Additional file 1 of Comparative proteomics reveals different protein expression in platelets in patients with alcoholic liver cirrhosis

Additional file 1. 2D gel of healthy control. Exemplary two-dimensional gel of a healthy control

Effects of Increased Von Willebrand Factor Levels on Primary Hemostasis in Thrombocytopenic Patients with Liver Cirrhosis

<div><p>In patients with liver cirrhosis procoagulant and anticoagulant changes occur simultaneously. During primary hemostasis, platelets adhere to subendothelial structures, via von Willebrand factor (vWF). We aimed to investigate the influence of vWF on primary hemostasis in patients with liver cirrhosis. Therefore we assessed in-vitro bleeding time as marker of primary hemostasis in cirrhotic patients, measuring the Platelet Function Analyzer (PFA-100) closure times with collagen and epinephrine (Col-Epi, upper limit of normal ≤165 s) or collagen and ADP (Col-ADP, upper limit of normal ≤118 s). If Col-Epi and Col-ADP were prolonged, the PFA-100 was considered to be pathological. Effects of vWF on primary hemostasis in thrombocytopenic patients were analyzed and plasma vWF levels were modified by adding recombinant vWF or anti-vWF antibody. Of the 72 included cirrhotic patients, 32 (44.4%) showed a pathological result for the PFA-100. They had mean closure times (± SD) of 180±62 s with Col-Epi and 160±70 s with Col-ADP. Multivariate analysis revealed that hematocrit (<i>P</i> = 0.027) and vWF-antigen levels (<i>P</i> = 0.010) are the predictors of a pathological PFA-100 test in cirrhotic patients. In 21.4% of cirrhotic patients with platelet count ≥150/nL and hematocrit ≥27.0%, pathological PFA-100 results were found. In thrombocytopenic (<150/nL) patients with cirrhosis, normal PFA-100 results were associated with higher vWF-antigen levels (462.3±235.9% vs. 338.7±151.6%, <i>P</i> = 0.021). These results were confirmed by multivariate analysis in these patients as well as by adding recombinant vWF or polyclonal anti-vWF antibody that significantly shortened or prolonged closure times, respectively. In conclusion, primary hemostasis is impaired in cirrhotic patients. The effect of reduced platelet count in cirrhotic patients can at least be partly compensated by increased vWF levels. Recombinant vWF could be an alternative to platelet transfusions in the future.</p></div

Effect of von Willebrand factor on primary hemostasis in thrombocytopenic patients with liver cirrhosis.

<p>Increased levels of vWF were associated with maintained normal primary hemostasis in patients with liver cirrhosis and reduced platelet count (<150/nL). vWF-antigen and vWF-activity were higher in patients with normal results for PFA-100 after measuring with Col-Epi and Col-ADP (A+B). Substitution of recombinant vWF (r-vWF) resulted in improved primary hemostasis as compared to unmodified samples (C). Addition of a 66-µg or 165-µg polyclonal anti-vWF-antibody levels led to prolonged closure times compared to unmodified control samples (D+E). Horizontal lines represent the upper limit of normal for closure times with Col-Epi or Col-ADP [**: <i>P</i><0.01, *: <i>P</i><0.05].</p

Results for univariate analysis of predictors of a pathological PFA-100 test result.

<p>Univariate analysis of the following variables was performed to identify predictors of a pathological PFA-100 test results in the whole study cohort and in thrombocytopenic cirrhotic patients. Multivariate analysis was then performed with variables showing a <i>P</i>-value ≤0.1 in univariate analysis.</p><p>Results for univariate analysis of predictors of a pathological PFA-100 test result.</p

Results for multivariate analysis of predictors of a pathological PFA-100 test result.

<p>Variables with a P≤0.1 in univariate analysis were included in multivariate analysis, which was separately performed for the whole study cohort and for patients with a platelet count y 150/nL.</p><p>Results for multivariate analysis of predictors of a pathological PFA-100 test result.</p

Selection of the study population.

<p>The enrolment details of the study population are shown, including the number of patients excluded because of the use of antiplatelet drugs or because there was no information on use of these drugs. For all subgroups, the number of available test results for PFA-100 with Col-Epi and Col-ADP is given.</p

Analysis of the effect of vWF on results of PFA-100 in thrombocytopenic patients.

<p>Comparison of vWF-antigen and vWF-activity in thrombocytopenic (<150/nL) patients with liver cirrhosis and the effect of vWF on results of PFA-100 measured with Col-Epi and Col-ADP (norm.  =  normal test result, path.  =  pathological test result).</p><p>Analysis of the effect of vWF on results of PFA-100 in thrombocytopenic patients.</p

Results for PFA-100 in cirrhotic patients.

<p>Overall, mean closure times were above the upper limit of normal for Col-Epi (>165 s) and Col-ADP (>118 s) in cirrhotic patients. There were no significant differences between patients with different Child-Turcotte-Pugh scores.</p

Results for PFA-100, full blood count, and vWF.

<p>Results for PFA-100 and basic laboratory values are grouped by Child-Turcotte-Pugh classification. The ‘<i>P</i><0.05’-column indicates significant differences between groups according to one-way ANOVA or Fisher's exact test. For example, A/C indicates a significant difference between CTP groups A and C.</p><p>Results for PFA-100, full blood count, and vWF.</p

Characteristics of patients with liver cirrhosis.

<p>Description of baseline parameters such as age, sex, and etiology of patients with liver cirrhosis and labMELD-score; grouped by Child-Turcotte-Pugh score.</p>a<p>Including primary sclerosing cholangitis and biliary atresia.</p>b<p>Including Wilson disease and polycystic liver disease.</p><p>Characteristics of patients with liver cirrhosis.</p