THE STUDY OF PROTHROBOTIC STATE IN PATIENTS WITH TYPE 2 DIABETES MELLITUS AND CARDIOVASCULAR DISEASES Oana Bădulescu 1, Codruţa Bădescu 2, Manuela Ciocoiu 1, Magda Bădescu 1 1 DEPARTMENT OF PATHOPHYSIOLOGY; 2 DEPARTMENT OF INTERNAL MEDICINE, UNIVERSITY OF MEDICINE AND PHARMACY "GRIGORE T. POPA" IASI, ROMANIA Summary Epidemiologic studies consider vascular complications as the first cause of death in diabetic patients. Persistent hyperglycemia is responsible for the affection of large vessels, causing severe diseases, such as myocardial infarction, cerebrovascular accident or diabetic gangrene. The objective of this study was to determine the thrombotic risk in patients with type 2 diabetes mellitus (DM) with or without cardiovascular disease (CVD) compared with non-diabetic patients with cardiovascular disease. The study comprises 18 patients admitted to the Geriatric Ward of the C. I. Parhon Hospital from June 1, 9 to December 1, 9. The patients were distributed in following groups: diabetic patients without ischemic cardiopathy-related disorders (DM), diabetic patients with clinical or off-clinical (EKG, Echocord) ischemic cardiopathy-related disorders (DM+IC), non-diabetic patients with ischemic cardiopathy-related disorders (IC); the patients were investigated by determining three factors of coagulation profile: von Willebrand Factor (vwf), factor X (Stuart-Power), DD-imers. The highest levels of the three studied parameters were found in the diabetic groups (DZ+CI) (p<,1) and (DZ-CI) (p<,5). The elevated levels of vwf in diabetic patients, reflect the effects of hyperglycemia on endothelium and the existence of plateletvwf interaction, which would explain the accelerated atherosclerosis in this patients. But diabetics vascular pathology it is also based on a lack of balance between the thrombosisthrombolysis processes, to the benefit of thrombosis, followed by the occurrence of ischemic events Key words. coagulation, fibrinolysis, hyperglycaemia, insulin resistance. Introduction Diabetes mellitus is a medical condition associated with evolving atherosclerosis (ATS) and with a high prevalence of micro- and macro-vascular diseases, which together are the main cause of morbidity and mortality in diabetic patients. Type 2 diabetes mellitus is characterized by fluctuating hyperglycemia, and as a consequence the development to varying degrees of protein glycation. Some of the established effects of hyperglycemia on oxidative stress, endothelial cell dysfunction, extracellular matrix formation and apoptosis are considered to play an important role in diabetes-related vascular 81 damage. (Bădescu et al., ). The insulin resistance is accompanied by the development of a chronic inflammatory, atherothrombotic phenotype which appears to underpin the strong association with both type 2 diabetes and cardiovascular diseases. Haemostatic mechanisms are altered both in response to insulin resistance itself, with further changes secondary to abnormal glucose metabolism as impaired glucose tolerance and frank type 2 diabetes develop. The abnormalities in haemostasis, in insulin resistance and diabetes seem co-ordinated to generate a prothrombotic phenotype. Evidence indicates that the use of antiplatelet regimens in both primary and
secondary prevention, leads to a fall in morbidity and mortality. Equally, in the acute setting, low molecular weight heparins and fibrinolytic agents have beneficial effects on outcomes. One of the earliest vascular changes associated with insulin resistance is the development of endothelial dysfunction, with suppression of phosphoinositide 3- kinase-mediated nitric oxide (NO) generation. This has a number of adverse effects on the vasculature, including the loss of control of platelet activation, normally afforded by NO. At the same time, insulin resistance is accompanied by increased levels of a number of proteins involved in the fluid phase of coagulation. Material and methods The study comprises 18 patients who were followed for 6 month, from June 1, 9 to December 1, 9. The patients were distributed in the following groups: - group 1 Diabetic women with clinical or off-clinical disorders (EKG, Echocord) related to ischemic cardiopathy (DM+IC) - group 2 Diabetic women without ischemic cardiopathy-related disorders (DM- IC) - group 3 Diabetic men with clinical or off-clinical ischemic cardiopathyrelated disorders (DM+IC) - group 4 Diabetic men without ischemic cardiopathy-related disorders (DM- IC) - group 5 Non-diabetic women with ischemic cardiopathy-related disorders (IC) - group 6 Non-diabetic men with ischemic cardiopathy-related disorders (IC) The major objective of our study was: the determination of the thrombotic risk in patients with type 2 diabetes mellitus with or without cardiovascular disease compared with non-diabetic patients with cardiovascular disease. Results and disscussions In the studied group, we followed the values of three parameters (von Willebrand factor, fibrinogen, DD-imers), in correlation with the duration of the diabetes mellitus. Fibrinogen In women, fibrinogen had the most homogeneous individual values in the group of diabetic women without ischemic cardiopathy (DM-IC) (4,14CV%). in diabetic women with cardiovascular diseases associated (DZ+IC), the fibrinogen values is directly, but week, correlated with the duration of the disease (r=,29); in diabetic women without cardiovascular diseases (DZ-IC), the fibrinogen values are indirect and weak correlated with the duration of the disease (r = -,25). In men, comparing the fibrinogen values, it was observed the same aspects as in women s groups. Fibrinogen had the most homogeneous individual values in the group of diabetic men with ischemic cardiopathy (DM+IC) (8,95CV%). - in group of diabetic men with ischemic cardiopathy (DM+IC), there was no correlations between fibrinogen values and the duration of the disease (r=,8) ; - in diabetic patients without cardiovascular diseases (DZ-IC) the elevated values of fibrinogen are associated in percentage of 43% with the duration of the disease. Regarding the mean values of fibrinogen, these did not show a significant differences between genders or between the studied groupes. 82
7 r=.29 5 1 15 diabetic women with IC 48 46 44 42 38 36 DM+IC DM-IC nondm+ic Male Female 7 r= -.25 5 1 15 diabetic women without IC Fig.1: Correlations between fibrinogen values () and the duration of the DM, in women r=.8 5 1 15 diabetic men with IC r=.43 5 1 15 diabetic men without IC Fig.2: Correlations between fibrinogen values () and the duration of the DM, in men Fig.3: The mean values of fibrinogen () in the studied groups von Willebrand Factor In women, vwf had the most homogeneous individual values in group of diabetic women with ischemic cardiopathy ( DM+IC) (13,3CV%). it was observed a direct correlation (r=,86) with the duration of the disease in diabetic women with ischemic cardiopathy associated (DM+IC) ; an indirect correlation (r =-,22) was observed in the group of diabetic women without ischemic cardiopathy (DM-IC); the highest values of vwf were found in recently diagnosed DM. In men, comparing the values of von Willebrand factor, it was observed the same aspects as in women groups: von Willebrand factor had the most homogeneous individual values in the group of diabetic men with ischemic cardiopathy (DM+IC) (28,47CV%). a direct, but weak correlation (r=,21), was observed in the group of diabetic men with ischemic cardiopathy (DM+IC); a moderate direct correlation between elevated values of vwf and the duration of the disease (r =,59) was observed in the group of diabetic men without ischemic cardiopathy (DM-IC). Regarding the mean values of vwf, these did not show a significant differences between genders or between the studied groupes. 83
35 25 15 5 35 25 15 5 r=.86 5 1 15 2 diabetic women with IC r= -.22 5 1 15 2 diabetic women without IC Fig.4: Correlations between Von Willebrand factor values (%) and the duration of the DM, in women 35 25 15 5 35 25 15 5 r=.21 5 1 15 diabetic men with IC r=.59 5 1 15 diabetic men withoutic Fig.5 :Correlations between Von Willebrand factor values (%) and the duration of the DM, in men 25 15 1 5 DM+IC DM-IC nondm +IC Male Female Fig. 6: The mean values of von Willebrand factor (%) in the studied groups DD-imers In women, DD-imers had the most homogeneous individual values in the group of diabetic women without ischemic cardiopathy ( DM-IC) (15,91CV%). it was observed an indirect correlation (r=-,65) with the duration of the disease in diabetic women with ischemic cardiopathy (DM+IC) ; in the group of diabetic women without ischemic cardiopathy (DM-IC) it was observed a direct correlation (r =,24) with the duration of the disease; the DD-imers' values increase with the history of DM. In men, it was observed that DD-imers had the most homogeneous individual values in group of diabetic men with ischemic cardiopathy (DM+IC)(8,39CV%). a direct, but weak, correlation with the duration of the disease (r=,23) it was observed in group of diabetic men with ischemic cardiopathy (DM+IC); an indirect correlation (r = -,38) it was observed in diabetic men without cardiovascular disease (DZ-CI); the elevated values of DD-imers are associated with the recently diagnosed DM. Regarding the mean values of DD-imers', these presented the following differences: there were not significant differences between genders ; in women, diabetic patients with cardiovascular diseases (DZ+CI) have significantly higher mean values than diabetic patients without cardiovascular diseases (DZ+CI) (p<,5) ; in men, no significant differences were recorded in mean values of DD-imers, in the studied groups. 84
8 r= -.65 5 1 15 diabetic women with IC 8 r=.24 5 1 15 duration of the disease (years) Fig.7: Correlations between DD-imers values () and the duration of the DM, in women. 8 8 r=.23 5 1 15 duration of the disease (years) r= -.38 5 1 15 duration of the disease (years) Fig.8: Correlations between DD-imers values () and the duration of the DM, in men 85 1 8 DM+IC DM-IC nondm+ic Female Male Fig. 9: The mean values of DD-imers () in the studied groups The elevated levels of vwf are an indication of endothelial cell damage and are correlated with other cardiovascular risk factors (Blann., 1991). Many studies have reported an association between elevated FVIII and/or vwf levels, the presence of atherosclerotic disease (Egeberg, 1962) and the risk of future cardiovascular events in patients with angina and previous myocardial infarction (Jansson et al., 1991). The mechanisms linking the elevated levels of vwf FVIII to insulin resistance and type 2 diabetes may be related to the presence of underlying endothelial dysfunction and or inflammation, both of which are involved in the development of insulin resistance. It was shown that the fibrinogen plays a part in increasing the sensitivity of platelets to aggregating agents. The fibrinogen is linked to the platelet receptors and may play an agonist part in the in vitro platelet aggregation. It was also shown that after the stimulation with agonists, the fibrinogen adhesion to platelets in diabetic subjects increases. The level of the plasmatic fibrinogen is high in the diabetics and this level is in direct relation to glycemia regulation. Therefore, it is natural to say that in diabetes the interaction fibrinogen thrombocytes takes place, which contributes to the increase of the sensitivity of thrombocytes by stimulating it with the agonists and may play a part in the acceleration of the process of atherosclerosis in diabetics. Plasma levels of fibrinogen influence thrombogenesis, affecting the rheology of blood flow, blood viscosity and platelet aggregation, and elevated levels have been shown to be a strong and independent cardiovascular risk factor in prospective epidemiological studies.
Hyperglycemia per se has been implicated in the development of prothrombotic changes in clamp studies (Stegenga et al., 6) where under conditions of euinsulinaemic hyperglycemia, twofold increases in thrombin antithrombin complexes and circulating soluble tissue factor were documented. Hyperinsulinemia in the presence of euglycaemia led to increases in PAI-1, and the authors made the observation that glucose modulated thrombotic processes whilst insulin regulated fibrinolysis. In vitro, glycated albumin has been reported to increase tissue factor expression in both monocytes and umbilical vein endothelial cells to indicate a mechanism by which glycation might initiate coagulation processes. Association studies in nondiabetic subjects have indicated correlations between serum advanced glycation end products (AGE) and both PAI-1 and fibrinogen levels to support the concept that AGE accumulation may stimulate prothrombotic changes in man. In addition, higher levels of glycation were associated with endothelial cell apoptosis which might contribute to vascular damage. Multiple coagulation pathways are abnormal in individuals with type 2 DM. There is impaired fibrinolysis, in part due to elevated PAI-1 levels. Increased PAI-1 levels are associated with thrombotic disorders, including myocardial infarction and are an important predictor of the development of type 2 DM (Stegenga et al., 6). Several components contribute to this elevation of PAI-1: one is insulin resistance and resulting hyperinsulinemia, possibly independent of hyperglycemia, as there is an insulin promoter site on the PAI-1 gene. (Stegenga et al., 6). Another factor promoting increases in PAI-1 are the elevations in triglycerides and very-low density lipoprotein cholesterol, which also promote PAI-1 synthesis. Furthermore, PAI-1 is also produced by platelets in excessive amounts by visceral adipocytes. Hypercoagulation may also be promoted by tumor necrosis factor α (TNF-α), adiponectin and interleukin-6. There is also increased activation of factor VII as well as increased levels of fibrinogen and vwf. Abnormalities of visceral fat and its metabolic function may provide a common precursor insulin resistance, lipid abnormalities and abnormalities in coagulation/fibrinolysis. Visceral fat produces proinflammatory cytokines such as interleukin-6 and TNF-α, which in turn elevate fibrinogen levels. These cytokines are responsible for some of the endothelial dysfunction that causes an increase in vwf. There is also some experimental evidence to support the notion that the coagulopathy is driven by hyperglycemia rather than hyperinsulinemia (Stegenga et al., 6). Nevertheless, most studies have found a relation of either hyperinsulinism or hypertriglyceridemia to specific abnormalities in coagulation. Conclusions The assessment of the blood flow, vascular reactivity and certain endothelial dysfunction markers has been shown to be associated with an unfavorable cardiovascular prognosis. In DM, hyperglycemia and the metabolic syndrome components directly or indirectly cause endothelial dysfunction. The development of endothelial dysfunction, in pacients with type 2 diabetes mellitus, promotes vasospasm, thrombosis and inflammation, processes that may all be involved in the early stages of atherosclerotic disease, a view supported by clinical studies. DM is by itself a major atherogenesis risk factor. The risk of developing cardiovascular events is higher in DM patients, because hyperglycemia determines subsequent metabolic disorders, by forming AGE and then by triggering quantitative and qualitative changes in serum lipoproteins, mechanisms which would explain the atherosclerosis acceleration in these patients. References Bădescu, M., Roşca M., Bohotin, C.: Fiziopatologie Generală. Vol. 2, chap. 1, 38-353,. Edited by M. Badescu, Ed. Cantes, Iasi Blann A., Increased circulating levels of von Willebrand Factor antigen in smokers may be due to lipid peroxides. Med. Sci. Res. 1991; 19: 535 6. Egeberg O.,Clotting factor levels in patients with coronary atherosclerosis. Scand. J. Clin. Lab. Invest. 1962; 14: 253 8. Jansson J., Nilsson T., Johnson O. von Willebrand factor in plasma: a novel risk factor for recurrent myocardial infarction and death. Br. Heart J. 1991; 66: 351 5. Stegenga M.E., van der Crabben S.N., Levi M., Hyperglycemia stimulates coagulation, whereas hyperinsulinemia impairs fibrinolysis in healthy humans. Diabetes. 6;55:187 1812. 86