible population[27]. The vast majority of the participants were Caucasian. Between 1989 and 2000 the number of VIP participants in the Umeå region without diabetes type 1 or 2 at the time of their health examination was 28 736. A study of selection bias showed small differences between participants and non-participants[28].

Of all VIP participants examined between 1989 and 2000, 237 were diagnosed with T2DM after their examination and before January 2001 (Figure 1). T2DM was diagnosed according to World Health Organisation (WHO) criteria 1999[29]. Participants were excluded if fasting plasma glucose (FPG) levels exceeded 6.9 mmol/L or 2-hour plasma glucose (2hPG) levels exceeded 12.1 mmol/L (n=11), if the participants had chosen not to donate blood (n=34), if the samples were prioritized to other studies (n=25), if data were missing (n=8), or if there were no matching referents (n=2). Two referents with stored plasma samples and without T2DM according to the registry were randomly selected for each case and matched for sex, age, and year of health examination. Thus, we included 157 cases with T2DM diagnosis during follow-up and 277 referents.

Body mass index (BMI) was calculated as measured weight in kilograms divided by the square of the height in metres (kg/m²). Smoking was categorized into three groups: non-smokers, ex-smokers, or daily smoking. Participants who reported that they smoked irregularly were categorised as ex-smokers. Participants were considered to have a family history of diabetes if they reported having a parent or sibling with T2DM. Physical activity was categorised into three levels based on the questionnaire about self-reported leisure time exercise and commuting habits during winter, as physical activity during the rest of the year was presumed to be at least as high as the activity during winter: 1) sedentary, never exercise, cycle and/or walk during their leisure time less than 2–3 times per month, take bus or car to work, or cycle and/or walk to work less than 2 km; 2) moderate, exercise now and then but not regularly or at most once a week, cycle and/or walk during their leisure time at least 2–3 times per week, or cycle and/or walk to work 2–5 km; 3) active, moderately active person who trains at least 2–3 times/week, or cycle or walk to work more than 5 km.

Hypertension was defined as systolic blood pressure ≥ 140 mmHg and/or diastolic blood pressure ≥ 90 mmHg and/or reported use of antihypertensive medication during the 14 days prior to the health examination. Blood pressure was measured with a mercury sphygmomanometer with the participant in the supine position after a 5-min rest.

Participants were instructed to fast from midnight until the time of blood sampling. Venous blood samples were drawn with minimum stasis in a sitting position into evacuated glass tubes containing 1/100 volume of 0.5% EDTA. The sample tube was centrifuged at 1500 x g; the plasma was immediately frozen at −20°C and stored at −80°C in the Northern Sweden Medical Research Bank until analysis. Oral glucose tolerance tests (OGTTs) were performed with a 75-g glucose load according to WHO standards. Fasting glucose concentrations were measured in venous plasma and glucose concentrations 2 hours after glucose intake in capillary plasma on a Reflotron bench-top analyser (Boehringer Mannheim GmbH, Mannheim, Germany). Triglycerides were analysed in thawed frozen samples via routine methods at the Department of Clinical Chemistry at Umeå University Hospital. ELISA reagent kits for tPA antigen, PAI-1 antigen, and the tPA/PAI-1 complex were purchased from Biopool (Umeå, Sweden). The inter-assay coefficients of variation (CVs) were: tPA 10.8%, PAI-1 7.1%, and tPA/PAI-1 complex 8.3%. Reagent kits with identical batch numbers were used for each analysis. The VWF levels were analysed with reagents purchased from DAKO (Copenhagen, Denmark). The CV for VWF was 6.5%. CRP was measured with an automated chemiluminescent immunoassay using the IMMULITE® analyser from DPC (Los Angeles, CA, USA). The CV for CRP was 3.8%. All samples were thawed in a 37°C water bath and analysed directly afterward. Samples from cases and their matched referents were analysed together in a random order. Measurements were made in 2005 by laboratory staff unaware of each participant’s disease status. Storage time has been shown to have a negligible impact on laboratory measurements of frozen samples[30].

Conditional logistic regression was used to calculate age- and sex-matched odds ratios (ORs) comparing the risk of incident T2DM in cases and referents. Natural logarithmic transformation was used for CRP, triglycerides, VWF, tPA, PAI-1, and tPA/PAI-1 measurements because these variables were not normally distributed. The ORs with 95% confidence intervals (CIs) were calculated per 1 SD increment for continuous variables. Adjustments were performed for each haemostatic risk factor and potential confounders in three models with complete data sets: 1) BMI, smoking, family history of diabetes, and physical activity; 2) BMI, smoking, CRP, systolic blood pressure, and triglycerides; and 3) model 2 plus FPG and 2hPG levels.

To investigate whether haemostatic factors deteriorate prior to the increase in plasma glucose, cases were divided into two groups: 59 cases with normal glucose values (FPG <6.1 mmol/L and 2hPG <8.9 mmol/L at baseline) and their matched referents, and 98 cases with elevated glucose values (FPG = 6.1-6.9 mmol/L and/or 2hPG = 8.9-12.1 mmol/L at baseline) and their matched referents. Multivariate conditional logistic regression was performed for each fibrinolytic factor, BMI, family history of diabetes, physical activity, smoking, CRP, systolic blood pressure, and triglycerides. ORs with 95% CIs were calculated per 1 SD increment for continuous variables.

This study protocol was approved by the Research Ethics Committee of Umeå University. All participants provided informed consent.

Conditional univariate and multivariate analysis was performed separately for the subgroups of participants with normal glucose levels and elevated glucose levels at baseline (Table 4). Multivariate analysis was carried out with adjustments for BMI, smoking, family history of diabetes, physical activity, CRP, systolic blood pressure, triglycerides, and each haemostatic variable (Figure 2). As the subgroups were based on normal or elevated glucose levels, no adjustments were made for glucose levels.

For participants with normal glucose levels, all haemostatic variables were significantly associated with incident diabetes in the univariate analysis (tPA: OR = 2.43, 95% CI 1.50-3.91; PAI-1: OR=2.66, 95% CI 1.66-4.25; tPA/PAI-1 complex: OR=3.23, 95% CI 1.93-5.41; VWF: OR=1.66, 95% CI 1.13-2.46). In multivariate analysis, only PAI-1 remained significant (OR=2.06, 95% CI 1.10-3.86). For participants with elevated glucose levels, all haemostatic variables were significantly associated with incident diabetes in the univariate analysis (tPA: OR=3.05, 95% CI 2.02-4.61; PAI-1: OR=2.20, 95% CI 1.57-3.07; tPA/PAI-1 complex: OR=4.80, 95% CI 2.90-7.92; VWF: OR=1.86, 95% CI 1.35-2.58). After adjustments, tPA (OR=1.82, 95% CI 1.11-2.98) and tPA/PAI-1 complex (OR=4.01, 95% CI 1.90-8.46) remained significant.

The major advantage of this study is the prospective nested case-referent study design within a well-defined population-based intervention programme, the VIP. Men and women age 30 to 60 years underwent an extensive baseline health examination regarding cardiovascular risk factors at their local health centre. Diabetic patients were defined by FPG and OGTT. Participants who developed T2DM were examined years before the manifest diagnosis.

The number of missing cases is high, as 80 of the original 237 cases did not have blood samples available for laboratory analysis of the haemostatic variables. Baseline data for 60 cases without blood samples available showed that no significant difference existed in diastolic and systolic blood pressure, FPG levels, or BMI compared to the cases in the study. As data on the cases without blood samples was similar to the data on those included in the study, selection bias is unlikely. Lifestyle changes recommended in the initial health examination, such as weight loss and smoking cessation, may have influenced the results. In a 10-year follow-up study[44], VIP participants with baseline glucose intolerance or T2DM were the most likely to avoid weight gain, but this occurrence most likely affected the incidence of T2DM and not the relationship between baseline fibrinolytic data and the risk of developing diabetes in a given individual. The vast majority of participants were Caucasian, limiting the possibility of generalising the results to other ethnicities. A risk of regression dilution bias exists because only a single baseline blood sample was used, which may decrease the estimations of the observed associations.