thromboplastin and Liver-Failure--Acute

Microparticles (MPs), membrane fragments of 0.1-1.0 μm, are derived from many cell types in response to systemic inflammation. Acute liver failure (ALF) is a prototypical syndrome of systemic inflammatory response syndrome (SIRS) associated with a procoagulant state. We hypothesized that patients with ALF develop increased procoagulant MPs in proportion to the severity of systemic complications and adverse outcome. Fifty patients with acute liver injury (ALI), 78% of whom also had hepatic encephalopathy (HE; ALF), were followed until day 21 after admission. MPs were characterized by Invitrox Sizing, Antigen Detection and Enumeration, a light-scattering technology that can enumerate MPs as small as 0.15 μm, and by flow cytometry. Procoagulant activity was assessed by a functional MP-tissue factor (MP-TF) assay. Sixteen patients (32%) died and 27 (54%) recovered without liver transplantation (LT). Total MPs (0.15-1.0 μm) were present in nearly 19-fold higher concentrations in ALI/ALF patients, compared to healthy controls (P < 0.0001). MP-TF assays revealed high procoagulant activity (9.05 ± 8.82 versus 0.24 ± 0.14 pg/mL in controls; P = 0.0008). MP concentrations (0.28-0.64 μm) were higher in patients with the SIRS and high-grade HE, and MPs in the 0.36-0.64-μm size range increased in direct proportion to SIRS severity (P < 0.001) and grade of HE (P < 0.002). Day 1 MPs (0.28-0.64 μm) correlated with laboratory predictors of death/LT (higher phosphate and creatinine; lower bicarbonate), and day 1 and 3 MPs were higher in patients who died or underwent LT, compared to spontaneous survivors (P ≤ 0.01). By flow cytometry, 87% of patients had circulating CD41(+) MPs, indicating platelet origin.. Highly procoagulant MPs of specific size ranges are associated with the SIRS, systemic complications, and adverse outcome of ALI/ALF. MPs may contribute to the multiorgan system failure and high mortality of ALF.

Topics: Adult; Cell-Derived Microparticles; Female; Hepatic Encephalopathy; Humans; Liver Failure, Acute; Male; Middle Aged; Platelet Membrane Glycoprotein IIb; Systemic Inflammatory Response Syndrome; Thromboplastin

Our studies and those of many others have implicated hepatocyte necrosis and apoptosis mediated by fibrinogen-like protein-2 (fgl2) prothrombinase and tumor necrosis factor receptor (TNFR) in the development of fulminant viral hepatitis, a disease with a mortality rate greater than 80% in cases lacking immediate organ transplantation. This study was designed to explore the efficacy of dual short hairpin RNA (shRNA) interference with fgl2 and TNFR1 in the treatment of murine hepatitis virus strain 3 (MHV-3)-induced fulminant hepatitis in mice. Plasmids p-mfgl2shRNA and p-mTNFR1shRNA, complementary to the sequences for mfgl2 and mTNFR1, were constructed. Plasmids pEGFP-mfgl2 and pEGFP-mTNFR1 expressing mfgl2-EGFP (enhanced green fluorescent protein) and mTNFR1-EGFP fusion proteins were also constructed to screen the inhibitory effect of p-mfgl2shRNA and p-mTNFR1shRNA on mfgl2 and mTNFR1 expression. Cotransfection of individual shRNA plasmids and pcDNA3.0-mfgl2 and pcDNA3.0-mTNFR1 expression constructs into Chinese hamster ovary (CHO) cells significantly inhibited mfgl2 and mTNFR1 gene expression, as evidenced by fluorescence microscopy, reverse transcription-polymerase chain reaction, and Western blotting. In vivo hydrodynamic delivery of dual-interference shRNA plasmids for mfgl2 and mTNFR1 significantly decreased mfgl2 and mTNFR1 expression; markedly ameliorated fibrin deposition, hepatocyte necrosis, and apoptosis; and prolonged survival against fulminant viral hepatitis induced by MHV-3 in BALB/cJ mice compared with mfgl2 or TNFR1 single-gene interference. These results indicate that in vivo interference with genes for more than one key target provides superior treatment efficacy compared with single-gene interference.

Topics: Animals; Apoptosis; Blotting, Western; CHO Cells; Cricetinae; Cricetulus; Disease Models, Animal; Female; Fibrinogen; Gene Expression; Hydrodynamics; Liver Failure, Acute; Mice; Mice, Inbred BALB C; Microscopy, Fluorescence; Murine hepatitis virus; Receptors, Tumor Necrosis Factor, Type I; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Thromboplastin; Transcription, Genetic

Acetaminophen (N-acetyl-p-aminophenol [APAP]) is one of the leading causes of acute liver failure, and APAP hepatotoxicity is associated with coagulopathy in humans. We tested the hypothesis that activation of the coagulation system and downstream protease-activated receptor (PAR)-1 signaling contribute to APAP-induced liver injury. Fasted C57BL/J6 mice were treated with either saline or APAP (400 mg/kg intraperitoneally) and were euthanized 0.5-24 hours later. Hepatotoxicity and coagulation system activation occurred by 2 hours after administration of APAP. Treatment with APAP also caused a rapid and transient increase in liver procoagulant activity. In addition, significant deposition of fibrin was observed in the liver by 2 hours, and the concentration of plasminogen activator inhibitor-1 in plasma increased between 2 and 6 hours. Pretreatment with heparin attenuated the APAP-induced activation of the coagulation system and hepatocellular injury and diminished hepatic fibrin deposition at 6 hours. Loss of hepatocellular glutathione was similar in APAP-treated mice pretreated with saline or heparin, suggesting that heparin did not diminish bioactivation of APAP. In mice deficient in tissue factor, the principal cellular activator of coagulation, APAP-induced liver injury, activation of coagulation, and hepatic fibrin deposition were reduced at 6 hours. Formation of the tissue factor-factor VIIa complex leads to the generation of thrombin that can activate cells through cleavage of PAR-1. Mice lacking PAR-1 developed less injury and hepatic fibrin deposits at 6 hours in response to APAP than control mice.. Activation of the coagulation system and PAR-1 signaling contribute significantly to APAP-induced liver injury.

Topics: Acetaminophen; Analgesics, Non-Narcotic; Animals; Anticoagulants; Blood Coagulation; Dose-Response Relationship, Drug; Factor VIIa; Fibrin; Heparin; Liver; Liver Failure, Acute; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Receptor, PAR-1; Thromboplastin

The mechanisms leading to the hemostatic changes of acute liver injury are poorly understood. To study these further we have assessed coagulation and immune changes in patients with acute paracetamol overdose and compared the results to patients with chronic cirrhosis and normal healthy controls. The results demonstrate that in paracetamol overdose coagulation factors (F)II, V, VII and X were reduced to a similar degree and were significantly lower than FIX and FXI (mean levels 0.28, 0.16, 0.13, 0.19, 0.51 and 0.72 IU mL(-1), respectively). In cirrhosis, by contrast, FII, FV, FVII, FIX and FX were equally reduced whilst FXI was lower than the other factors (mean levels 0.64, 0.69, 0.62, 0.60, 0.66 and 0.40 IU mL-1, respectively). FVIII was raised in paracetamol overdose patients but normal in those with cirrhosis (mean levels 1.95 and 1.01 IU mL(-1), respectively). Interleukin-6 and tumor necrosis factor-alpha levels were raised in both patient groups, but higher levels were found in paracetamol overdose, compared to cirrhosis. Thrombin-antithrombin and soluble tissue factor levels were higher in those with acute liver injury but normal in cirrhosis. Antithrombin levels were reduced in both acute liver injury and cirrhosis. From these data we put forward a novel mechanism for the coagulation changes in acute paracetamol induced liver injury. We propose that immune activation leads to tissue factor-initiated consumption of FII, FV, FVII and FX, but that levels of FIX and FXI are better preserved because antithrombin inhibits the thrombin induced positive feedback loop that activates these latter factors.

Topics: Acetaminophen; Adolescent; Adult; Aged; Aged, 80 and over; Antithrombin III; Blood Coagulation; Blood Coagulation Factors; Case-Control Studies; Humans; Interleukin-6; Liver Cirrhosis; Liver Failure, Acute; Middle Aged; Models, Biological; Peptide Hydrolases; Thromboplastin; Tumor Necrosis Factor-alpha

Prothrombin time (PT) is a universal indicator of liver disease severity. However, variability in thromboplastin reagents leads to large interlaboratory differences in PT results. The aim of this study was to determine whether the use of the international normalized ratio (INR) or other modes of expression might achieve PT standardization in patients with liver failure. PT was measured with seven thromboplastin reagents with different sensitivities in plasmas from 27 patients with miscellaneous chronic and acute liver failure and, as a control population, 29 patients on oral anticoagulation therapy. PT was expressed in seconds, ratio, activity percentage, and INR. In patients with liver failure, only activity percentage expression eliminated variability in PT results obtained with the seven thromboplastins while INR, seconds, and ratio values remained significantly different (P < .01). In patients on oral anticoagulant therapy, only INR normalized PT results. We conclude that, in patients with liver failure, INR fails to yield a PT expression independent of the thromboplastin used and only activity percentage expression may provide a common international scale of PT reporting.

Topics: Analysis of Variance; Anticoagulants; Humans; Liver Failure; Liver Failure, Acute; Prothrombin Time; Recombinant Proteins; Reference Values; Reproducibility of Results; Sensitivity and Specificity; Thromboplastin