key: cord-1037793-q3tud33g authors: Xing, Yueyi; Jiang, Yueping; Xing, Shichao; Mao, Tao; Guan, Ge; Niu, Qinghui; Zhao, Xianzhi; Zhou, Jianrui; Jing, Xue title: Neutrophil extracellular traps are associated with enhanced procoagulant activity in liver cirrhosis patients with portal vein thrombosis date: 2022-04-18 journal: J Clin Lab Anal DOI: 10.1002/jcla.24433 sha: 1ac5edc77b61370e2bf9cc62f3bd9a45a539eb4a doc_id: 1037793 cord_uid: q3tud33g OBJECTIVE: Patients with liver cirrhosis (LC) commonly exhibit hypercoagulability and tend to develop thrombosis. Neutrophil extracellular traps (NETs) are associated with a variety of thrombotic conditions, but their possible value in portal vein thrombosis (PVT) is not known. We assessed whether NETs promote thrombosis and contribute to the procoagulant state in patients with LC. METHODS: The circulating levels of NETs markers (myeloperoxidase, neutrophil elastase, citrullinated histone H3) were measured in 72 patients (median age, 55 years; 48 [66.7%] men) with LC from September 2020 to February 2021. Then they were divided into two groups: patients with or without PVT. NETs procoagulant activity was assessed based on thrombin–antithrombin complex (TAT complex) and Factor X. The levels of plasma markers were determined by ELISA. RESULTS: There were 28 patients with PVT and 44 patients without PVT. The levels of NETs markers and hypercoagulability markers in the plasma of cirrhosis patients with PVT were significantly higher than those of cirrhosis patients without PVT (p < 0.05). Additionally, the levels of the NETs markers correlated with TAT complex and Factor X (Spearman correlation rho >0.73, p < 0.0001). CONCLUSIONS: Neutrophil extracellular traps seem to enhance procoagulant activity in LC patients with PVT; thus, they may be a practical predictor of PVT as well as a rapid and easy‐to‐use diagnostic and treatment guide for PVT in patients with cirrhosis. compared with patients without PVT due to hepatic dysfunction. 4 Therefore, there is an urgent requirement for validated biomarkers that might predict the development of PVT. For the past few years, terms such as "thromboinflammation," "immunothrombosis," and "immunohemostasis" have been used to describe responses/mechanisms associated with both thrombosis and inflammation. 5 Neutrophil extracellular traps (NETs) are extracellular DNA fibers comprising histones and neutrophil antimicrobial proteins released from activated neutrophils. 6 NETs stimulate thrombosis and coagulation, which are abundant in venous, 7, 8 arterial, and cancer-associated thrombosis. 9,10 Therefore, NETs have been identified to play a major role in immune thrombosis. 11, 12 Recently, several studies have suggested that NETs are potential contributors to hypercoagulability in cancers. 13, 14 However, there have been no reports as to whether there is a correlation between NETs and a hypercoagulable state in LC patients with PVT. The aim of this study was to assess whether NETs are related to hypercoagulability and whether they can predict PVT formation in patients with LC. Demographic status, etiology, and clinical laboratory test data were available through the electronic medical records at the Affiliated Hospital of Qingdao University from September 2020 to February 2021. The Model of End-Stage Liver Disease (MELD) and Child-Pugh scores were used to evaluate the severity of LC. Patients with primary or secondary hepatic malignancy, hematologic diseases, Budd-Chiari syndrome, or inflammatory diseases were excluded. Thrombosis in Liver Cirrhosis (2020, Shanghai), 1 the patients were F I G U R E 1 Flowchart of this study divided into two groups, PVT group (n = 28) and non-PVT group (n = 44). The flowchart of this study is shown in Figure 1 . After obtaining informed consent from all the patients, blood samples were taken from the antebrachial vein and collected into vacuum tubes containing EDTAK2 on the day of admission to the hospital and were then centrifuged for 15 min at 1000 g. The plasma was frozen and stored at a temperature of −80°C for further analysis. Enzymelinked immunosorbent assays (ELISA) were used for measurement of the plasma levels of TAT complex, Factor X, MPO, NE, CitH3, endotoxin, and tissue factor (Enzyme-linked Biotechnology Co.). The current study was approved by the Clinical Trials (NCT05012501) and Ethics Committee of the Affiliated Hospital of Qingdao University, China (QYFYWZLL26363). Quantitative variables are expressed as mean ± standard deviation, and significance of difference was determined using Student's t test. Categorical variables are expressed as frequencies and percentages, and significance of difference was determined using the χ² or Fisher's exact test. Non-normally distributed variables are expressed as median and interquartile range, and significance of difference was de- After screening by inclusion and exclusion criteria, 28 LC patients with PVT and 44 cirrhosis patients without PVT were chosen. The basic characteristics of the patients with PVT (n = 28) and those without PVT (n = 44) are presented in Table 1 . The study participants in the PVT group had a mean age of 55.61 ± 7.01 years and 60.7% were men; in the non-PVT cohort, the mean age was 54.43 ± 11.19 years and 70.5% were men. There were no significant differences in age, gender, or history of smoking and alcohol consumption between the two groups. According to MELD score and Child-Pugh level, liver function was worse in the non-PVT group as shown in Table 1 . We analyzed the laboratory data of the PVT group and non-PVT group ( Table 2) Plasma levels of MPO, NE, and CitH3 in LC patients with PVT were all significantly higher than those in non-PVT patients ( Figure 2 ). We used ROC curves to obtain the cutoff points and found that patients with PVT had significantly higher levels of NETs markers compared with those without PVT ( Table 4 ). The levels of PT, APTT, fibrinogen, TT, and antithrombin III activity were not different between patients with or without PVT (Table 2) . However, D-dimer level was significantly higher in PVT patients than in non-PVT patients (p < 0.0001). Moreover, the plasma levels of Factor X and TAT complex were significantly higher in patients with PVT ( Figure 4 ). The results of Spearman's correlation analyses indicated significant correlations between the levels of circulating NETs markers and TAT complex or Factor X ( Figure 5 and Table 4 ). Plasma levels of TF and endotoxin were significantly higher in PVT patients than in non-PVT patients (p = 0.046, p = 0.045, respectively; Figure 6 ). Moreover, except for D-dimer, the results showed strong correlations between the levels of circulating NETs markers, hypercoagulability markers, and TF/endotoxin ( Table 5 ). the PVT group (n = 28) were significantly higher than those in the non-PVT group (n = 44). *p < 0.05 "thromboinflammation," "immunothrombosis," and "immunohemostasis" have been shown to be associated with both thrombosis and inflammation. 5 It has been proposed that the systemic activation of the coagulation system is a response to the dysregulation of inflammatory markers. 15 The current study showed that the systemic inflammatory marker, NLR, was an independent risk factor for PVT. Additionally, NETs have been identified as an essential mediator of thrombosis. 16, 17 Activated neutrophils activate NE, MPO, and protein-arginine deiminase type 4 (PAD4) to catalyze citrullination of histones. Then, reactive oxygen species (ROS) promote NETosis through the release of chromatin outside the cell; a final release of DNA, histones, and other intracellular granules forms the extracellular traps. 18 NETs adhere to red blood cells, platelets, and platelet adhesion molecules, such as fibrinogen, von Willebrand factor (VWF), and fibronectin, to form a scaffold that can trigger platelet activation and blood coagulation. 19 Moreover, mainly via the activity of neutrophil serine proteases, NETs can promote both the intrinsic 20 and the extrinsic coagulation pathway. 19 In this study, we found that the levels of plasma NETs markers were significantly higher in This study also revealed that NETs were more abundant in patients with PVT than in those non-PVT. In addition, NET formation promoted the hypercoagulable state in patients with PVT, suggesting that NETs may be a predictor and a potential new target for the treatment of PVT. Patients with enhanced TF and endotoxin are more likely to suffer from PVT. Our study has some strengths and limitations should be acknowledged. As far as we know, the present study is the first report on circulating NETs markers in patients with PVT. However, as a single-center study, it lacks representativeness and a sufficient number of participants. Additionally, this is a cross-sectional study, which cannot claim causality between NETs markers and PVT formation. Causality can be confirmed in longitudinal studies or in animal models in the future. Moreover, prospectively designed studies are necessary to confirm these results. Further research is needed to determine whether NETs can be used as PVT-targeted therapy. In our cohort, we observed a strong correlation between PVT and increased plasma levels of NETs markers. NETs may be a practical predictor of PVT and may be helpful to clinicians as a rapid and easyto-use diagnostic indicator, possibly guiding the treatment of PVT in patients with cirrhosis. No authors had conflict of interest. Y-yX made the major contribution to the conception and design of the study, performed the experiments, and drafted the manuscript. Y-pJ obtained institutional review board approval. S-cX directed the completion of experiments. TM contributed to the data acquisition. GG, Q-hN, X-zZ, and J-rZ assisted in blood specimen collection. XJ revised the manuscript. XJ is the guarantor of the study. All authors read and approved the final manuscript prior to the submission. Raw data can be obtained by contacting the corresponding author. 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