Significant Association between Serum Levels of Von Willebrand Factor ( vWF ) Antigen with Stages of Cirrhosis

Objective: Von Willebrand factor (vWF) is a mediator that increases endotoxemic medium like in cirrhosis. In this study we evaluated the association of serum VWF antigen (Ag) level with the stage of cirrhosis (according to Child-Pugh classification). Materials and Methods: We included 82 cirrhotic patients (Female/ Male (F/M): 26/56) and 86 healthy subjects (F/M: 44/42) in the study. Ages of the both groups of patients were not different (P= 0.095). We excluded possible other reasons that may cause VWF level increase. Diagnosis of cirrhosis was made on the basis of biopsy in 7 patients and with clinical and laboratory parameters in 75 patients. VWF Ag level was determined by immunoturbidimetric test. The stage of cirrhosis was defined with Child-Pugh classification. Data were analysed by using Statistical Package for the Social Sciences (SPSS) 10.0 software program. Results: VWF Ag level was significantly higher in cirrhotic patients compared to control group (220±90 and 87±38, P<0.001, respectively). We observed significant increase of VWF Ag level with the increasing stages of cirrhosis according to Child-Pugh score (VWF Ag level for Child A-B-C 156.4±54/215±45/284.8±93, respectively; P values for Child A-B/A-C/B-C; <0.001/<0.001/0.006, respectively). Conclusion: Serum VWF Ag level increases in cirrhotic patients and this is more pronounced with higher stages of cirrhosis.


Introduction
Patients with cirrhosis have an endotoxemic medium due to intestinal flora changes, decreased hepatic clearance of endotoxins and transition of bacteria to mesenteric lymph nodes from intestinal barrier.This fact was shown in animal experiments with spontaneous bacterial peritonitis.It was also demonstrated that this process can be suppressed by lactulose and antibiotics [1][2][3].Serum Von Willebrand factor (vWF) increases in a linear ratio with the intensity of endotoxemia.So we hypothesized a significant correlation between serum VWF antigen and Child-Pugh score used in staging and prognosis of cirrhosis.
The aim of our study was to compare the VWF antigen level with the stage of cirrhosis according to Child-Pugh score and each parameter used in this scoring system and try to determine whether vWF level can be an additional alternative parameter to Child-Pugh score.

Materials and Methods
82 patients in different stages of cirrhosis were included into the study.56 patients were male and 26 women.Their ages were between 17-74 years.They had different signs of cirrhosis but their blood pressure, pulse rate and body temperature measurements were in normal ranges.Other possible factors (diabetes, hypertension, cardiac failure, renal dysfunction, gestation, hyperlipidaemia etc.) associated with high VWF Ag levels were excluded.With regards to the knowledge of increasing VWF Ag levels as an acute phase reactant in sepsis and infections (including spontaneous bacterial peritonitis), patients with infectious symptoms at the time of sampling were not included into the study.
Diagnosis of 75 patients was made with clinical, laboratory and radiological (ultrasonography, hepatic Doppler ultrasonography) means, rest of the patients had a liver biopsy.In screening for hepatitis B, HBsAg and anti-Hbc IgM; for hepatit-C, HCV Ab; and for delta virus HDV Ab was used.Tests for viruses and autoimmune parameters were negative in patients accepted as cryptogenic cirrhosis.Patients classified as alcoholic cirrhosis had negative viral serologic tests and had a history of alcohol consumption at least 160 g/day for 8-10 years [4].For autoimmune hepatitis AMA, ASMA and anti-LKM tests were used.In addition, increased transaminase levels, hyperbilirubinemia, hypoalbuminemia, anaemia, leukopenia thrombocytopenia and hypergammaglobulinemia were used as supportive laboratory tests for cirrhosis diagnosis.In order to define Wilson cirrhosis, copper level of 24 hour urine and serum ceruloplasmin levels were measured.Hepatocellular carcinoma was excluded by normal serum alpha feto protein level and absence of any mass in ultrasonography.D-dimer levels were normal when measured in order to exclude systemic hyperfibrinolysis.Child-Pugh classification was used for staging [5,6].There were 30 patients with child-A cirrhosis, 21 child-B and 31 child-C.Number of patients with different stages and aetiologies were shown in Table 1.
From 86 patients included in the control group, 42 were male, 44 were women and their ages ranged between 24-66 years.Control group consisted of healthy patients who do not have any disease and abnormal laboratory tests including liver function tests.Their physical examination was normal and they were not using any medicine.We excluded patients with hepatocellular carcinoma, spontaneous bacterial peritonitis and other infectious diseases, thrombotic event history, diabetes mellitus, systemic hyperfibrinolysis (D-dimer >216) and hypertension.

Methods
Peripheral blood samples were obtained without using a tourniquet to a citrated tube and were transferred to the labora-tory with an ice-bag.The tubes were centrifuged for 4 minutes at 4000 cycle.Plasma was collected and was stored at -20ºC until the test time.VWF antigen level was measured using a Behring BSC device by immunoturbidimetric method [7][8][9].

Statistical Analysis
The study data were analysed in SPSS 10.0 for Windows.The parametric comparison of two groups was performed by Student-t test and non-parametric comparison by Mann Whitney U test.The parametric comparison of three or more groups was performed by ANOVA and non-parametric comparison by Kruskal Wallis test.The post-hoc analysis was performed by Bonferroni correction.All the hypotheses were constructed as two tailed and an alpha critical value of 0.05 was accepted as significant.

Results
There was not any significant difference between demographic features and ages of the patients and controls (p=0.095).Cirrhotic patients had higher serum VWF Ag levels (82 patients; 220±89.8)compared to the control group (86 individuals; 87±38) (p<0.001).Serum VWF Ag levels of cirrhotic patients in different stages according to Child Pugh classification were shown in Table 2.Even patients in Child-A group had statistically higher serum vWF Ag levels compared to control group.
When the data were analysed in terms of parameters included in Child-Pugh classification the results were as follows: VWF Ag level was 177±61.2 in patients with an albumin level of >3.5g/dL; 236.7±108 for albumin levels between 2.8-3.5g/dL and 266.3±83.4 for an albumin level under 2.8g/dL.All of the three groups had higher vWF levels in comparison to control group (p<0.001).vWF level was negatively corre- Prothrombin time (PT) longer than 14 seconds (in 65 patients) was associated with a higher vWF Ag level (234.8±81.1)compared to PT≤14 sn (17 patients; 163.4±57.7) in cirrhotic patients.When these two goups were compared with the control goup there was a significant difference (P <0.001).
Cirrhotic patients with ascites (31 patients; 237±94.2) had significantly higher vWF Ag levels than patients without ascites (51 patients; 191±74.7)(p=0.032).There was also a significant difference between the control group and the mean vWF Ag level of these groups.
The ages of patients were not statistically different in the formed subgroups above according to Child Pugh classification.

Discussion
Our study shows the significant increase of vWF Ag level in cirrhotic patients and that this rise is correlated to the stage of cirrhosis.Data and studies about endotoxemia and higher levels of vWF Ag relevant to this state are limited.In those studies data supporting the endotoxemic medium and its association with the stage of cirrhosis was obtained [10][11][12][13][14][15].However the limited number of patients in those studied necessitated larger studies in order to confirm the results.
In our study we observed significant increase of vWF Ag level in every stages of cirrhosis compared to control group.This finding supports the existence of endotoxemia even in early stages of cirrhosis.Moreover we found that vWF increases as a marker of endotoxemia with higher stages of cirrhosis according to Child Pugh classification and child-C cirrhosis patients had the highest levels.This data shows the increase of endotoxemia in correlation with hepatocellular failure in cirrhosis.
In this study we also tried to determine the relation of vWF Ag increase with the synthesis functions of the liver.Albumin is a protein synthesized in liver and decreases with hepatic failure.Therefore it is in the lowest levels in Child-C patients according to Child Pugh classification.In our study vWF Ag increased significantly when the hypoalbuminemia was more pronounced (p<0.001).This shows a negative correlation between endotoxemia and albumin level thus synthesis functions of liver.
As hepatocellular failure develops and progresses, synthesis of clotting factors decrease and eventually prothrombin time increases.In our study we found that cirrhotic patients with prolonged prothrombin time have significantly higher plasma vWF Ag in comparison to cirrhotic patients with normal prothrombin time (p<0.001).This result is also an evidence of negative correlation between synthesis functions of liver and vWF Ag level.
With higher stages of cirrhosis (except cholestatic liver diseases) bilirubin levels increase.High bilirubin levels accompany higher damage in liver and therefore this causes an increased endothelial dysfunction.vWF Ag level is a reliable marker of endothelial dysfunction and it was significantly high in cirrhotic patients with increased bilirubin levels in comparison to patients with normal bilirubin levels (p< 0.001).This finding shows a positive correlation between endothelial cell dysfunction and bilurubin levels in cirrhotic patients.
As mentioned above, VWF is in a strong correlation with each of the liver synthesis functions (albumin, bilirubin, PT) which are at the same time part of the Child-Pugh classification.These findings support the significant association of vWF AG level with hepatocellular insufficiency.In addition, cirrhotic patients with ascites had significantly higher values of vWF Ag than other cirrhotic patients (p=0.032).Ascites is a part of portal hypertension however hepatocellular failure contributes to its development.One of the causes of ascites formation is decreased oncotic pressure in the case of hypoalbuminemia.Nevertheless we do not know the association between vWF Ag level and the existence of ascites is due to portal hypertension, hepatocellular failure or both.
Ferro et al. [13] investigated the existence and mechanism of endothelial dysfunction in cirrhosis in a study with 32 patients.In the analysis, vWF level showed a strong association with endotoxemia.In that study patients had treatment with non-absorbable antibiotics or non-absorbable antibiotics plus steroids for seven days and subsequently a significant decrease of both endotoxin levels and vWF antigen was observed.These results show the presence of endothelial dysfunction in cirrhosis and that endotoxemia plays a major role in that situation, both supporting the data of our study.
Albornoz et al. [16] investigated whether vWF Ag level can be a marker of endothelial dysfunction in another study with 27 cirrhotic patients.Results of the study showed the positive correlation of vWF Ag level and NO degradation enzymes (nitrite-nitrate) which are known to be markers of endothelial dysfunction.They reported more prominent increase of these parameters with higher stages of cirrhosis.These results support the existence of endotoxemia in cirrhosis.Findings of our study are consistent with that study.Our study demonstrated these findings in higher number of patients and confirmed the results of that study.In that study, increase in the levels of vWF Ag and NO degradation enzymes were analysed in different stages of cirrhosis, however in our study we also analysed the data according to the each parameter used in Child-Pugh classification and showed the significant correlation.These results strengthen the evidence of relation between vWF Ag level and endotoxemia in cirrhosis.
It is obvious that there is a strong correlation between hepatocellular failure and vWF Ag level in cirrhosis and synthesis function of liver is negatively correlated with vWF [17,18].Strength of the statistical significance in our study supports these findings.Both Child-Pugh score and each of the parameters of this scoring system are significantly associated with vWF.The fact that underlying mechanism of haemodynamic and haemostatic changes in cirrhosis is endothelial dysfunction makes vWF more important.While parameters used in staging show one side of deterioration in cirrhosis, changes in levels of vWF demonstrate a more complex injury of the liver.
In conclusion, serum VWF Ag level increases in correlation with the stage of cirrhosis and indicates the existence of endotoxemic medium in cirrhosis.

Table 1 . Number of patients with different stages and aetiologies
HBV: Hepatitis B virus; HCV: Hepatitis C virus; HDV: Hepatitis D virus Eurasian J Med 2015; 47: 21-5 lated with albumin level.The difference was not significant only between cirrhotic patients with an albumin level of 2.8g/ dL and 2.8-3.5g/dL(P=0.236).