the risk of stroke and myocardial infarction is considerably increased in subjects with diabetes . already at the time of diagnosis of type 2 diabetes , many patients have manifest cardiovascular disease ( cvd ) .
this could be due to a long presymtomatic period with increased glucose level as evident by the increased cvd risk already present with impaired glucose tolerance ( igt ) .
thus , interest has focused on factors , not only glucose levels , in the prediabetic state that would increase the atherothrombotic process . a common , possibly genetic , antecedent of both type 2 diabetes and cvd has been proposed as the " common soil " hypotheses .
recently , markers of inflammation , such as highly sensitive c - reactive protein , have been found to predict type 2 diabetes in long - time follow up of population samples , although adjusting for measurements of obesity attenuates the relationship [ 8 - 12 ] .
such markers are weakly related to variables reflecting endothelial function such as adhesion molecules and von willebrant factor . in a large population - based study , also increased plasma levels of plasminogen activator inhibitor-1 ( pai-1 ) were strongly related to the development of diabetes independent from insulin resistance and obesity .
the possibility of elevated pai-1 being a very early risk marker of the insulin resistance syndrome and diabetes was raised . furthermore , haemostatic variables related to endothelial function , such as von willebrant factor and factor viii , also predicted diabetes , especially in women . data are lacking regarding other aspects of the fibrinolytic system and the development of diabetes , such as the activities of pai-1 and tissue plasminogen activator ( tpa ) or the mass concentration of the endothelial - derived tpa ( " tpa antigen " ) , factors related to cvd [ 15 - 19 ] thus , there are two questions related to fibrinolytic variables and the development of diabetes .
firstly , do subjects with normal glucose tolerance , who later convert to diabetes , have disturbances in the fibrinolytic system and endothelial function that could explain their increased risk of cvd even before diabetes ensues ?
if so , do these changes predict future diabetes , independent of the influence of the insulin resistance syndrome , thereby pointing to other pathways to the diabetic state such as endothelial dysfunction ?
we studied fibrinolytic activity , as measured by tpa and pai-1 activity , and a marker of endothelial dysfunction , as measured by tpa antigen , in a population sample with normal glucose tolerance at baseline and analysed data according to conversion to diabetes or not over nine years of follow - up .
this study was performed within the framework of the northern sweden monica study . in 1990
a total of 2,000 randomly selected subjects aged 25 to 64 years were invited . in all , 1,583 persons participated ( 79.2 % ) .
a 75-gram oral glucose tolerance test ( ogtt ) was performed after an overnight fast in a randomly selected subset of subjects without known diabetes .
venous plasma glucose samples were analysed by the hexokinase method ( boehringer mannheim automated analysis for bm / hitachi system 717 ) .
glucose tolerance was classified according to who criteria from 1999 : normal glucose tolerance ( ngt )  fasting glucose < 7 mmol / l and post load glucose < 7.8 mmol / l , impaired glucose tolerance ( igt )  fasting glucose < 7 mmol / l and post load glucose 7.811.0 mmol / l , and diabetes  fasting glucose 7.0
weight was measured with a balance scale to the nearest 0.2 kg , and height was measured to the nearest centimetre .
bmi was calculated as total body weight ( kg ) / height ( m ) .
waist circumference was measured midway between the lower rib margin and the iliac crest , which in most occasions was identical with the level of the umbilicus .
all measurements were done in a standing position while breathing normally , and participants were asked to wear light underwear and remove shoes . for the measurement of waist circumference , the recording was done after a gentle breath - out .
tpa and pai-1 activities were determined by chromogenic assays , spectrolyse / fibrin and spectrolyse / pl kit ( biopool ab , ume , sweden ) , respectively .
tpa antigen was measured by an enzyme - linked immunosorbent assay method ( tintelize tpa , biopool ab ) .
serum insulin was determined by radio immunoassay with a double - antibody solid - phase technique ( phadaseph insulin ria , pharmacia diagnostics ab , uppsala , sweden ) in 1999 , all participants , alive and still living in the area , were recalled for a follow - up examination . the same questionnaire and anthropometrical measurements as in the baseline survey were used .
altogether 1,148 ( 72.5 % ) subjects turned up for re - examination . subjects who previously had performed an ogtt with normal or impaired glucose tolerance , and answered no to the question "
participants , who denied having diabetes in the baseline survey and answered affirmative to the question " do you have diabetes mellitus ? " at follow up , were defined as incident cases of known diabetes mellitus according tho the who criteria from 1999 . in november 2002 , a questionnaire exploring details related to the diagnosis of diabetes , and to the type and duration of current hypoglycaemic treatment was sent to them .
case records were scrutinised to confirm diabetes diagnosis and time for start of insulin therapy .
subjects that started their insulin treatment within two years of diagnosis were classified as type 1 .
however , for subjects fulfilling these criteria but with a age of 40 or above at onset , current bmi had to be < 27 to be classified as type 1 .
the research ethics committee of ume university and the national computer data inspection board have approved the northern sweden monica study .
means or medians are given for those who remained non - diabetic and those who converted to diabetes . because the distributions for serum triglycerides , insulin and the fibrinolytic variables are highly skewed , we used logarithmically transformed values ( i.e. geometric means ) .
the risk of incident diabetes was compared across quartiles of the fibrinolytic variables , calculated from the total sample .
logistic regression was used for calculation of odds ratios and 95% confidence intervals ( ci ) for the development of diabetes comparing the quartile with highest risk with the other three pooled quartiles .
stepwise adjustment for age and sex , waist , serum insulin , triglycerides and diastolic blood pressure was used .
five hundred and fifty - one subjects who initially had normal glucose tolerance returned for re - examination . a new ogtt was done in 477 of those with normal glucose tolerance and without clinically diagnosed diabetes .
thus , there was no repeated ogtt in 13 % , mostly due to logistic reasons such as not being able to turn up in the morning after an over night fast . among persons with initially normal glucose tolerance , in all 15 subjects ( 2.7 % ) had type 2 diabetes diagnosed , either clinically ( n = 4 ) or by ogtt ( n = 11 ) after a 9-year follow - up or 4,959 person years .
fifty - one subjects ( 10,7 % ) worsened from normal glucose tolerance to impaired glucose tolerance .
converters from ngt to diabetes were 7 years older and had higher bmi and waist circumference .
diastolic blood pressure did not differ but both fasting triglycerides , insulin , fasting and post load plasma glucose were higher in subjects with subsequent diabetes .
plasma levels of tpa antigen and pai-1 activity were higher and tpa activity was lower .
baseline characteristics of subjects with normal glucose tolerance according to diabetes or not during 9 years of follow - up data are means ( sd ) , median ( interquartile range ) and [ geometric means ] .
the risk of developing diabetes increased with increasing quartiles of tpa antigen and pai-1 activity and decreased in a similar way for tpa activity ( figure 1 ) . in the highest quartile of tpa antigen ( above 8.7
g / l ) nearly 9 % had incident diabetes compared to none in the lowest quartile .
incidence of type 2 diabetes over nine years in subjects with normal glucose tolerance according to baseline quartiles of fibrinolytic variables .
the number of cases in each quartile for tpa activity was 7 , 4 , 3 and 1 .
corresponding numbers for tpa antigen was 0 , 1 , 2 and 12 and for pai-1 activity 2 , 1 , 3 and 9 , respectively .
p values for x(linear by linear association ) were 0.026 for tpa activity , < 0,001 for tpa antigen and 0,007 for pai-1 activity .
logistic regression was performed with an initial model adjusting for age and sex . in a second model
, waist circumference was added and in a third model , also diastolic blood pressure , serum insulin and triglycerides were added .
quartiles 13 of tpa antigen and pai-1 activity were contrasted with the fourth quartile and , for tpa activity , the first quartiles with quartile 24 ( table 2 ) . logistic regression analysis for fibrinolytic variables at baseline with incident type 2 diabetes as the dependent variable .
the lowest quartile of tpa activity was associated with a 3-fold increase in the risk of diabetes , which was attenuated after adjustment for waist circumference .
after further adjustment for diastolic blood pressure , fasting insulin and triglycerides , no prediction from low tpa activity on diabetes development was seen .
subjects in the highest quartile of tpa antigen experienced more than a ten - fold increase in risk , although the confidence intervals were wide due to few cases .
taking waist circumference into account , the odds ratio diminished to 6.7 and diminished further to 5.5 when adjusting for blood pressure , triglycerides and insulin .
this lead to somewhat higher age- and gender adjusted odds ratio ( 7.4 ; ci 1.830 , p value 0.005 ) . exchanging diastolic with systolic blood pressure did not change the findings .
furter adjustment for leisure time physical activity reduced the odds ratio from 5.5 to 5.1 ( p value 0.03 ) .
high pai-1 activity increased the risk of diabetes four - fold , although the result was attenuated to an insignificant or of 2.0 when all factors were adjusted for simultaneously .
the risk of developing diabetes or igt during follow up ( n = 66 ) increased stepwise across quartiles of tpa antigen , from 7.8% to 19.8% ( p = 0,003 , test for linear trend ) . when adjusted for age and sex , the odds ratio for the highest vs. the three lower quartiles of tpa antigen was 1.9 ( ci 1.011.6 , p = 0.04 ) but diminished further when also waist circumference was adjusted for , or = 1.3 ( ci 0.636.5 , p = 0.5 ) .
subjects with normal glucose tolerance , who subsequently develop type 2 diabetes over the ensuing nine years , are already at baseline characterised by impaired fibrinolysis and endothelial dysfunction .
low tpa activity and high pai-1 activity is mainly explained by the presence of abdominal obesity but also by other metabolic disturbances characteristic of the insulin resistance syndrome , such as high serum levels of triglycerides and insulin .
a novel finding , in this context , is that a high plasma level of the endothelial - derived tpa is only slightly explained by such factors .
thus , the development of type 2 diabetes is preceded by many years of perturbations in fibrinolytic and endothelial function , which increases the risk of atherothrombotic disease long before overt diabetes is present . pa1 - 1 is partly synthesised in fat cells and its activity is strongly related to abdominal obesity as mirrored by high waist circumference or whr .
high circulating levels of pai-1 inhibit tpa released from the vessel walls and lead to low levels of free tpa , i.e. low tpa activity and impaired capability of thrombolysis .
the only report of an association between fibrinolysis and the development of diabetes is a recent study where pai-1 antigen was an independent predictor of diabetes in american - mexicans that were followed up to 5 years .
there is no known pathway whereby impaired fibrinolysis would increase insulin resistance or decrease insulin release and our finding that high pai-1 and low tpa activity precedes diabetes probably mirrors the strong relationship with intraabdominal fat and circulating levels of free fatty acids .
a recent experimental study show an almost three fold increase in the level of pai-1 antigen with an infusion of triacylglycerol in healthy subjects without any impact on tpa .
this effect was noted with constant levels of insulin and glucose and also increased levels of soluble vascular cell adhesion molecules-1 was found .
this support our findings that adjustment for baseline triglyceride levels , which were increased in converters , did not significantly change the relationships noted for tpa antigen but did decrease those for pai-1 .
it is thus possible that also tpa concentration is an inflammatory marker although we found low correlations between tpa antigen and fibrinogen   and highly sensitive crp ( own unpublished data ) .
recently , it was described in type 2 diabetes patients , that increased levels of tpa antigen , among other endothelial markers , predicted the development of urinary albumin excretion , a strong marker for cvd risk .
these findings should be related to recent reports where inflammatory markers predict the development of diabetes in longitudinal studies . in most cases ,
pro - inflammatory substances derived from abdominal fat tissue may be the common background for these findings .
we therefore find it plausible that the relation between high tpa and diabetes is a marker of some early disturbance in the vascular wall that operates independent of obesity and inflammation .
already borderline hypertension is associated with high concentrations of tpa antigen , even after adjustment for abdominal obesity , insulin and triglycerides .
thus , tpa mass may mirror an endothelial dysfunction that is not causally related to the insulin resistance syndrome but acts by a parallel pathogenetic mechanism leading to atherothrombosis [ 30 - 32 ] .
insulin resistance , on the other hand , is associated with endothelial dysfunction and impaired vasodilatation due to defects in endothelium - derived nitric oxide . taken together , these findings put focus on the role of the vessel walls in the pathogenesis of type 2 diabetes .
the relevance of impaired fibrinolysis , i.e. low activity of tissue plasminogen activator ( tpa ) or high activity of its inhibitor ( pai-1 ) , as independent risk factors for cvd is unclear , as these variables seem to mirror most components of the insulin resistance syndrome . in some studies , decreased fibrinolysis , as measured by high pai-1 activity , has been associated with cardiovascular events .
high levels of tpa antigen independently predict cardiovascular events both in a healthy population   and in patients with prevalent coronary disease .
the major shortcoming of our study is the small number of incident cases of diabetes leading to wide confidence intervals . on the other hand , the odds ratios for tpa antigen are high and consistent and based on a truly representative population sample where basal normal glucose tolerance was well defined , as were the incident cases of diabetes .
thus , the risk of bias due to selection or case ascertainment should be low .
admittedly , the lack of a renewed ogtt in 13 % of the subjects could contribute to some misclassifiaction and dilute the results somewhat .
the early prediabetic state , with normal glucose tolerance , is characterised by both impaired fibrinolysis and endothelial dysfunction years before glucose levels increase .
changes in endothelial function are independent from the insulin resistance syndrome and point towards a distinctly different pathway both for cardiovascular disease and for the development of diabetes . to answer a classical question from 1990    yes
, the clock for coronary heart disease seems to start ticking before the onset of clinical diabetes !
bmi = body mass index ci = confidence interval crp = c - reactive protein cvd = cardiovascular disease igt = impaired glucose tolerance monica = monitoring of trends and determinants of cardiovascular disease ngt = normal glucose tolerance ogtt = oral glucose tolerance test pai-1 = plasminogen activator inhibitor-1 tpa = tissue plasminogen activator
me drafted the research on fibrinolysis within the monica project , performed the statistical analysis and drafted the manuscript .
bs and bl participated in the design of northern sweden monica study and participated in its design and coordination .
supported by grants from norrbotten and vsterbotten counties , by the joint committee of northern sweden health care region , the swedish public health institute and the swedish medical research council .
 backgroundimpaired fibrinolysis is found in impaired glucose tolerance and type 2 diabetes , associated with components of the metabolic syndrome . 
 there are no data concerning fibrinolysis in subjects with normal glucose tolerance that convert to diabetes.methodswe studied the activities of tissue plasminogen activator ( tpa ) and plasminogen activator inhibitor-1 ( pai-1 ) and the levels of tpa antigen ( a marker of endothelial dysfunction ) in 551 subjects with normal glucose tolerance in 1990 in relation to incident diabetes during nine years of follow-up.resultssubjects with diabetes at follow - up ( n = 15 ) had significantly lower baseline tpa activity and higher pai-1 activity and tpa antigen than non - converters . 
 the risk of diabetes increased linearly across quartiles of pai - activity ( p = 0.007 ) and tpa antigen ( p < 0.001 ) and decreased across quartiles of tpa activity ( p = 0.026 ) . 
 the risk of diabetes with low tpa activity or high pai-1 activity persisted after adjustment for age and sex but diminished to a non - significant level after further adjustments . 
 the odds ratio of diabetes for high tpa antigen was 10.4 ( 95% confidence interval 2.740 ) adjusted for age and sex . 
 after further adjustment for diastolic blood pressure , waist circumference , insulin , triglycerides , fasting and post load glucose the odds ratio was 6.5 ( 1.333 , p = 0.024).conclusionsimpaired fibrinolysis and endothelial dysfunction are evident in subjects with normal glucose tolerance who later develop diabetes . 
 high tpa antigen is predictive of future diabetes independent from the metabolic syndrome . 
Background
Methods
Statistics
Results
Discussion
Conclusions
Abbreviations
Competing interests
Authors' contributions
Acknowledgements