The role of neutrophil extracellular trap formation in kidney transplantation: Implications from donors to the recipient.

Kidney transplantation remains the gold standard for patients with end-stage renal disease, but severe donor organ shortage has led to long waiting lists. The utilization of expanded criteria donor kidneys within the category of deceased donors has enlarged the pool of available kidneys for transplantation; however, these grafts often have an increased risk for delayed graft function or reduced graft survival following transplantation. During brain or circulatory death, neutrophils are recruited to the vascular beds of kidneys where a proinflammatory microenvironment might prime the formation of neutrophil extracellular traps (NETs), web-like structures, containing proteolytic enzymes, DNA, and histones. NETs are known to cause tissue damage and specifically endothelial damage while activating other systems such as coagulation and complement, contributing to tissue injury and an unfavorable prognosis in various diseases. In lung transplantation and kidney transplantation studies, NETs have also been associated with primary graft dysfunction or rejection. In this review, the role that NETs might play across the different phases of transplantation, already initiated in the donor, during preservation, and in the recipient, will be discussed. Based on current knowledge, NETs might be a promising therapeutic target to improve graft outcomes.

Acquired bleeding disorders.

Acquired bleeding disorders can develop in previously healthy people irrespective of age or gender but are particularly common in patients with certain underlying conditions. Here, we review recent advances in the management of acquired haemophilia A (AHA), acquired von Willebrand syndrome (AVWS), and patients with hemostatic abnormalities due to chronic liver disease (CLD). Patients with AHA can now benefit from prophylaxis with emicizumab, a therapeutic antibody that mimics the function of activated coagulation factor VIII. The treatment of AVWS remains challenging in many situations and requires careful consideration of the underlying condition. Haemostatic abnormalities in CLD are often compensated by proportional reduction in pro and anti-haemostatic factors resulting in sustained or even increased thrombin generation. Consequently, bleeding in CLD is rarely caused by haemostatic failure and infusion of plasma or coagulation factor concentrates may not be effective.

Recommendations for the safe use of direct oral anticoagulants in patients with cirrhosis based on a systematic review of pharmacokinetic, pharmacodynamic and safety data.

PURPOSE: The popularity of direct oral anticoagulants (DOACs) is increasing among patients with cirrhosis. Cirrhosis has a major impact on the pharmacokinetics of drugs, potentially increasing adverse events. Safe use of drugs in cirrhosis requires a diligent risk-benefit analysis. The aim of this study is to develop practice recommendations for safe use of DOACs in cirrhosis based on a systematic review of pharmacokinetic, pharmacodynamic and safety data. METHODS: We conducted a systematic literature search to identify studies on pharmacokinetics, pharmacodynamics and safety of DOACs in cirrhosis. Data were collected and presented in summary tables by severity of cirrhosis using the Child-Turcotte-Pugh (CTP) classification. A multidisciplinary expert panel evaluated the results and classified the DOACs according to safety. RESULTS: Fifty four studies were included. All DOACs were classified as 'no additional risks known' for CTP A. For CTP B, apixaban, dabigatran and edoxaban were classified as 'no additional risks known'. Apixaban and edoxaban showed fewer adverse events in patients with cirrhosis, while dabigatran may be less impacted by severity of cirrhosis based on its pharmacokinetic profile. Rivaroxaban was classified as 'unsafe' in CTP B and C based on significant pharmacokinetic alterations. Due to lack of data, apixaban, dabigatran and edoxaban were classified as 'unknown' for CTP C. CONCLUSION: DOACs can be used in patients with CTP A cirrhosis, and apixaban, dabigatran and edoxaban can also be used in CTP B. It is recommended to avoid rivaroxaban in CTP B and C. There is insufficient evidence to support safe use of other DOACs in CTP C cirrhosis.

Generation of neutrophil extracellular traps in patients with acute liver failure is associated with poor outcome.

BACKGROUND AND AIMS: Acute liver failure (ALF) is characterized by significant changes in the hemostatic system and by systemic inflammation. The formation of neutrophil extracellular traps (NETs), in which an activated neutrophil expels its DNA, histones, and granular enzymes, such as myeloperoxidase (MPO), has been associated with immune-mediated and thrombotic diseases. We hypothesized that formation of NETs in patients with ALF contributes to progression of disease. APPROACH AND RESULTS: A total of 676 patients with ALF (international normalized ratio [INR], ≥1.5) or severe acute liver injury (ALI; INR, ≥2.0) were recruited from the U.S. ALF Study Group Registry between 2011 and 2018, of whom 308 patients (45.6%) had acetaminophen-induced ALF. Up to 21 days after admission, 483 patients (71.5%) survived without liver transplantation (LT). Levels of cell-free DNA (cfDNA) and the specific NET marker MPO-DNA complexes were measured in plasma samples obtained on admission and compared to levels in healthy controls. In addition, liver tissue obtained at transplantation of 20 ALF patients was stained for NETs. Levels of cfDNA were 7.1-fold, and MPO-DNA complexes 2.5-fold, higher in patients with ALF compared to healthy controls. cfDNA levels were not associated with 21-day transplant-free survival, but were higher in those patients with more-severe disease on admission, as reflected by various laboratory and clinical parameters. MPO-DNA levels were 30% higher in patients with ALF who died or required urgent LT. Liver tissue of ALF patients stained positive for NETs in 12 of 18 evaluable patients. CONCLUSIONS: Here, we provide evidence for NET formation in patients with ALF. Elevated plasma levels of MPO-DNA complexes in patients with ALF were associated with poor outcome, which suggests that NET formation contributes to disease progression.

Nonmalignant portal vein thrombi in patients with cirrhosis consist of intimal fibrosis with or without a fibrin-rich thrombus.

BACKGROUND AND AIM: Portal vein thrombosis (PVT) is a common complication of cirrhosis. The exact pathophysiology remains largely unknown, and treatment with anticoagulants does not lead to recanalization of the portal vein in all patients. A better insight into the structure and composition of portal vein thrombi may assist in developing strategies for the prevention and treatment of PVT. APPROACH AND RESULTS: Sixteen prospectively and 63 retrospectively collected nonmalignant portal vein thrombi from patients with cirrhosis who underwent liver transplantation were included. Histology, immunohistochemistry, and scanning electron microscopy were used to assess structure and composition of the thrombi. Most recent CT scans were reanalyzed for thrombus characteristics. Clinical characteristics were related to histological and radiological findings. All samples showed a thickened, fibrotic tunica intima. Fibrin-rich thrombi were present on top of the fibrotic intima in 9/16 prospective cases and in 21/63 retrospective cases. A minority of the fibrotic areas stained focally positive for fibrin/fibrinogen (16% of cases), von Willebrand factor (VWF; 10%), and CD61 (platelets, 21%), while most of the fibrin-rich areas stained positive for those markers (fibrin/fibrinogen, 100%; VWF, 77%; CD61, 100%). No associations were found between clinical characteristics including estimated thrombus age and use of anticoagulants and presence of fibrin-rich thrombi. CONCLUSION: We demonstrate that PVT in patients with cirrhosis consists of intimal fibrosis with an additional fibrin-rich thrombus in only one-third of cases. We hypothesize that our observations may explain why not all portal vein thrombi in patients with cirrhosis recanalize by anticoagulant therapy.

Von Willebrand factor is an independent predictor of short-term mortality in acutely ill patients with cirrhosis.

BACKGROUND AND AIMS: Levels of von Willebrand factor (VWF) are elevated in patients with cirrhosis, and correlate well with disease severity. In patients with decompensated cirrhosis (DC), plasma VWF is associated with mortality. The value of VWF in predicting short-term mortality risk in patients with acute-on-chronic liver failure (ACLF) is, however, unclear. METHODS: We included patients with DC (n = 111) and ACLF (n = 105). We measured VWF levels and correlated these with other laboratory parameters and prediction models for mortality. Also, we assessed the predictive value of VWF in the prediction of 90- and 30-day mortality in patients with DC and ACLF, respectively, and compared this to the predictive value of clinically used prediction models. Finally, we determined the optimal cut-off value for VWF in patients with ACLF. RESULTS: Sixteen of 111 (14%) patients with DC and 35 of 105 (33%) with ACLF died within 90 and 30 days, respectively. VWF was associated with mortality and correlated closely with other prediction models. In patients with ACLF, VWF levels had a discrimination for 30-day mortality comparable with these models and accurately identified ACLF patients with high 30-day mortality risk. CONCLUSIONS: Levels of VWF associate closely with risk of mortality in patients with DC and ACLF, and may have predictive utility as a laboratory marker of prognosis. Further research is warranted to assess the additional value of VWF in the prediction of mortality and associated complications in chronic liver failure syndromes.

Enhanced in vivo and ex vivo thrombin generation after lower-leg trauma, but not after knee arthroscopy.

BACKGROUND: There is room for improvement of prevention of venous thromboembolism (VTE) after lower-leg cast application or knee arthroscopy. Information about the mechanism of clot formation in these patients may be useful to identify new prophylaxis targets. We aimed to study the effect of 1) lower-leg injury and 2) knee arthroscopy on thrombin generation. METHODS: A cross-sectional study was conducted using plasma samples of POT-(K)CAST trials to measure ex vivo thrombin generation (Calibrated Automated Thrombography [CAT]) and plasma levels of prothrombin fragment 1 + 2 (F1 + 2), thrombin-antithrombin (TAT), fibrinopeptide A (FPA). Plasma was obtained shortly after lower-leg trauma or before and after (< 4 h) knee arthroscopy. Participants were randomly selected from those who did not develop VTE. For aim 1, samples of 88 patients with lower-leg injury were compared with 89 control samples (i.e., preoperative samples of arthroscopy patients). Linear regression was used to obtain mean differences (or ratios if ln-retransformed because of skewedness) adjusted for age, sex, body mass index, comorbidities. For aim 2, pre- and postoperative samples of 85 arthroscopy patients were compared, for which mean changes were obtained. RESULTS: In patients with lower-leg injury (aim 1), endogenous thrombin potential, thrombin peak, velocity index, FPA and TAT were increased as compared with controls. In arthroscopy patients (aim 2), pre- and postoperative levels were similar for all parameters. CONCLUSION: Lower-leg trauma increases thrombin generation both ex vivo and in vivo, in contrast to knee arthroscopy. This may imply that the pathogenesis of VTE is different in both situations.

The use of prothrombin complex concentrate in chronic liver disease: A review of the literature.

Patients with chronic liver disease (CLD) and cirrhosis present a rebalanced hemostatic system in the three phases of haemostasis. This balance is however unstable and can easily tip towards bleeding or thrombosis. Management of both spontaneous bleeding and bleeding during invasive procedures remains a challenge in this patient population. Transfusion of blood products can result in circulatory overload and thereby worsen portal hypertension. As an alternative to fresh frozen plasma (FFP), prothrombin complex concentrates (PCC) may have merit in patients with liver disease because of their low volume. The impact of PCC in in-vitro spiking experiments of cirrhotic plasma is promising, but also warrants cautious use in light of thromboembolic risk. The majority of existing studies carried-out in CLD patients are retrospective or do not have an adequate control arm. A prospective study (the PROTON trial) was set up in 2013 to investigate the utility of PCC in patients undergoing liver transplantation. However, the study has never recruited the planned number of patients. Robust data on PCC safety in CLD is also required. The limited existing evidence does not seem to indicate an excessive thromboembolic risk. Currently, the utilisation of PCC in CLD cannot be routinely recommended but can provide an option for carefully selected cases in which other measures were not sufficient to control bleeding and after delicately weighing risks and benefits.

Antithrombotic Management in Adult Kidney Transplantation: A European Survey Study.

In kidney transplantation (KTx), renal graft thrombosis (RGT) is one of the main reasons for early graft loss. Although evidence-based guidance on prevention of RGT is lacking, thromboprophylaxis is widely used. The aim of this survey was to obtain a European view of the different thromboprophylactic strategies applied in KTx. An online 22-question survey, addressed to KTx professionals, was distributed by email and via platforms of the European Society for Organ Transplantation. Seventy-five responses (21 countries, 51 centers) were received: 75% had over 10 years' clinical experience, 64% were surgeons, 29% nephrologists, and 4% urologists. A written antithrombotic management protocol was available in 75% of centers. In 8 (16%) centers, respondents contradicted each other regarding the availability of a written protocol. Thromboprophylaxis is preferred by 78% of respondents, independent of existing antithrombotic management protocols. Ninety-two percent of respondents indicated that an anticipated bleeding risk is the main reason to discontinue chronic antithrombotic therapy preoperatively. Intraoperatively, 32% of respondents administer unfractionated heparin (400-10,000 international units with a median of 5,000) in selected cases. Despite an overall preference for perioperative thromboprophylaxis in KTx, there is a high variation within Europe regarding type, timing, and dosage, most likely due to the paucity of high-quality studies. Further research is warranted in order to develop better guidelines.

Haemostatic alterations and management of haemostasis in patients with cirrhosis.

Patients with cirrhosis frequently acquire complex changes in their haemostatic system including a decreased platelet count and decreased levels of various haemostatic proteins. Although historically patients with cirrhosis were thought to have a haemostasis-related bleeding tendency, it is now widely accepted that the haemostatic system of patients with cirrhosis remains in balance as a result of simultaneous changes in pro- and anti-haemostatic systems. The concept of rebalanced haemostasis has led to changes in clinical management, although firm evidence from well-designed clinical studies is largely lacking. For example, many invasive procedures in patients with cirrhosis and a prolonged prothrombin time are now performed without prophylaxis with fresh frozen plasma. Conversely, clinicians have become more aware of the need for anti-thrombotic therapy, even in those patients with abnormal routine coagulation tests. This paper will outline recent advances in pathogenesis, prevention and treatment of both bleeding and thrombotic complications in patients with cirrhosis. Among other topics, we will discuss the haemostatic status of acutely ill patients with cirrhosis, the various causes of bleeding in patients with cirrhosis, and how best to prevent or treat bleeding. In addition, we will discuss the hypercoagulable features of patients with cirrhosis, new insights into the pathogenesis of portal vein thrombosis, and how best to prevent or treat thromboses.

Factor VIII/protein C ratio independently predicts liver-related events but does not indicate a hypercoagulable state in ACLD.

BACKGROUND & AIMS: It has been suggested that the ratio of procoagulant factor VIII to anticoagulant protein C (FVIII/PC) reflects the hemostatic equilibrium. Moreover, FVIII/PC predicted decompensation/death in a small study not accounting for portal hypertension severity. We investigated (i) the prognostic value of FVIII/PC (outcome-cohort) and (ii) whether FVIII/PC reflects the hypercoagulable state (assessed by thrombomodulin-modified thrombin generation assay [TM-TGA]) or the risk of bleeding/thrombotic events in patients undergoing hepatic venous pressure gradient (HVPG) measurement during follow-up. METHODS: (i) The outcome-cohort comprised 576 patients with evidence of advanced chronic liver disease (liver stiffness measurement ≥10 kPa and/or HVPG ≥6 mmHg). (ii) TM-TGA-cohort patients (n = 142) were recruited from the prospective VIenna CIrrhosis Study (VICIS: NCT03267615). RESULTS: (i) FVIII/PC significantly increased across clinical stages (p <0.001) as well as HVPG (p <0.001) and MELD score (p <0.001) strata and remained independently associated with decompensation/liver-related death (adjusted hazard ratio 1.06; 95% CI 1.01-1.11; p = 0.013), even after multivariable adjustment. It was also associated with acute-on-chronic liver failure (ACLF) development (adjusted hazard ratio 1.10; 95% CI 1.02-1.19; p = 0.015) in patients with decompensated cirrhosis. (ii) FVIII/PC showed a weak positive correlation with endogenous thrombin potential (Spearman's ρ = 0.255; p = 0.002), but this association disappeared after adjusting for the severity of liver disease. FVIII/PC was not associated with the development of bleeding (p = 0.272) or thrombotic events (p = 0.269). However, FVIII/PC correlated with biomarkers of different pathophysiological mechanisms that promote liver disease progression. CONCLUSION: FVIII/PC provides prognostic information regarding hepatic decompensation/death and ACLF, independently of established prognostic indicators. However, this is not evidence that hypercoagulability drives disease progression, as the correlation between FVIII/PC and thrombin generation is confounded by liver disease severity and FVIII/PC was not associated with thrombosis. Therefore, FVIII/PC does not reflect coagulation and results from previous studies on FVIII/PC require re-interpretation. CLINICAL TRIAL NUMBER: NCT03267615 (in part). LAY SUMMARY: A balanced coagulation system is essential for preventing bleeding episodes and blood clot formation (thrombosis). Blood of patients with advanced liver disease may have increased coagulation potential, possibly promoting the worsening of liver disease via thrombosis in the blood vessels of the liver. The ratio between the results of 2 blood tests (procoagulant factor VIII to anticoagulant protein C) has been suggested to reflect these increases in coagulation potential. Our study demonstrates, on the one hand, that this ratio is a versatile predictor of the development of complications of cirrhosis, yet on the other hand, that it is unrelated to coagulation.

Long-term normothermic machine preservation of human livers: what is needed to succeed?

Although short-term machine perfusion (≤24 h) allows for resuscitation and viability assessment of high-risk donor livers, the donor organ shortage might be further remedied by long-term perfusion machines. Extended preservation of injured donor livers may allow reconditioning, repairing, and regeneration. This review summarizes the necessary requirements and challenges for long-term liver machine preservation, which requires integrating multiple core physiological functions to mimic the physiological environment inside the body. A pump simulates the heart in the perfusion system, including automatically controlled adjustment of flow and pressure settings. Oxygenation and ventilation are required to account for the absence of the lungs combined with continuous blood gas analysis. To avoid pressure necrosis and achieve heterogenic tissue perfusion during preservation, diaphragm movement should be simulated. An artificial kidney is required to remove waste products and control the perfusion solution's composition. The perfusate requires an oxygen carrier, but will also be challenged by coagulation and activation of the immune system. The role of the pancreas can be mimicked through closed-loop control of glucose concentrations by automatic injection of insulin or glucagon. Nutrients and bile salts, generally transported from the intestine to the liver, have to be supplemented when preserving livers long term. Especially for long-term perfusion, the container should allow maintenance of sterility. In summary, the main challenge to develop a long-term perfusion machine is to maintain the liver's homeostasis in a sterile, carefully controlled environment. Long-term machine preservation of human livers may allow organ regeneration and repair, thereby ultimately solving the shortage of donor livers.

Effects of Inflammation on Hemostasis in Acutely Ill Patients with Liver Disease.

Patients with liver diseases are in a rebalanced state of hemostasis, due to simultaneous decline in pro- and anticoagulant factors. This balance seems to remain even in the sickest patients, but is less stable and might destabilize when patients develop disease complications. Patients with acute decompensation of cirrhosis, acute-on-chronic liver failure, or acute liver failure often develop complications associated with changes in the hemostatic system, such as systemic inflammation. Systemic inflammation causes hemostatic alterations by adhesion and aggregation of platelets, release of von Willebrand factor (VWF), enhanced expression of tissue factor, inhibition of natural anticoagulant pathways, and inhibition of fibrinolysis. Laboratory tests of hemostasis in acutely-ill liver patients may indicate a hypocoagulable state (decreased platelet count, prolongations in prothrombin time and activated partial thromboplastin time, decreased fibrinogen levels) due to decreased synthetic liver capacity or consumption, or a hypercoagulable state (increased VWF levels, hypofibrinolysis in global tests). Whether these changes are clinically relevant and should be corrected with antithrombotic drugs or blood products is incompletely understood. Inflammation and activation of coagulation may cause local ischemia, progression of liver disease, and multiorgan failure. Anti-inflammatory treatment in acutely-ill liver patients may be of potential interest to prevent thrombotic or bleeding complications and halt progression of liver disease.

VWF/ADAMTS13 Imbalance, But Not Global Coagulation or Fibrinolysis, Is Associated With Outcome and Bleeding in Acute Liver Failure.

BACKGROUND AND AIMS: Recent studies of acute liver failure (ALF) in man and animals have suggested that rebalanced hemostasis occurs, with distinct hypercoagulable features clinically evidenced by a low risk of bleeding. Rodent models have shown a link between intrahepatic microthrombus formation and progression of ALF. We sought to confirm these earlier findings in a large series of patients with well-characterized ALF from the Acute Liver Failure Study Group. APPROACH AND RESULTS: Citrated plasma samples taken on admission from 676 patients with ALF or acute liver injury (international normalized ratio ≥2.0 without hepatic encephalopathy) were used to determine levels of von Willebrand factor (VWF), a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) activity, thrombomodulin-modified thrombin generation, and clot lysis time (CLT) and compared with the levels in 40 healthy controls. Patients had 3-fold increased VWF levels, 4-fold decreased ADAMTS13 activity, similar thrombin generating capacity, and 2.4-fold increased CLT, compared with controls. Increasing disease severity was associated with progressively more elevated VWF levels as well as hypofibrinolysis. Patients who died or underwent liver transplantation within 21 days of admission had higher VWF levels, lower ADAMTS13 activity, but similar thrombin generation and a similar proportion of patients with severe hypofibrinolysis, when compared with transplant-free survivors. Likewise, patients with bleeding complications had higher VWF levels and lower ADAMTS13 activity compared to those without bleeding. Thrombin generation and CLT did not differ significantly between bleeding and nonbleeding patients. CONCLUSIONS: Rebalanced hemostatic status was confirmed in a large cohort of patients with acute liver injury/ALF, demonstrating that VWF/ADAMTS13 imbalance is associated with poor outcome and bleeding. The association between VWF/ADAMTS13 imbalance and bleeding suggests that bleeding in ALF relates more to systemic inflammation than a primary coagulopathy.

Coagulopathy, Bleeding Events, and Outcome According to Rotational Thromboelastometry in Patients With Acute Liver Injury/Failure.

BACKGROUND AND AIMS: Patients with acute liver injury or failure (ALI/ALF) experience bleeding complications uncommonly despite an abnormal hemostatic profile. Rotational thromboelastometry (ROTEM), which assesses clot formation in whole blood, was used to determine the nature of abnormal hemostasis and whether it contributes to bleeding events, illness severity, or survival. APPROACH AND RESULTS: A total of 200 patients were recruited from sites of the ALF Study Group. Blood collected daily for up to 5 days was analyzed using ROTEM delta devices. Consistent with standard laboratory evidence of hypocoagulability (median international normalized ratio = 2.9 and platelet count = 144 × 109 /L), patients frequently exhibited ROTEM parameters outside the normal range (73% and 62% had abnormalities in clot formation from extrinsic and intrinsic clotting cascades, respectively); however, measures of clot stability were generally normal. Eighteen patients (9%) experienced bleeding events, in whom clot initiation, assembly, and firmness were more severely deranged than patients without bleeding. Abnormal ROTEM parameters were more frequently observed in patients with non-acetaminophen ALI/ALF than those with acetaminophen ALI/ALF (clot initiation [P < 0.001], assembly [P = 0.02], firmness at 10 minutes [P = 0.05], and maximal firmness [P = 0.06]). Patients with more severe systemic complications (high-grade hepatic encephalopathy and need for renal replacement therapy) also had a higher incidence of abnormal ROTEM parameters. Finally, more hypocoagulable ROTEM parameters (clot initiation (P = 0.005), stiffness at 10 minutes (P = 0.05), and maximal stiffness by fibrin assembly (P = 0.004)) were observed in patients who died or underwent liver transplantation than those who survived with their native liver. CONCLUSIONS: In patients with ALI/ALF, abnormal ROTEM parameters are frequent and proportional to disease severity. Whether the increased bleeding risk associated with abnormal ROTEM indicates hemostatic failure or is a proxy for disease severity requires additional study.

Acquired bleeding disorders.

Acquired bleeding disorders can accompany hematological, neoplastic, autoimmune, cardiovascular or liver diseases, but can sometimes also arise spontaneously. They can manifest as single factor deficiencies or as complex hemostatic abnormalities. This review addresses (a) acquired hemophilia A, an autoimmune disorder characterized by inhibitory autoantibodies against coagulation factor VIII; (b) acquired von Willebrand syndrome in patients with cardiovascular disorders, where shear stress abnormalities result in destruction of von Willebrand factor; and (c) liver function disorders that comprise complex changes in pro- and anti-hemostatic factors, whose clinical implications are often difficult to predict. The article provides an overview on the pathophysiology, diagnostic tests and state-of-the-art treatment strategies.

Fibrin clot quality in acutely ill cirrhosis patients: Relation with outcome and improvement with coagulation factor concentrates.

BACKGROUND & AIMS: Patients with liver disease may acquire substantial changes in their hemostatic system, which are most pronounced in patients who are critically ill. Changes in the quality of the fibrin clot in critically ill patients have not been studied in detail. Here we assessed markers of fibrin clot quality and effects of coagulation factor concentrates in patients with acutely decompensated (AD) cirrhosis and acute on chronic liver failure (ACLF). METHODS: We measured plasma levels of fibrinogen, factor XIII, prothrombin and performed thrombin generation assays in 52 AD patients, 58 ACLF patients and 40 controls. In addition, we examined the effects of coagulation factor concentrates on functional assays of fibrin quality. RESULTS: We found increased thrombin generating capacity in both AD and ACLF in comparison with healthy controls. Plasma levels of prothrombin, fibrinogen, and factor XIII were lower in patients compared to controls, appeared lower in ACLF compared to AD patients, and were related to clinical outcomes. Fibrinogen concentrate, but not factor XIII or prothrombin complex concentrate, improved clot quality in vitro. Prothrombin complex concentrate increased the resistance of the clot to break down. CONCLUSIONS: We have demonstrated elevated thrombin generation but decreased plasma levels of prothrombin, fibrinogen and FXIII in acutely ill patients with cirrhosis. In addition, we showed that fibrinogen concentrate and PCCs, but not factor XIII concentrate, improve clot properties in patient plasma. Whether there is true clinical benefit from coagulation factor concentrates in prevention or treatment of bleeding requires further study. LAY SUMMARY: Patients with liver diseases are at risk of bleeding, but mechanisms involved in this bleeding risk are incompletely understood. We studied components that determine the stability of the blood clot and found that concentrations of certain proteins involved in clot stability are present in low levels in acutely ill patients with liver disease. We furthermore demonstrated that some clinically available drugs improve the stability of blood clots from these patients in a test tube.

Patients With COVID-19 Have Elevated Levels of Circulating Extracellular Vesicle Tissue Factor Activity That Is Associated With Severity and Mortality-Brief Report.

OBJECTIVE: Patients with coronavirus disease 2019 (COVID-19) have a high rate of thrombosis. We hypothesized that severe acute respiratory syndrome coronavirus 2 infection leads to induction of TF (tissue factor) expression and increased levels of circulating TF-positive extracellular vesicles (EV) that may drive thrombosis. Approach and Results: We measured levels of plasma EV TF activity in 100 patients with COVID-19 with moderate and severe disease and 28 healthy controls. Levels of EV TF activity were significantly higher in patients with COVID-19 compared with controls. In addition, levels of EV TF activity were associated with disease severity and mortality. Finally, levels of EV TF activity correlated with several plasma markers, including D-dimer, which has been shown to be associated with thrombosis in patients with COVID-19. CONCLUSIONS: Our results indicate that severe acute respiratory syndrome coronavirus 2 infection induces the release of TF-positive EVs into the circulation that are likely to contribute to thrombosis in patients with COVID-19. EV TF activity was also associated with severity and mortality.

Circulating Markers of Neutrophil Extracellular Traps Are of Prognostic Value in Patients With COVID-19.

OBJECTIVE: The full spectrum of coronavirus disease 2019 (COVID-19) infection ranges from asymptomatic to acute respiratory distress syndrome, characterized by hyperinflammation and thrombotic microangiopathy. The pathogenic mechanisms are poorly understood, but emerging evidence suggest that excessive neutrophil extracellular trap (NET) formation plays a key role in COVID-19 disease progression. Here, we evaluate if circulating markers of NETs are associated with COVID-19 disease severity and clinical outcome, as well as to markers of inflammation and in vivo coagulation and fibrinolysis. Approach and Results: One hundred six patients with COVID-19 with moderate to severe disease were enrolled shortly after hospital admission and followed for 4 months. Acute and convalescent plasma samples as well as plasma samples from 30 healthy individuals were assessed for markers of NET formation: citrullinated histone H3, cell-free DNA, NE (neutrophil elastase). We found that all plasma levels of NET markers were elevated in patients with COVID-19 relative to healthy controls, that they were associated with respiratory support requirement and short-term mortality, and declined to those found in healthy individuals 4 months post-infection. The levels of the NET markers also correlated with white blood cells, neutrophils, inflammatory cytokines, and C-reactive protein, as well as to markers of in vivo coagulation, fibrinolysis, and endothelial damage. CONCLUSIONS: Our findings suggest a role of NETs in COVID-19 disease progression, implicating their contribution to an immunothrombotic state. Further, we observed an association between circulating markers of NET formation and clinical outcome, demonstrating a potential role of NET markers in clinical decision-making, as well as for NETs as targets for novel therapeutic interventions in COVID-19.

Proceedings of ESPGHAN Monothematic Conference 2020: "Acute Liver Failure in Children": Diagnosis and Initial Management.

OBJECTIVES: The Hepatology Committee of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) aims to educate pediatric gastroenterologists, members of ESPGHAN and professionals from other specialties promoting an exchange of clinical expertise in the field of pediatric hepatology. Herewith we have concentrated on detailing the recent advances in acute liver failure in infants and children. METHODS: The 2020 ESPGHAN monothematic three-day conference on pediatric hepatology disease, entitled "acute liver failure" (ALF), was organized in Athens, Greece. ALF is a devastating disease with high mortality and most cases remain undiagnosed. As knowledge in diagnosis and treatment of ALF in infants and children has increased in the past decades, the objective was to update physicians in the field with the latest research and developments in early recognition, curative therapies and intensive care management, imaging techniques and treatment paradigms in these age groups. RESULTS: In the first session, the definition, epidemiology, various causes of ALF, in neonates and older children and recurrent ALF (RALF) were discussed. The second session was dedicated to new aspects of ALF management including hepatic encephalopathy (HE), coagulopathy, intensive care interventions, acute on chronic liver failure, and the role of imaging in treatment and prognosis. Oral presentations by experts in various fields are summarized highlighting key learning points. CONCLUSIONS: The current report summarizes the major learning points from this meeting. It also identifies areas where there is gap of knowledge, thereby identifying the research agenda for the near future.