CHANGES IN THE HAEMOSTASIS SYSTEM UNDER THE INFLUENCE OF TREATMENT OF PATIENTS WITH ALCOHOLIC LIVER CIRRHOSIS IN COMBINATION WITH OBESITY USING ADEMETHIONINE, ARGININE GLUTAMATE AND ROSUVASTATIN

Introduction. The urgency of the problem of liver cirrhosis (LC) is caused by the in­ crease in morbidity, prevalence, life­threatening complications, disability and increasing mortality of able­bodied population. The aim of the study was to examine the effect of complex treatment with ademethionine, arginine glutamate and rosuvastatin on chang­ es in the haemostasis systemin patients with alcoholic liver cirrhosis (ALC) in combina­ tion with obesity. Research methods. The study included 105 patients diagnosed with ALC in combination with obesity. The assessment of the effectiveness of a three­month treatment regimen with ademethionine, arginine glutamate and rosuvastatin in obese patients with alcoholic liver cirrhosis (ALC) included indicators of synthetic liver func­ tion and hemostasis (total protein, albumin, fibrinogen, platelet count, factor Von Wil­ lebrand factor, activated partial thromboplastin time (APTT), thrombin time (TT), in­ ternational normalized ratio (INR), prothrombin index (PI), D-dimer, tissue plasminogen activator (tPA), 1 plasminogen activator (PAI-1), tPA/PAI-1 index, asymmetric dimethy­ larginine (ADMA)), as well as liver cirrhosis severity (Child-Pugh score) and 3-month MELD mortality score. Results. Decreased levels of total protein, albumin, fibrinogen, PI, platelet count and increased levels of Von Willebrand factor, prothrombin time (PT), APTT, TT, INR, D­dimer, tPA and PAI­1, ADMA were revealed. Such changes worsened with increasing liver cirrhosis decompensation and were accompanied by an increase in the Child-Pugh and MELD scores (P < 0.05). There was a more pronounced increase in levels of PAI-1 than tPA, that was accompanied by a decrease in tPA/PAI-1 index. A number of researchers indicate that an increase in PAI­1 levels can cause a hyperco­ agulable state, so its increase with a decrease in tPA/PAI-1 index in patients with ALC in combination with obesity indicates a risk of thrombogenic conditions. This is also evidenced by the increasing number of D-dimers. Therefore, the fibrinolytic/antifibrino­ lytic factors should be considered in the treatment of such patients to prevent LC com­ plications. Conclusions. The inclusion of ademethionine, arginine glutamate and rosu­ vastatin in the treatment regimen for 3 months improved the levels of total protein, albumin, fibrinogen, PI, platelet count, Von Willebrand factor, PT, APTT, TT, INR, D­ dimer, tPA and PAI­1, ADMA, which was accompanied by a decrease in Child­Pugh severity score and MELD 3­month mortality score.

disease is characterized by a defective hepatic synthesis of clotting factors and thrombocytopenia, largely caused by portal hypertension and by hyperfibrinolysis that may further contribute to alterations in hemostasis and may cause intra-and extrahepatic thrombogenic conditions [2,3,10,11].
Standard coagulation tests, such as international normalized ratio (INR), pro thrombin time (PT), activated partial thromboplastin time (APTT) are not sufficient in prediction of bleeding or thrombotic complications of LC [1,4,14].
The regulation of normal blood flow is performed by a fibrinolytic system, the main enzyme of which is plasmin. It is made up of plasminogen with serine prote ases, such as tissue or urokinase plasminogen activator (t-PA and u-PA, respec tively). All proteins involved in fibrinolysis, except t-PA andtype 1 plasminogen activator (PAI-1), are synthesized in the liver. The concentration of plasminogen, α2-antiplasmin, factor XIII and inhibitor of Thrombin Activatable Fibrinolysis (TAFI) is reduced at LC. Fibrinolysis is inhibited by PAI-1 and TAFI, the amount of which increases in the blood [5,8,9]. It is proved that the increase of PAI-1, which is considered as a key component of the system of fibrinolysis, in liver diseases cor relates with the parameters of the systemic inflammatory process, endothelial dys function and fibrogenesis [6,13].
Due to the disorder of the hemostasis system the growing incidence of ALD, methods are being sought to prevent the progression of the pathological process in the liver, the occurrence of complications, to improve the liver function and to im prove the quality of life of such patients.
The aim of the study was to examine the effect of complex treatment with ade methionine, arginine glutamate and rosuvastatin on changes in the haemostasis sys temin patients with alcoholic liver cirrhosis (ALC) in combination with obesity.
Research methods. The study included 105 patients diagnosed with ALC in combination with obesity, including 18 women and 138 men aged (46.1 ± 8.5) years and a median duration of disease (5.3 ± 2.6) years. Patients were divided into sub groups depending on the stage of Child-Pugh decompensation: class A (n = 39), class B (n = 34), class C (n = 32). Depending on the applied treatment, all patients were divided into groups: patients of group I (IA (n = 22), IB (n = 18) and IC (n = 16)) received basic therapy; patients of group II -IIA (n = 17), IIB (n = 16) and IIC (n = 16), additionally received ademethionine, arginine glutamate and rosuvastatin.
Group IІА in addition to the basic treatment, received intravenously 500 mg of ademethionine per day during two weeks, followed by oral administration of 500 mg of ademethionine, 1500 mg of arginine glutamate per day for 12 weeks.
Group IІB in addition to the basic treatment, received intravenously 1000 mg of ademethionine per day for two weeks, followed by oral administration of 1000 mg of ademethionine and 3000 mg of arginine glutamate for 12 weeks.
Group IІC in addition to their basic treatment, received intravenously 1000 mg of ademethionine per day for two weeks, followed by oral administration of 1500 mg of ademethionine and 4500 mg of arginine glutamate per day for 12 weeks.
Groups IIA and IIB received additionally 20 mg of rosuvastatin orally per day for 12 weeks, and patients of group ІІС received an additional 10 mg of rosuvastatin orally per day for 12 weeks.
Diagnosis was verified using clinical and laboratory-instrumental methods in ac cordance with the order of the Ministry of Health of Ukraine No. 826 dated Novem ber 6, 2014, adapted clinical guidelines "Non-Alcoholic Fatty Liver Disease", 2014, adapted clinical guidelines "Alcoholic Liver Disease", 2014, adapted clinical guidelines " Liver Cirrhosis, 2017 (State Expert Centre of the Ministry of Health of Ukraine, Ukrainian Gastroenterology Association, Kyiv), recommendations of the European Association for the Study of Liver, Diabetes and Obesity (EASL-EASD-EASO, 2016).
Exclusion criteria were liver cirrhosis of the viral, toxic and autoimmune genesis, metabolic diseases of the liver, oncological diseases, and the lack of indi vidual consent of the patient to conduct the study. All patients were matched ac cording to age and sex. The research was carried out in accordance with the ethical principles of conducting scientific research and principles of the Helsinki Declaration.
The protein-synthetic function of the liver was determined by the level of albu min, fibrinogen in blood, prothrombin index (PI) and INR, which were measured using conventional methods. PT, APTT, thrombin time (TT) and fibrinogen were measured using conventional methods. The content of plasmin degradation product of the insoluble D-dimer product fibrin (DD) was measured by immunoassay using "Technozym D-dimer ELISA" ("Technoclone", Austria). The Willebrand factor was determined by the method based on the stimulation of ristomycin aggregation (sus pension of fixed donor platelets by the Willebrand factor of the study without plate let plasma). Calculation was carried out on a calibration curve built on logarithmic paper. For its construction, 20 donor platelet-poor plasmas were diluted 1 : 1, 1 : 2, 1 : 4, 1 : 5, corresponding to a concentration of 100 %, 50 %, 25 %, 12.5 % of the Willebrand factor in blood plasma. To increase the accuracy of the analysis, two parallel samples were tested, followed by an average value calculation. The t-PA level in the blood was determined by the immunoassay method using the t-PA Com bi Actibind ELISA kits ("Technoclone GmbH", Austria). The PAI-1 level in the blood was determined by immunoassay using human PAI-1 Antigen ELISA kits ("Technoclone", Austria). The endothelial dysfunction was studied by content of asymmetric dimethylarginine (ADMA) in the blood, determined by the immune enzymatic method using ADMA High Sensitive ELISA (Biovendor, Czech Republic).
The severity of the LC was assessed using the Child-Pugh score and the MELD score (Mayo Endstage Liver Disease, 2001). The control group consisted of 20 healthy individuals, who were age and gender matched. Assessment of patients was per formed before and after 3 months from the beginning of treatment.
Statistical processing of the obtained results was carried out using the software package Statistica v. 12.0 (StatSoft, USA, trial) and Microsoft Exсel. The average values are presented in the form (M ± m), where "M" is the average value of the indicator, "m" is the standard error of the average. Student's ttest was used to de termine the significance of differences between groups in a distribution close to normal. Differences at P < 0.05 were considered statistically significant.
Results and discussion. In patients with LC in combination with obesity, chang es in synthetic liver function and hemostasis are characterized by decreased levels of total protein, albumin, fibrinogen, PI, platelet count, accompanied by an increase in Von Willebrand factor, PT, APTT, TT, INR, D-dimer, tPA and PAI-1 and ADMA. Such rates deteriorated with increasing LC decompensation, which is accompanied by an increase in the Child-Pugh and MELD scores (P < 0.05) (Tabl. [1][2][3]. After the course of treatment in patients of group II at the stage of compensa tion, the rates of total protein, albumin, fibrinogen, platelet count, Von Willebrand factor, PT, APTT, TT, INR, PI, D-dimer, tPA, PAI-1, tPA/PAI-1, ADMA, Child-Pugh and MELD scores improved significantly (P < 0.05), while in patients of group I they worsened, but no significant difference compared to baseline was found (P > 0.05). In patients of group II at the stage of compensation after treatment, Von Willebrand factor, D-dimer, tPA, PAI-1 and ADMA differed from those in the con trol group (P < 0.05), indicating a higher sensitivity of such indicators to disorders in the system of blood clotting compared to routine rates.
At the stage of subcompensation after treatment in patients of group II, the total protein, albumin, fibrinogen, platelet count, Von Willebrand factor, PT, APTT, TT, INR, PI, D-dimer, tPA, PAI-1, tPA/PAI-1, ADMA, Child-Pugh and MELD scores significantly improved, but differed from those in the control group (P < 0.05). In patients receiving basic treatment, they worsened, but no significant difference com pared to baseline was found (P > 0.05).      At the stage of decompensation after treatment in patients of group II, the total protein, albumin, fibrinogen, platelet count, Von Willebrand factor, PT, APTT, TT, INR, PI, D-dimer, tPA, PAI-1, tPA/PAI-1, ADMA, Child-Pugh and MELD scores significantly improved and differed from those in the control group (P < 0.05). At the stage of decompensation after treatment, in patients of group I indicators of total protein, albumin, fibrinogen, platelet count, Von Willebrand factor, PT, APTT, TT, INR, PI, D-dimer, tPA, PAI-1, tPA/PAI-1, ADMA, Child-Pugh and MELD scores significantly deteriorated compared to baseline, indicating profound progressive impairment of synthetic liver function and hemostasis system, which can be considered prognostically unfavorable (P < 0.05).
The liver has a number of hemostatic roles including the production of most coagulation factors and inhibitors, as well as fibrinolytic factors.This stability is disrupted by the effects of advanced liver disease in the form of decreased syn thesis of coagulation factors, inhibitors, abnormal clotting factors, abnormalities of fibrinolytic activity, disseminated intravascular coagulation and platelet function defects. In end-stage liver disease several pathophysiological mechanisms, espe cially profound endothelial dysfunction (ED), may be responsible for a hyperco agulable state with the potential for severe thrombotic complications. ED is re sponsible for increased production of liver independent coagulation factors such as von Willebrand factor, factor VIII), and PAI-1. These alterations, in combination with changes in the balance of coagulation/anticoagulation and fibrinolytic/antifibrinolytic factors, affect all levels of the hemostatic system. Standard laboratory coagulation tests are unable to predict bleeding and are inadequate for the assess ment of hemostatic status in these patients.
The assessment of the effectiveness of a three-month treatment regimen with ademethionine, arginine glutamate and rosuvastatin in obese patients with ALC included indicators of synthetic liver function and hemostasis (total protein, albumin, fibrinogen, platelet count, Von Willebrand factor, APTT, TT, INR, PI, D-dimer, tPA, PAI-1, tPA/PAI-1, ADMA), as well as LC severity (Child-Pugh score) and 3-month MELD mortality score.
Decreased levels of total protein, albumin, fibrinogen, PI, platelet count and in creased levels of Von Willebrand factor, PT, APTT, TT, INR, D-dimer, tPA and PAI-1, ADMA were revealed. Such changes worsened with increasing LC decompensation and were accompanied by an increase in the Child-Pugh and MELD scores (P < 0.05).
There was a more pronounced increase in levels of PAI-1 than tPA, that was accompanied by a decrease in tPA/PAI-1 index. A number of researchers indicate that an increase in IAP-1 levels can cause a hypercoagulable state, so its increase with a decrease in tPA/PAI-1 index in patients with ALC in combination with obe sity indicates a risk of thrombogenic conditions. This is also evidenced by the in creasing number of D-dimers. Therefore, the fibrinolytic/antifibrinolytic factors should be considered in the treatment of such patients to prevent LC complications.
The inclusion of ademethionine, arginine glutamate and rosuvastatin in the treat ment regimen for 3 months improved the levels of total protein, albumin, fibrinogen, PI, platelet count, Von Willebrand factor, PT, APTT, TT, INR, D-dimer, tPA and PAI-1, ADMA, which was accompanied by a decrease in Child-Pugh severity score and MELD 3-month mortality score.
Conclusions. Patients with ALC compared with obesity showed a decrease in total protein, albumin, fibrinogen, PI, platelet count and increased levels of Von Willebrand factor, PT, APTT, TT, INR, D-dimer, tPA and PAI-1, ADMA, which was accompanied by an increase in the Child-Pugh and MELD scores. A more pronounced increase in levels of PAI-1 and a decrease in tPA/PAI-1 index was noted, which indicates the risk of thrombogenic conditions in obese patients with LC. Inclusion of ademethionine, arginine glutamate and rosuvastatin in the treatment regimen of patients with LC in combination with obesity for 3 months improved total protein, albumin, fibrinogen, PI, platelet count, Von Willebrand factor, PT, APTT, TT, INR, D-dimer, tPA та PAI-1, ADMA, which was accompanied by a decrease in the Child-Pugh severity score and MELD 3-month mortality score.

Conflict of interest.
There is no conflict of interest regarding the research, authorship and/or publication of this article.