Coagulation factors IX and XI activities are significantly enhanced in type 2 diabetic patients

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1 Original article Coagulation factors IX and XI activities are significantly enhanced in type 2 diabetic patients Nermina Babić 1*, Nesina Avdagić 1, Asija Začiragić 1, Suzana Tihić-Kapidžić 2, Sabina Zukić 3, Amela Dervišević 1, Miralem Musić 4, Izeta Aganović-Mušinović 5, Almira Hadžović-Džuvo 1 1 Department of Human Physiology, Faculty of Medicine University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 2 Department of Clinical Chemistry and Biochemistry, Clinical Center University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 3 Family Medicine, Community Health Center Banovići, Banovići, Bosnia and Herzegovina 4 Department of Pathophysiology, Faculty of Medicine University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 5 Department of Immunology, Faculty of Medicine University of Sarajevo, Sarajevo, Bosnia and Herzegovina, Submitted: / Accepted: Abstract Objectives: The aim was to examine whether plasma coagulation factors activities are increased in patients with diabetes mellitus type 2 (DM2). Also, we aimed to assess whether any association exists between plasma coagulation factors and cardiometabolic risk factors in these patients. Methods: This cross-sectional study included 30 DM2 patients and 30 healthy subjects as control group. Plasma fibrinogen concentration and activities of coagulation factors II, V, VII, IX, X, XI and XII were measured. Results: Plasma factor IX activity was significantly higher in DM2 patients compared to control subjects (145.51±5.27 vs ±3.86% of norm; p<0.0005), as was factor XI activity (136.38±5.08 vs ±5.95% of norm; p=0.001) and fibrinogen concentration [10.5 ( ) vs. 8.8 ( ) mmol/l; p=0.001]. Plasma activities of factors II, V, VII, X and XII were not significantly different in diabetic patients compared to control subjects. A significant positive correlation between fasting blood glucose and factors IX, X and XI, was observed in DM2 patients. In the same group significant positive correlation was determined between factors II, VII, IX, X and triglycerides and between factor II and total cholesterol. Conclusion: Procoagulant state in DM2 as evidenced by enhanced activation of coagulation factors IX and XI and elevated fibrinogen concentration, may contribute to the increased risk of thrombosis and vascular complications in DM2 patients. Furthermore, in the prevention of thrombotic complications in patients with diabetes mellitus it is necessery to keep blood glucose and lipids under control. Key words: coagulation factors, patients with DM2, thrombosis, lipids 2016 Folia Medica Facultatis Medicinae Universitatis Saraeviensis. All rights reserved. *Corresponding author Nermina Babić Department of Human Physiology, Faculty of Medicine, University of Sarajevo, Čekaluša 90, Sarajevo, Bosnia and Herzegovina nermina.babic@mf.unsa.ba Introduction Diabetes mellitus, as complex condition characterized by metabolic and vascular abnormalities, is associated with increased risk of cardiovascular diseases. Atherosclerotic lesions have been recognized as the main cause for the development of cardiovascular events, but recent data suggest that cardiovascular risk in diabetic patients is determined also by the vulnerable blood and the vulnerable myocardium [1,2]. Moreover, atherothrombotic complications are the most common cause of mortality in patients with diabetes mellitus [3,4]. Previous studies have shown that the plasma activities of many coagulation factors are elevated in diabetes, and that diabetes mellitus is a hypercoagulable, pro-thrombotic state. This state includes an increased coagulability and decreased fibrinolytic activity, with prolongation of clot lysis time. Hyperglycemia, as occurs in diabetes mellitus, contributes to endothelial injury through irreversible glycation proteins and lipids, forming advanced glycation end products [5,6]. The products of non-enzymatic glycation accumulate in the vascular wall bind to surface receptors on variety of cells contributing in oxidative stress and inflammatory response. They also reduce nitric oxide activity [7]. Disturbances in endothelial functions lead to increased platelet adhesion and aggregation. Activated platelets are associated with an enhanced expulsion of granules which leads to amplification of their aggregation and it is directly related to formation of thrombi [3,8]. Hyperglycemia may cause glycation of coagulation factors, altering their activity. The glycation of fibrinogen leads to the formation of a rigid and tight fibrin clot that is resistant to fibrinolysis. Previous studies have suggested that elevated levels of coagulation factors are associated with an increased risk of thrombosis. Higher levels of factors II, VIII, IX and 70

2 XI have been associated with an increased risk of venous thrombosis, while elevated levels of fibrinogen, factor V, VII and von Willebrand factor have primarily been associated with an increased risk of arterial thrombosis [9]. The study by Cushman et al. [10] reported that among several procoagulant factors (IX XIII), only elevated factor XI was independently associated with increased risk of future venous thromboembolism. Participants in that study who developed venous thromboembolism had statistically significantly higher mean levels of factor IX and XI than participants who did not developed venous thromboembolism. Recent study by Heikal et al. [11] reported that elevated factor IX activity is associated with transient ischemic attack (TIA) /stroke. Their finding suggests an association between elevated factor IX and both arterial and venous thrombotic events. Data on coagulation status of Bosnian patients with diabetes mellitus are scarce. In our previous study, in which we examined only factor VIII and fibrinogen, we found that diabetic patients (type 1 and type 2) had elevated plasma coagulation factor VIII activity and increased plasma fibrinogen concentration [12]. The aim of this study was to examine the coagulation status of patients with diabetes mellitus type 2 (DM2). This study was designed to examine whether plasma coagulation factors activities are increased in diabetic patients type 2. Also, we aimed to assess whether any association exists between plasma coagulation factors and cardiometabolic risk factors in these patients. Subjects and methods Subjects This cross-sectional study included 30 Type 2 (DM2) diabetic patients and 30 gender- and age-matched healthy subjects as control. Patients were selected from both outpatient and inpatient departments of Clinic of Endocrinology, Diabetes Mellitus and Metabolic Diseases, University Clinical Center of Sarajevo. Diabetes mellitus was previously diagnosed using criterion defined by the American Diabetes Association. Control subjects were recruited from volunteers without clinical and biochemical signs and symptoms of disease. Each subject provided a written consent after the explanation of the study procedure. Subjects with illness and subjects receiving medications that could affect the plasma coagulation factors were excluded from the study. The study protocol was approved by the Ethic Committee of the Faculty of Medicine, University of Sarajevo, and it complied with the Helsinki Declaration. Biochemical analysis Blood samples were collected after an overnight fasting. Fasting blood glucose (FBG) and glycated hemoglobin (HbA1c) levels were determined by standard methods. Serum total cholesterol, triglycerides and high density lipoprotein cholesterol (HDL-C) were assessed by enzymatic method. The low density lipoprotein cholesterol (LDL-C) concentrations were calculated by using Friedewald s formula. The normal ranges for some of the measured parameters were: fasting blood glucose mmol/l, HbA1c<6%, total cholesterol ( mmol/l), triglycerides ( mmol/l), HDL-C ( mmol/l), LDL-C ( mmol/l). Biochemical analyses were done on the Dimension clinical chemistry system (Siemens Healthcare Diagnostics Ltd., Camberley, UK). The activity of plasma coagulation factors were determined by coagulometric methods on automated coagulation analyzer, Siemens Healthcare Diagnostics. The normal ranges for various parameters measured were: Plasma fibrinogen ( mmol/l), Factor II, VII, IX, X, XI (70-120%), Factor V (70-140%), Factor XII (70-150%). Physical examination Anthropometric measurements were obtained, including height, weight and minimum waist circumference. Body mass index (BMI) was calculated by dividing weight (kg) by square of height (m 2 ). Blood pressures were measured twice in the left arm in a sitting position, after a 30-minute rest using a mercury-column sphygmomanometer. Hypertension was defined by a systolic blood pressure 140 mmhg and a diastolic blood pressure 90 mmhg. Statistical Analysis All statistical calculations were performed using SPSS software for Windows (version 13.0; SPSS, Chicago, IL, USA). The Shapiro-Wilk test of normality was used to test the distribution of variables. The Student s independent test or Mann-Whitney U-test was used to compare the differences between the groups, as appropriate. The correlations between the variables were assessed by Spearman rank sum test or Pearson correlation test. Probability value of <0.05 was considered statistically significant. Normally distributed data are presented as mean±sem and skewed variables as median and interquartile ranges. Results The baseline characteristics of DM2 patients and control subjects are summarized in Table 1. The mean duration of diabetes was 5.13±1.24 years. There was no statistically significant difference between the study groups with respect to the age. Diabetic patients were older than healthy subjects, but this should not be considered as clinically relevant. Waist circumference, BMI, systolic and diastolic blood pressure in DM2 patients were significantly higher compared to the control group (p<0.0005; p=0.001)(table 1). 71

3 Table 1. Baseline characteristics of diabetic patients and healthy subjects Variables Data are presented as mean ± SEM. DM2, type 2 diabetic patients; Control, healthy subjects. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; p-probability; - not significant As shown in Table 2, serum triglycerides concentration was significantly higher in DM2 compared to control group (p=0.001). On the other hand, serum HDL cholesterol concentration in diabetic patients was significantly lower compared to the control group (p=0.005). Total cholesterol and LDL-cholesterol concentration in DM2 patients were higher, but not significantly in comparison with healthy subjects. As expected, the diabetic patients had significantly higher fasting blood glucose levels and HbA 1c than healthy subjects (p<0.0005; p=0.001). Variables Total cholesterol HDL-cholesterol LDL-cholesterol Triglycerides DM2 6 (4.85-6,39) 1.02 ( ) Control 5.5 ( ) 1.39 ( ) p p= ± ± ( ) 1.13 ( ) p=0.001 FBG 9.85± ±0.08 p< HbA 1 c DM2 (n=30 ) 8.8 ( ) Control Age (yrs) 49.9± ±1.35 DM duration (time since diagnosis) (yrs) Waist circumference (cm) 5.13± ± ±2.57 p< BMI (kg/m 2 ) 29.88± ±0.85 p< SBP (mmhg) ± ±2.13 p< DBP (mmhg) 84.38± ±1.3 p=0.001 Table 2. Lipid profile and glycemic status of diabetic patients and healthy subjects 5.4 ( ) p=0.001 Data are presented as mean ± SEM or as median and interquartile range (25th 75 th percentile). DM2, type 2 diabetic patients; Control, healthy subjects. HDL, highdensity lipoprotein cholesterol; LDL, low-density lipoprotein cholesterol; FBG, fasting blood glucose; HbA1c, glycated haemoglobin A1c; p-probability; - not significant p Plasma factor IX activity was significantly higher in DM2 patients compared to control subjects (145.51±5.27 vs ±3.86% of norm; p<0.0005), as was factor XI activity (136.38±5.08 vs ±5.95% of norm; p=0.001) and fibrinogen concentration [10.5 ( ) vs. 8.8( ) mmol/l; p=0.001] (Table 3). Table 3. Coagulation factors in diabetic patients and healthy subjects Variables Fibrinogen (mmol/l ) Factor II activity Factor V activity Factor VII activity Factor IX activity Factor X activity Factor XI activity Factor XII activity DM ( ) Control 8.8 ( ) Plasma activities of coagulation factors II, V, VII, X and XII were not significantly different in DM2 patients compared to the control group. The plasma coagulation factor II activity was significantly positively associated with plasma total cholesterol concentration (r=0.498; p=0.011) and plasma triglycerides (r=0.415; p=0.044) in DM2 diabetic patients (Table 4.). In same group, significant positive association was observed between plasma coagulation factor VII and plasma triglycerides (r=0.43; p=0.05). The coagulation factor IX and factor X were significantly positively associated with FBG (r=0.566; p=0.003 and r=0.432; p=0.035) and plasma triglycerides (r=0.597; p=0.002 and r=0.685; p<0.0005), while the factor XI was significantly positively associated only with FBG (r=0.47; p=0.027). There were no any associations between plasma coagulation factors and DM duration, HbA 1 c and BMI. p p= ± ± ± ± ± ± ± ±3.86 p< ± ± ± ±5.95 p= ± ±3.18 Data are presented as mean ± SEM or as median and interquartile range (25th 75 th percentile). DM2, type 2 diabetic patients; Control, healthy subjects; p-probability; - not significant 72

4 Table 4. Association between plasma coagulation factors and the cardiometabolic risk factors in DM2 group Fibrinogen (g/l) Factor II Factor V Factor VII Factor IX Factor X Factor XI Factor XII DM duration (yrs) FBG HbA 1 c ( %) BMI (kg/m2 ) Total cholesterol Triglycerides r=0498 p=0.011 r=0.415 p=0.044 r= p=0.003 r= p=0.035 r=0.470 p=0.027 r=0.43 p=0.05 r=0.597 p=0.002 r=0.685 p< FBG, fasting blood glucose; HbA1c, glycated haemoglobin A1c; BMI, body mass index; r- coeficient correlation, p-probability; - not significant Discussion Many studies have shown that diabetes is a procoagulant state as indicated by increased coagulability and impaired fibrinolysis [5,6]. Hypercoagulability as evidenced by enhanced activation of some plasma cloting factors may contribute to higher risk of aterothrombotic events in diabetic patients. The major diabetic complications involve thrombotic mechanisms [13]. Results of our study have showed that plasma activities of coagulation factors IX and XI in type 2 diabetic patients were significantly higher compared to age and sex matched healthy subjects. Plasma activities of coagulation factors II, V, VII and X were higher, while plasma activity of coagulation factor XII was lower in diabetic patients than in control subjects, but those differences were not statistically significant. Mard-Soltani et al. [14] reported significant increase in plasma activities of coagulation factors II, IX, XI and significant decrease in an activity of factor VIII in patients with non-insulin dependent diabetes mellitus. In explanation of their results the authors indicated that intrinsic pathway is more responsible for hypercoagulable state in diabetes mellitus than extrinsic patway because more increment in factors IX and XI than factors II and X. In our study we did not investigate plasma activity of factor VIII, but we have previously reported its increased activity in type 1 and type 2 diabetic patients [12]. Our results are not consistent with the findings of Madan et al. [6]. These authors investigated haemostatic parameters and whether any their relationship exists with microvascular complications in type 2 diabetes mellitus. Results of that study have shown that factor V, VIII and IX activity did not differ significantly between diabetic patients and healthy controls or between diabetic patients with and without microvascular complications. These authors concluded that diabetes is a procoagulant state as evidenced by increased fibrinogen levels, increased von Willebrand factor activity, increased PAI-1 levels and decreased protein S levels. In study of Erem et al. [15] coagulation factors V, VII, VIII and X did not differ between diabetic patients and control subjects. In our study, plasma fibrinogen concentration in diabetic patients was significantly higher than in controls. Similarly to our results Zhao et al. [16] observed that there was significantly higher fibrinogen level in the diabetes group when compared with the euglycemic group. We have previously reported increased plasma fibrinogen concentrations in both, type 1 and type 2 diabetic patients compared to healthy control subjects [12]. An increased plasma fibrinogen concentration is considered as independent risk factor for cardiovascular disease. Observed fibrinogen increase in our diabetic patients might be explained by chronic low-grade inflammation which is characterized by elevated interleukin-6 (IL-6) levels. Same effect has adipose tissue which cells produce IL-6. This cytokine stimulates hepatic fibrinogen synthesis. Our diabetic patients were on the border of adiposity and they had abdominal obesity. Besides, in diabetes mellitus type 2 hepatic fibrinogen production rises in response to insulin in oppose to healthy subjects and type 1 diabetic patients [4]. 73

5 There are very few data concerning correlation between coagulation factors and cardiometabolic factors in type 2 diabetic patients, in the available literature. In our study we observed significant positive correlation between fasting blood glucose and coagulation factors IX, X and XI, in diabetic patients. The hemostatic abnormalities observed in diabetic patients were caused by hyperglycemia. Hyperglycemia is often accompanied by hiperinsulinemia which also play a role in the pathogenesis of prothrombotic state in type 2 diabetes. Several thrombotic conditions have been described as being accompanied by stress induced acute hyperglycemia which is often transient [17]. There were no any associations between fibrinogen and cardiometabolic risk factors in our diabetic patients. Obtained results are not in the accordance with results of Kafle et al. [18] who did find significant association between total cholesterol and fibrinogen and inverse association between HDL and fibrinogen in diabetic patients. These authors did not find any significant association between LDL, triglyceride and fibrinogen. A correlation between plasma glucose and fibrinogen concentrations has been documented, but we did not find significant association between these two variables. Our results have established highly significant positive correlation between coagulation factors II, VII, IX, X and plasma triglycerides and positive correlation between factor II and total cholesterol in diabetic patients. Kim et al. [19] in their study in which were included 448 normal individuals with hypercholesterolemia or triglyceridemia reported significant association of global coagulation test results with blood lipid levels. Furthermore, significant correlation was found between total cholesterol and triglyceride with coagulation factor II, VII, IX and X except fibrinogen, implying that elevation of procoagulant factors may contribute to shorter prothrombin time in subjects with high blood lipids. In our study, the serum concentration of triglycerides was significantly higher in DM2 than in control group. The serum concetrations of total cholesterol and LDL-cholesterol in the same group were higher, but not significantly in comparison with healthy subjects. We did not find any associations between plasma coagulation factors and HbA 1 c, DM duration and BMI. Future prospective studies are required to evaluate relationship between coagulation factors and cardiometabolic risk factors in patients with diabetes mellitus. of thrombosis and vascular complications in type 2 diabetic patients. Declaration of interest The authors declare no conflict of interest. Conclusions Results of the present study suggest that procoagulant state, as evidenced by enhanced activation of plasma cloting factors IX and XI and elevated plasma fibrinogen concentration, may contribute to the increased risk 74

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