Richard Todd Stravitz

The transglutaminase coagulation factor XIII (FXIII) is critical for the stability and function of intravascular fibrin clots. Prorepair extravascular fibrin(ogen) deposits are potentially subject to cross-linking by FXIII and other transglutaminases not typically resident in plasma. However, the impact of these alternative modifiers on fibrin(ogen) structure and function is not known. We tested the hypothesis that tissue transglutaminase (TG2) modifies FXIII-directed fibrin(ogen) cross-linking in vitro and within injured tissue. Global proteomic analysis after experimental acetaminophen (APAP)-induced acute liver injury revealed that intrahepatic fibrin(ogen) deposition was associated with hepatic TG2 levels that exceeded that of FXIII. Mass spectrometry-based cross-link mapping of in vitro fibrin matrices uncovered, to our knowledge, the first evidence of synergistic fibrin(ogen) α-α cross-linking catalyzed by both transglutaminases. Fibrin(ogen) cross-linking was increased in livers from patients with APAP-induced acute liver failure. APAP-challenged TG2−/− mice displayed an altered pattern of FXIII-dependent fibrin(ogen)-γ and fibrin(ogen)-α chain cross-linking aligned with the impact of TG2 on fibrin cross-linking in vitro. This shift in fibrin(ogen) cross-linking exacerbated pathologies including hepatic necrosis and sinusoidal congestion. The results, to our knowledge, are the first to indicate that TG2 impacts FXIII-directed fibrin(ogen) cross-linking, both in vitro and in vivo. The results suggest that TG2 functions to dynamically alter the structure of extravascular fibrin(ogen) to mitigate liver damage, a novel mechanism likely applicable across types of tissue injury.

INTRODUCTION: Acute viral hepatitis (AVH) comprises 11% of acute liver failure (ALF) in North America while acetaminophen (APAP) toxicity represents 46%. The use of APAP to treat prodromal hepatitis symptoms is common. It is unknown if concurrent APAP use impacts liver injury in AVH-induced ALF. METHODS: In this prospective, multicenter cohort study, 356 patients meeting criteria for AVH including hepatitis A, B, Epstein-Barr virus, and herpes simplex virus, all leading to ALF (hepatic encephalopathy after acute illness, international normalized ratio ≥1.5), or acute liver injury (acute liver injury, international normalized ratio >2.0, no hepatic encephalopathy) were reviewed for evidence of APAP use: APAP ingestion history or measurement of serum APAP level or APAP-CYS adducts, a specific biomarker released into blood with APAP injury. Patients were grouped by APAP exposure level, from high (measurable APAP levels or toxic APAP-CYS), medium (therapeutic APAP-CYS), low (history of APAP ingestion only and/or barely detectable APAP-CYS), or no exposure recorded. RESULTS: Two hundred five of 356 patients (57.5%) with AVH-ALF had evidence of APAP use: 87 out of 356 (24%) demonstrated high or medium exposures. The aminotransferase and bilirubin levels of high/medium group resembled a mixed APAP-viral injury. Mortality was the highest (51.6%, 21.4%, 28.8%, and 30.5%), and transplant-free survival was the lowest (22.6%, 44.6%, 41.5%, and 40.4%) in the high exposure group compared with medium, low, and no exposure groups. However, the specific comparisons of mortality and transplant-free survival between the high exposure and no exposure groups were not statistically different even after adjusting for baseline patient characteristics differences. DISCUSSION: APAP use in AVH-ALF is common and may negatively impact outcomes compared with little or no APAP exposure. Prospective studies of the safest and effective dose of APAP to use in patients with AVH are needed.

Autoimmune hepatitis is a common cause of acute liver failure. Treatment includes steroids for acute liver injury and liver transplantation in those who fail to respond or develop acute liver failure. The aim of this study is to further characterize acute liver failure secondary to autoimmune hepatitis and identify variables that predict 21-day transplant-free survival. This study included adults hospitalized with acute liver failure enrolled in the Acute Liver Failure Study Group Registry between 1998 and 2019 from 32 centers within the US. The etiology of all cases was reviewed by the Adjudication Committee, and all cases identified as autoimmune hepatitis were included. Acute liver injury was defined as an INR ≥2.0 without encephalopathy and acute liver failure as INR ≥ 1.5 with encephalopathy. Laboratory and clinical data were reviewed. Variables significantly associated with 21-day transplant-free survival were used to develop a multivariable logistic regression model. A total of 193 cases of acute liver failure secondary to autoimmune hepatitis were identified and reviewed. There were 161 patients (83.4%) diagnosed with acute liver failure on enrollment, and 32 (16.6%) developed acute liver failure during hospitalization. At 21 days, 115 (59.6%) underwent liver transplantation, 28 (14.5%) had transplant-free survival, and 46 (23.8%) died before liver transplantation. Higher admission values of bilirubin, INR, and coma grade were associated with worse outcomes. A prognostic index incorporating bilirubin, INR, coma grade, and platelet count had a concordance statistic of 0.84. Acute liver failure secondary to autoimmune hepatitis is associated with a high short-term mortality. We developed a model specifically for autoimmune hepatitis that may be helpful in predicting 21-day transplant-free survival and early identification of patients in need of expedited liver transplant evaluation.

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.

Background and Aims: The ¹³C‐methacetin breath test (MBT) is a noninvasive, quantitative hepatic metabolic function test. The aim of this prospective, multicenter study was to determine the utility of initial and serial ¹³C‐MBT in predicting 21‐day outcomes in adults with acute liver failure (ALF) and non‐acetaminophen acute liver injury (ALI). Approach and Results: The ¹³C‐MBT BreathID device (Exalenz Biosciences, Ltd.) provided the percent dose recovery (PDR) for a duration of 60 minutes after administration of ¹³C‐methacetin solution as the change in exhaled ¹³CO₂/¹²CO₂ compared with pre‐ingestion ratio on study days 1, 2, 3, 5, and 7. Results were correlated with 21‐day transplant‐free survival and other prognostic indices. A total of 280 subjects were screened for enrollment between May 2016 and August 2019. Median age of the 62 enrolled patients with adequate data was 43 years, 79% were Caucasian, 76% had ALF with the remaining 24% having ALI. The mean PDR peak on day 1 or day 2 was significantly lower in nonsurvivors compared with transplant‐free survivors (2.3%/hour vs. 9.1%/hour; P < 0.0001). In addition, serial PDR peaks were consistently lower in nonsurvivors versus survivors (P < 0.0001). The area under the receiver operating characteristic curve (AUROC) of the ¹³C‐MBT in the combined cohort was 0.88 (95% CI: 0.79‐0.97) and higher than that provided by King’s College (AUROC = 0.70) and Model for End‐Stage Liver Disease scores (AUROC = 0.83). The ¹³C‐MBT was well tolerated with only two gastrointestinal adverse events reported. Conclusions: The ¹³C‐MBT is a promising tool to estimate the likelihood of hepatic recovery in patients with ALF and ALI. Use of the PDR peak data from the ¹³C‐MBT point‐of‐care test may assist with medical decision making and help avoid unnecessary transplantation in critically ill patients with ALF and ALI.

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.

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor–dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK−/−) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK−/− mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

Background & Aims: Non‐medical factors which contribute to the severity of acute liver failure (ALF) remain poorly defined. The association of alcohol consumption on the severity of presentation and outcome were determined in patients with ALF and acute liver injury (ALI) in a large, multicentre registry.Methods: Alcohol consumption during the 6 months prior to study entry was analysed in 1170 patients enrolled in the ALF Study Group Registry. Consumption was categorized as none/minimal (P < .005). At least moderate alcohol consumption increased the odds of death by 45% (P = .01) across all aetiologies. In multivariate analysis, older age, non‐Caucasian race, peak INR, peak bilirubin and at least moderate alcohol consumption were significantly associated with death. Finally, in Kaplan‐Meier analysis of patients with all aetiologies, at least moderate alcohol consumption was associated with decreased time‐dependent survival (P = .002).Conclusion: Alcohol consumption adversely affects the presentation and outcome of both APAP‐ and non‐APAP‐induced ALI/ALF.