Correlation between coagulation and inflammation state in patients with diabetes mellitus type 2 in relation to gender differences: is there any impact of eight-week exercise training?

Background/Aim. The hypercoagulable state and inflammation state in diabetics has been widely studied by previous researchers, but there is a lack of research about a possible impact of exercise training on this relationship. The aim of this study was to assess and compare correlation between the coagulation and inflammation status in patients with type 2 diabetes mellitus taking into account the gender differences as well as an impact of the 8-week exercise training on the correlation coefficient and parameters of the inflammation and coagulation state. Methods. A total of 60 patients in stable clinical condition and well-regulated diabetic status passed through research demonstrated a weakening correlations in the males after intervention between D-dimer and hs-CRP ( p < 0.05, r = 0.378), between hs-CRP and FII ( p < 0.01, r = 0.501), FV ( p < 0.05, r = 0.298), FVII ( p < 0.05, r = 0.351) as well as between D-dimer and FII ( p < 0.01, r = 0.759), and FVII ( p < 0.05, r = 0.296). The increase of the FX values ( p < 0.05) in the females after intervention suggested the possible antiinflammatory effect of exercise training. Conclusion. According to previous research, the higher levels of vWF was associated with a risk of cardiovascular disease in people with type 2 diabetes mellitus and vWF may be a risk factor unique to these populations. We demonstrated that the 8-week exercise training can significantly reduce the value of vWF in the males and females, suggesting a potential beneficial effect on the endothelial function parameters. Our research demonstrated a stronger correlation between the coagulation and inflammation parameters as baseline in the males than in the females with type 2 diabetes mellitus. According to our results, the 8-week exercise training lead to a weakening of the strength of correlation between the coagulation and inflammation parameters in the males and complete disappearance of this correlation in the females, suggesting a unique effect of exercise training that should be explored in future research. fibrinogen level, thrombin time (TT), prothrombin time (PT), activated partial thromboplastin time (APTT) and coagulation factors: FII, FV, FVII and FX. The inflammation status was assessed by the high sensitivity C-reactive protein (hs-CRP) levels and leukocytes count. Both fibrinolytic and coagulation states were evaluated before and after EWET.


Conclusion:
According previously research, higher levels of vWF is associated with risk of cardiovascular disease in people with type 2 diabetes mellitus and vWF may be a risk factor unique to these populations.We demonstrated that eight-week exercise training can significantly reduce value of vWF in males and females, suggesting a potential beneficial effect on endothelial function parameters.Our research demonstrated stronger correlation between coagulation and inflammation parameters as baseline in males than females with diabetes mellitus type 2. According our results, eight-week exercise training leads to a weakening of the strength of the correlation between coagulation and inflammation parameters in males and complete disappearance of this correlation in females, suggesting a unique effect of exercise training that should be explored in future research.Ključnereči: fizički trening, koagulacioni status, inflamatorni status, dijabetesmelitustip 2, razlikepremapolu

Introduction
The number of diabetics worldwide estimates by 2025 will be 324 million with character of epidemic. 1Diabetes mellitus alters blood coagulation and platelet function which supports the suggestion that diabetes mellitus is a hypercoagulable state with changes in fibrinolysis, decreased fibrinolytic activity and increased thrombotic risk. 2,3Correlation between coagulation factors in diabetics is more evident than in health subjects and this may be the reason for the more hypercoagulable conditions stated in diabetics.Dayer et al. (2014)   obtained that path model or diagram for coagulation factors are more complicated in diabetic patients than in normal individuals and convey that a sudden increase in the synthesis of each coagulation factors or their activation may trigger the initiation of coagulation cascade, leading to vascular clot formation with myocardial consequences. 4ipose tissue release mediators that induce a chronic inflammation state and alterations in coagulation.5 The metabolic syndrome is frequently accompanied by a prothrombotic state.This includes elevated plasma levels Von Willebrand factor, coagulative factors VIII, VII and fibrinogen. 6D-dimer and fibrinogen are known to be thrombosis risk factors. 7,8Elevation of D-dimer may increase risk of future myocardial infarction, stroke, and peripheral vascular disease. 9dimer indicate a low grade of prothrombotic risk in patients with diabetes mellitus type 2, but a higher risk of vascular complications. 10In type 2 diabetic patients with or without vascular complications fibrinogen concentrations do not indicate remarkable difference and may not be an important causal factor for vascular complications, 11 and there is obtained that diabetic have more fibrinogen in blood than healthy subjects. 12vWF is an acute phase protein and its plasma level increases in systemic inflammation.Hemostatic imbalance may contribute to the development of cardiovascular disease in patients with diabetes mellitus type 2. In patients with diabetes mellitus there is a state of hypofibrinolysis and increased levels of vWF. 13 Elevated levels of vWF is correlated with atherosclerosis and is associated with endothelial dysfunction in type 2 diabetes, as well asthe development of diabetes in post-infarction patients. 14,15  recent research is shown that increased vWF and D-dimer levels are associated with renal dysfunction in patients with type 1 diabetes, suggesting that endothelial dysfunction and hypercoagulability are associated with nephropathy in type 1 diabetes. 16Regular exercise training has anti-inflammatory effects and can reduce the risk of future thrombotic events. 17The coagulation cascade plays a critical role in the development of cardiovascular disease.Exercise training is known to reduce cardiovascular disease risk and through improved coagulation profile may contribute to this reduction. 18Single application of physical training has thrombotic effect that results in an increase of the number and activity of platelets, but regular application of exercise training attenuates these effects and acting suppression of coagulation. 19The purpose of this study was to assess and compare correlation between coagulation and fibrinolytic state regarding to gender differences in diabetics with type 2 diabetes mellitus as well as impact of exercise training on both coagulation and fibrinolytic state and correlation between coagulation and fibrinolytic state.

Study design and study protocol
The study examined the impact of eight-week interval exercise training on markers of fibrinolysis, coagulation and inflammation in patients with well-regulated diabetes mellitus type 2 with regard to gender differences.The study was designed as longitudinal observational study.Study was conducted in accordance with the Declaration of Helsinki.
All the procedures were previously approved by Institutional Review Board and Ethics Committee of Faculty of Medicine, University of Belgrade (06-17512/62-12, No.22/XII-4-1 dd 22.03.2012).Sixty patients, 35 males and 25 females, were enrolled in the study according inclusions criteria.The purpose of the study was clearly explained to the patients.All the subjects were informed about study protocol and that were provided written consent before the beginning of study.Investigation was performed in accordance with the ethical standards and according to national and international guidelines.For the purpose of fibrinolytic and coagulation states assessment we analyzed D-dimer, vWF, fibrinogen level, TT, PT, APTT, fibrinogen and coagulative factors II, V, VII and X.
Inflammation status were assessed by hs-CRP and leukocytes levels.Both fibrinolytic and coagulation states were evaluated before and after eight week exercise training.

Physical exercise protocols
Training consisted of aerobic training as 30 minutes briskly walking with speed 5 km/h and exercise training program under supervision.The exercise training was of interval mode with intensity that was estimated according to 75% of maximal heart rate.The rest time between one exercise sessions was equivalent with the time of exercise itself.Patients walked and trained five times a week for eight week.Each exercise session was completed in 45 minutes and was consisted of warm up for 10 minutes at 50 % of maximum heart rate in particular individual, twenty-five minute intervals at 75% of maximum heart rate in particular individual and ten-minutes cool-down period at 50% of maximum heart rate in particular individual.Exercise program included flexibility exercise, balance exercise, stretching, circles going forward, circles going backward, hip flexors stretch, hip circles, arm circles, toe and heal walks, lunge and trunk rotation exercise.

The inclusions criteria
Patients were selected based on their medical history and 60 patients with well -regulated diabetes conducted all phases of the study.The inclusions criteria was age between 40 and 60 years, stable clinical condition and well-regulated diabetic status as revealed by HbA1c in the range 6,0 -8,0 % .

The exclusion criteria
Patients on warfarin or heparin which might affect APTT and fibrinogen were excluded for the study.The exclusion criteria was recent surgery or illness, cardiac arrhythmias, abnormal ECG during exercise treadmill testing before study, diabetic cardiomyopathy, uncontrolled hypertension, uncompensated heart failure, severe valvular heart disease and musculoskeletal conditions that would hinder safe completion of the proposed exercise protocols.

Coagulation tests
The analyzed parameters were evaluated by the samples of drown blood in patients before meal in mourning period before and after intervention (the first day of the beginning of study and one day after the last training session).All samples were assayed in duplicate.
Venous blood (4.5 ml) for the test of fibrinogen, D-dimer, FII, FV, FVII and FX coagulation, APTT, TT and PT was collected in a fasting state into cooled tubes (Vacutainer System) using 3.2 % trisodium citrate as an anticoagulant, after centrifugation at 2500 g for 15 minutes.All coagulation tests were performed using Beckman Coulter ACL Elite Pro, Coagulation Analyzer.APTT and TT wasexpressed directly in seconds, with normal range 24.3-35.0sec and 11.0-17.8sec.PT results were reported in seconds (11.8-15.1).Coagulative factors were expressed as ratio: FII (50.0%-150.0%),FV (62.0%-139.0%),FVII (50.0%-129.0%),FX (77.0%-131.0%).Reference value for D-dimer was 255ng/ml and for fibrinogen was from 2.0 to 4.8 g/l.Determination of vWF Ag was performed using automated hemostasis analyzer Siemens BCS-XP (Siemens Healthcare Diagnostics Inc. Marburg/Germany, von Willebrand reagent REF OUBD37.vWFAg result was reported in percentage of normality.Reference value for vWF was from 55% to 200%. All plasma samples were stored in polypropylene tubes at -80 0 C until used for measurement.Le-account with reference value 3.4-9.7 x 10 ^9/L was determined using ADVIA 120 Hematology System, Siemens.High sensitivity CRP values with reference value 0-5 mg / L were analyzed by commercial kits of enzyme immunosorbent assay (ELISA).

Statistical analysis
The evaluated parameters were presented as mean values (MV) with standard deviation (SD).Students t-test was used to assess statistical difference between mean values were distribution was shown to be normal and statistical significance was set at P >0.05.
Pearson correlation coefficient test was done in order to establish correlation between continuous variables.Student's t-test for paired and unpaired samples was used to assess statistical difference between mean values were distribution was shown to be normal.
The significance level of 0.05 was used for correlation tests.Statistical analyses were performed using the Statistical Package for Social Sciences (SPSS, USA), version 19.0.
Average reduction in value of vWF in male was 10.32 with 95% the confidence interval 3.488.Eta value square (0.26) showed large effect of intervention.
Average reduction in value of thrombin time in male was 0.828 with 95% the confidence interval 4.303.Eta value square (0.35) showed large effect of intervention.
Average increase in value of factor FVII coagulation in male was -11.45 with 95% the confidence interval -4.364.Eta value square (0.35) showed large effect of intervention.
Average reduction in value ofvWF in female was 15.88 with 95% the confidence interval 3.601.Eta value square (0.35) showed large effect of intervention.
Average reduction in value of TT in females was 0.92 with the confidence interval 3.401.Eta value square (0.32) showed large effect of intervention.
Average increase in value of factor VII coagulation in females was 17.924 with 95% the confidence interval -4.398.Eta value square (0.44) showed large effect of intervention.
Average increase in value of factor X coagulation in females was 6.74 whit 95% the confidence interval 2.685.Eta value square (0.23) showed large effect of intervention.

According analysis for correlation between baseline inflammation and coagulation
parameters in females before EWET (Table 6), there is demonstrated significant moderate positive correlation between hs-CRP and fibrinogen (r=0.52,P<0.05), fibrinogen and F II coagulation (r= 0.52, P< 0.01).
According analysis for correlation between inflammation and coagulation parameters in females after EWET (Table 8), there is demonstrated negative correlation between Ddimer and hs-CRP (r=-0.46,P<0.05).

Discussion
The present study was undertaken to explore the correlation between inflammation and coagulation state as well as impact of exercise training on this correlation in diabetics.Von Willebrand factor is associated with cardiovascular disease, type 2 diabetes mellitus and insulin resistance.Higher levels of vWF is associated with risk of cardiovascular disease in people with type 2 diabetes mellitus or insulin resistance, which suggests that vWF may be a risk factor unique to these populations.In addition, elevated levels of vWF is associated with increased risk of cardiovascular disease in a community-based sample, even after accounting for traditional cardiovascular disease risk factors. 20r study showed significant decrease for values of vWF in both males (P<0.005) and females (P<0.001) with diabetes mellitus type 2 after eight week training and these results correlate with recent researches.Creighton et al. explore the effects of an acute resistance exercise test on the primary hemostatic system in both resistancetrained and untrained individuals and obtained that reduced vWF in both groups may be attributed to training status. 21Androulakis et al. examined whether a high volume of training can lead to endothelial activation and/or dysfunction in professional soccer players due to exercise-induced oxidative stress and vWF antigen plasma levels were measured 1 day before and after 7 weeks of strenuous exercise.There is showed that mean vWF Ag plasma levels were significantly decreased from 95.1%±26% to 88.3%±27.2% at the end of the experimental period (P=0.018),suggesting a potential beneficial effect on the endothelium. 22According Jahangard et al. vWF showed significant reduction after 10 sessions of submaximal aerobic cycling in sedentary healthy postmenopausal women. 23We showed significant differences in mean values for vWF between males (106.25±32.61)and females (124.28±24.62)(P<0.05) as baseline with higher value in females within the reference value.
We showed significant decreases in values of TT in both males (P < 0,001) and females (P<0.005) after EWET.Our findings demonstrated no differences in APTT, PT and FII coagulation in diabetics are consistent with the results conducted by Lamprechts et al.
that showed no statistically significant differences between pre-and post a single bout of walking exercise in APTT, FII coagulationand PT in obese women. 24Short term exercise in healthy subjects is usually associated with a significant shortening of APTT, 25,26 but according our results there is no differences in APTT after regular eight week training.
According to Lockard et al. plasma prothrombin fragment was found to decrease significantly with exercise training three times a week in six months. 18 obtained that the values of fibrinogen were distinctly above normal in females both as baseline and after study and similar findings were reported byKafle and Shrestha. 12They obtained significantly higher fibrinogen in patients with diabetes than control and showed that fibrinogen was significantly higher in diabetic patients with coronary artery disease than whose patients who had only diabetes or coronary artery disease.We obtained no changes in fibrinogen concentration after intervention and these findings correlate with the research of Bizheh et al.which examined effect of single bout resistance exercise in sedentary middle aged men on fibrinogen. 27According a recent investigation, physical training reduces fibrinogen concentration in patients with coronary heart diseases who have long-term physical training applied.It was determined that fibrinogen concentration significantly increased after physical load in all the treatment stages in both coronary heart disease and control group, while fibrinogen concentration gradually decreased in the group of the trained patients after 1 year, 28 supportingthat long time physical training can effectually reduce fibrinogen concentration.
A recent study obtained correlation between hs-CRP and D-dimer in patients with pulmonary embolism. 29According our research there is obtained stronger baseline correlation between hs-CRP and D-dimer than between hs-CRP and fibrinogen in males as baseline.In females there is demonstrated strong positive correlation between hs-CRP and fibrinogen as baseline, but there is no correlation between those parameters after study.In females there is demonstrated negative correlation between hs-CRP and Ddimer both as baseline and after study.According the Speedwell Study it is shown a positive correlation between hs-CRP and D-dimer anda much stronger association between hs-CRP and fibrinogen in heart disease, 30 and these findings correlate with our findings as baseline.Also according a recent researchhs-CRP correlated positively with fibrinogen and D-dimer in hemodialysis patients. 31These findings correlate with our findings which showed strong positive correlation between hs-CRP and either D-dimer or fibrinogen in males as baseline.In addition, we demonstrated weakness of this correlation in males after EWET.
According to Long et al.D-dimer does not correlate positively with fibrinogen in diabetics. 32In our study there is shown none correlation between D-dimer and fibrinogen but only in females.We demonstrated strong correlation between D-dimer and fibrinogen in males both before and after study.FII coagulation is the main cause for hypercoagulable state and the last target of coagulation cascade either from intrinsic or extrinsic origin and play a determinant role in initiation of vascular complication in diabetics, while D-dimer indicate a higher risk of vascular complications in patients with diabetes mellitus type 2. According our study that is demonstrated that strong correlation between D-dimer and FII coagulation in males which becomes moderate after study.In females there is negative correlation between D-dimer and FII coagulation either baseline or after study.
We demonstrated no change in hs-CRP concentration in our study which correlates with research by Levingers et al. on 56 middle-aged men and women.They showed that 10 weeks of resistance training did not significantly alter hs-CRP expression. 33In our research, there is demonstrated a strong correlation between hs-CRP and FII coagulation in males as baseline that became moderate correlation after intervention suggesting potential benefit of eight week training on relationship between coagulation and inflammation.In addition, we showed no correlation between hs-CRP and FII coagulation in females either before or after study, pointing to a weaker association between coagulation and inflammation in females in comparison with males.
According analysis for correlation between baseline inflammation and coagulation parameters in males before our study, there is strong positive correlation between Ddimer and either FII coagulation or fibrinogen as baseline that stays strong after intervention, while those correlation have been negative in females both before and after study.We obtained weakness in correlation between hs-CRP and either fibrinogen, FII, FV, FVII, FX coagulation, leukocytes or D-dimer in males after study, supporting effectiveness of intervention.
A recent research by Dayer et al. obtained correlation between FV coagulation and vWF in diabetics which is absent in normal mode, 34 and in addition FV coagulation may bind to vWF, 4 and both findings correlate with our findings.We demonstrated moderate correlation between vWF and either FV, FII or FX coagulation but only in females, which completely disappear after study, suggesting potential effect of eight week training.We obtained moderate correlation between fibrinogen and FV coagulation in males as baseline becomes weak correlation after study suggesting both higher coagulative state and anti-coagulative effects of intervention in males, while there is no correlation between fibrinogen and FV coagulation in females either before or after study, suggesting a lower coagulative state in females.
A recent study which investigated the correlation of coagulation indicators with inflammatory markers for sepsis in hematologic malignancy patients demonstrated that the level of procalcitonin positively correlated with APTT and D-dimer level. 35Our study showed significant moderate positive correlation between hs-CRP and fibrinogen in both males and females as baseline as well as in males after eight weeks training, but no correlation between hs-CRP and fibrinogen in females after eight weeks training.Similar results to those reported by Thor and Swedenborg as a strong positive correlation between hs-CRP and fibrinogen particularly in diabetics which correlate with our findings. 36In addition, they reported a correlation between hs-CRP and either fibrinogen or vWF in diabetics.According them, there is no correlation between fibrinogen and other markers of hypercoagulability, thrombin-anti thrombin, prothrombin and D-dimer although the latter three correlated with each other.In our study there is no correlation between vFW and hs-CRP.We showed correlation between hs-CRP and fibrinogen in males both as baseline and after study and in addition we showed that positive correlation between hs-CRP and fibrinogen as baseline in females which becomes not significant after study, supporting anti-inflammatory effect of eight week training.
According Alehagen et al., patients with suspected heart failure and low plasma concentrations of FII, FVII and FXI coagulation had significantly higher mortality rates during the follow-up period of ten years as compared with those with higher plasma concentrations.Increasing in plasma concentrations of FII and FVII coagulation after exercise training may indicate a protective effect of training on mortality, and a protective effect of exercise training on hemorrhage. 37We demonstrated significant increase within the normal range of FVII coagulation in males and little above normal range in females after eight week training, suggesting a protective effect of training on mortality.According to Ruiz-Saez et al.afibrinogenemia, factor VII or factor XI deficiencies are those most commonly associated with venous or arterial thrombosis. 382 Gleeson et al. pointed out to the novel function concerning FX coagulation factor as an endogenous, receptor-associated protein-sensitive, proteaseactivated receptor 2-dependent regulator of myeloid cell pro-inflammatory cytokine production.Further, exposure to factor X significantly impairs pro-inflammatory cytokine production.Factor X inhibited nuclear factor-kappa B activation in THP-1 reporter cells requires phosphatidylinositol 3-kinase activity for its anti-inflammatory effect. 43We demonstrated significant increase of factor X level in females within the normal range after intervention.In addition, we demonstrated moderate positive correlation between hs-CRP and FX in males as baseline that become weak correlation after study, and none correlation between hs-CRP and FX in females.Increasing in plasma concentration of FX coagulation that is demonstrated from the results of our study after eight week training only in females could suggest the possible anti-inflammatory effect of exercise trainingand should be examined in future research.
The main limitation of the present study is the relatively small number of patients, but the study enrolled both men and women in an adequate number to obtain statistically significant results.The major advantage of the present study is investigation of correlation between inflammation and coagulation state between the two sexes with diabetes, which is insufficient in previous research.This longitudinal study may bring some new findings regarding effects of exercise training on treatment of patients with diabetes mellitus type 2.

Conclusion
We demonstrated a statistically significant reduction in mean vWF levels after eight weeks exercise training in both males and females with diabetes mellitus type 2, suggesting a potential beneficial effect on endothelial function parameters.According our research there is obtained stronger correlation between coagulation and inflammation parameters in males than females as baseline and in addition we obtained the weakening of correlation between coagulation and inflammation parameters in both males and females after intervention, suggesting anti-inflammatory and anticoagulant effect of intervention.
The effect of exercise training on correlation between coagulation and inflammation state in patients with both well-controlled and poorly-controlled diabetes mellitus type 2 should be explored in future.
Increasing of factor VII levels in our study may indicate possible reduction of risk for venous or arterial thrombosis after intervention.The function of coagulation factor FXa is unclear.Interesting results were recent reported by Kuet al.Expression level of secretory group IIA phospholipase A2 is elevated in inflammatory diseases and lipopolysaccharide upregulates the expression.FXa suppressed the activation of cytosolic phospholipase A2 and extracellular signal-regulated kinase by lipopolysaccharide. 41According a study by Bukowskaa et al.FXa mediates inflammatory signaling in atrial tissue by inducing an inflammatory signaling by activation of proteaseactivated receptors.