Hypercoagulability progresses to hypocoagulability during evolution of acetaminophen-induced acute liver injury in pigs

Increases in prothrombin time (PT) and international normalised ratio (INR) characterise acute liver injury (ALI) and failure (ALF), yet a wide heterogeneity in clotting abnormalities exists. This study defines evolution of coagulopathy in 10 pigs with acetaminophen (APAP)-induced ALI compared to 3 Controls. APAP administration began at 0 h and continued to ‘ALF’, defined as INR >3. In APAP pigs, INR was 1.05 ± 0.02 at 0 h, 2.15 ± 0.43 at 16 h and > 3 at 18 ± 1 h. At 12 h thromboelastography (TEG) demonstrated increased clot formation rate, associated with portal vein platelet aggregates and reductions in protein C, protein S, antithrombin and A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats–13 (ADAMTS-13) to 60%, 24%, 47% and 32% normal respectively. At 18 ± 1 h, INR > 3 was associated with: hypocoagulable TEG profile with heparin-like effect; falls in thrombin generation, Factor V and Factor VIII to 52%, 19% and 17% normal respectively; further decline in anticoagulants; thrombocytopenia; neutrophilia and endotoxemia. Multivariate analysis, found that ADAMTS-13 was an independent predictor of a hypercoagulable TEG profile and platelet count, endotoxin, Protein C and fibrinogen were independent predictors of a hypocoagulable TEG profile. INR remained normal in Controls. Dynamic changes in coagulation occur with progression of ALI: a pro-thrombotic state progresses to hypocoagulability.


Acetaminophen (APAP) treatment leads to acute liver injury (ALI) and failure (ALF) associated with elevation in International Normalised Ratio (INR). APAP treatment in 10 pigs resulted
in non-significant increases in INR (calculated from a bedside prothrombin time (PT CoagDx ) analyser) from 1.05 ± 0.02 at 0 h to 1.26 ± 0.05 at 12 h (p = 0.265) and to 1.34 ± 0.07 at 14 h (p = 0.137). However by 16 h, a significant increase in INR to 2.15 ± 0.43 (p < 0.001) was seen and at 18 ± 1 h INR exceeded 3 (p < 0.001), indicative of ' ALF' (Fig. 1). Total dose of APAP administered to achieve ' ALF' was 46.0 ± 2.9 g. In three Control pigs, placebo treatment resulted in no change in INR from 0 h to 20 h.
In the APAP group, APAP-induced hepatotoxicity at the time INR exceeded 3 was further confirmed by demonstrating significant elevation in total serum high-mobility group box-1 protein (HMGB1) 21 . In the APAP group total HMGB1 was 1.4 ± 0.1 ng/ml and 5.0 ± 1.0 ng/ml at 0 h and ' ALF' respectively (p = 0.003). Whereas, in the Control group total HMGB1 was 1.5 ± 0.1 ng/ml and 1.2 ± 0.1 ng/ml at 0 h and 20 h respectively. In addition, liver histopathology at death or sacrifice, which occurred within 20 h of ' ALF' , revealed acute centrilobular and midzonal hepatocyte degeneration and necrosis in all APAP pigs.
No bleeding complications were observed in any animal. However, the portal vein catheters thrombosed in 4 out of 10 APAP pigs by 12 h in 3 pigs and by 14 h in 1 pig.
Progression of ALI is associated first with a hypercoagulable thromboelastography (TEG) profile followed by a hypocoagulable TEG profile with a heparin-like effect. Thromboelastography was used to monitor global changes in coagulation in portal vein and arterial blood samples with progression of ALI. Results are given in Tables 1 and 2. Representative traces for APAP pigs are shown in Fig. 2. Overall TEG results were significantly affected by time (p < 0.001) and the use of heparinase (p < 0.001) for both arterial and portal vein blood. However, there was no significant difference in the TEG results from portal vein samples compared to arterial samples.
At 0 h TEG profiles without heparinase demonstrated significant differences from normal suggestive of hypocoagulability, but these differences were abrogated with heparinase (Tables 1 and 2). As time 0 h was prior to SCIeNTIFIC RepoRts | 7: 9347 | DOI:10.1038/s41598-017-09508-3 APAP dosing and the observed changes were no longer apparent by 12 h, the TEG profiles at 0 h probably represented contamination with exogenous heparin used during catheter placement.
At 12 h hypercoagulability was evidenced by significant reduction in time required for clot formation (K) in both arterial and portal blood and an increase in rapidity of fibrin build-up and cross-linking (α) in arterial blood. This was however, a transient state with progression to hypocoagulability being demonstrated as early as 12 h on analysis of ultimate strength of the fibrin clot (MA), which showed a significant steady decline from baseline to ' ALF' in both arterial and portal blood. Significant increase in time to initial fibrin formation (R) and reduction in α were only seen once INR exceeded 3 at ' ALF' in both arterial and portal blood. Addition of heparinase affected TEG results significantly in both arterial and portal blood at 14 h and at the time of ' ALF' . At 14 h mean K was 45% and 58% lower in the presence of heparinase for arterial and portal blood respectively and at ' ALF' mean R was 64% and 53% lower in the presence of heparinase for arterial and portal blood respectively. These results suggest an endogenous heparin-like effect component to hypocoagulability.
Progression of ALI is associated with dynamic changes in clotting times, thrombin generation, procoagulants (Factor V, Factor VIII, von Willebrand Factor) and anticoagulants (A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats-13, Protein C, Protein S and Antithrombin). Femoral arterial blood samples were collected and stored at 0 h, 12 h, 16 h and ' ALF' from 3 Controls and 8 APAP pigs. Additionally, portal vein blood samples were collected and stored from 2 Controls and 3 APAP pigs at the same times, except that portal vein blood was collected at 14 h instead of 16 h. Prothrombin time (PT IL ) and activated partial thromboplastin time (APTT) increased with progression of ALI in stored samples from APAP animals (p < 0.001), but not from Controls (Fig. 1). Changes in INR calculated from stored samples mirrored those calculated immediately from a bedside analyser. From 0 h to ' ALF' , there was a 3.9-fold increase in PT IL (p < 0.001) and 4.7-fold increase in APTT in arterial blood (p < 0.001). platelet count (expressed as a ratio of red blood cell count (platelet count/RBC)) with progression through acute liver injury to acute liver failure (ALF), which was defined as international normalised ratio (INR) of 3 or more and change in (c) prothrombin time (PT IL ) and (d) activated partial thromboplastin time (APTT) in stored samples at 0 h, 12 h, 16 h and ALF. Mean ( ± se) for each parameter is plotted for acetaminophen (APAP) treated pigs (solid lines) and control pigs (broken lines). P values are given above significant differences between the APAP and Control groups.
Endogenous thrombin potential (ETP) in arterial and portal blood decreased significantly from 0 h to ' ALF' in both the Control and APAP groups, but the decline in the APAP group was greater, resulting in significantly lower ETP in the APAP group compared to Controls at ' ALF' (p < 0.001, Fig. 3). At 0 h, ETP in arterial and portal blood from the Control group was 461 ± 16 nM min and 400 ± 56 nM min respectively and from the APAP group was 339 ± 23 nM min and 409 ± 21 nM min respectively. Whereas at ' ALF' , ETP in arterial and portal blood from the Control group was 347 ± 39 nM min and 340 ± 73 nM min respectively and from the APAP group was 177 ± 17 nM min and 137 ± 11 nM min respectively. Peak Height also decreased significantly in arterial and portal blood with progression to ' ALF 'resulting in significant differences between Control and APAP groups at 16 h and ALF (p ≤ 0.001, Fig. 3). At 0 h, Peak Height in arterial and portal blood from the Control group was 121 ± 9 nM and 108 ± 1 nM respectively and from the APAP group was 97 ± 8 nM and 106 ± 2 nM respectively. Whereas at ' ALF' , Peak Height in arterial and portal blood from the Control group was 93 ± 7 nM and 91 ± 17 nM respectively and from the APAP group was 23 ± 2 nM and 17 ± 1 nM respectively. A significant decrease in mean Factor V (FV) activities was seen between time 0 h and 12 h in Controls and APAP animals with no significant difference between groups (Fig. 4). Decrease in Controls was likely due to aforementioned hemodilution. Mean activities at 12 h compared to 0 h were 47% (p < 0.001), 45% (p < 0.001), 59% (p = 0.002) and 38% (p = 0.001) in arterial Controls, arterial APAP samples, portal vein Controls and portal vein APAP samples respectively. However, after 12 h, FV activities continued to decrease in APAP samples, but not in Controls, leading to significantly lower arterial FV activities at 16 h (p < 0.001) and ' ALF' (p < 0.001) in APAP animals compared to Controls. Mean activities at 16 h and 20 h compared to 0 h were 59% and 63% respectively in arterial Controls and 24% and 19% respectively in arterial APAP samples. A similar change in Factor VIII (FVIII) activities was seen (Fig. 3), leading to significantly lower (p < 0.001) arterial FVIII activities at ' ALF' in APAP animals (mean FVIII activity of 17% compared to 0 h) compared to Controls (mean FVIII activity of 70% compared to 0 h).
Overall there was a significant effect of time on arterial and portal vein vWF Ag concentrations (p < 0.001) evidenced as decline primarily between 0 h and 12 h (Fig. 4). However, vWF Ag concentration did not differ significantly between the APAP group and Controls at any time. For A Disintegrin and Metalloprotease with Thrombospondin type 1 repeats-13 (ADAMTS-13) activity there was also a significant decline between 0 and 12 h in the APAP group and Controls (Fig. 4). However, the decline in the arterial APAP samples was significantly greater than in the arterial Control samples at 12 h: in APAP and Control samples mean ADAMTS-13 activities at 12 h were 32% and 71% that at 0 h respectively (p = 0.022). This decline continued in the arterial APAP samples to mean activities at ' ALF' of 6% that at 0 h.
Activities of the natural anticoagulants, Protein C (PC), Protein S (PS) and Antithrombin (AT) all demonstrated similar patterns of decline in arterial and portal vein samples (Fig. 5). Whilst there was a decline in all 3 activities in Control and APAP samples between 0 h and 12 h, the decline in the APAP samples was significantly greater at 12 h (p < 0.005). At 12 h mean arterial Control activities of PC, PS and AT were 85%, 55% and 74% that at 0 h respectively. Whereas at 12 h mean arterial APAP activities of PC, PS and AT were 60%, 24% and 47% that  Table 1. Thromboelastography (TEG) results from acetaminophen (APAP) treated pigs with progression of acute liver injury to acute liver failure (ALF). Parameters listed include: R, time to initial fibrin formation and K, speed of clot formation. Times listed are: Normal, prior to any intervention; hours from onset of APAP dosing; and ' ALF' , time at which international normalised ratio exceeded 3. n represents sample number. p values given in same cell as data represent significant differences compared to Normal. p values given in column to the right of data represent significant differences between adjacent natural and heparinase cups.
SCIeNTIFIC RepoRts | 7: 9347 | DOI:10.1038/s41598-017-09508-3 at 0 h respectively. Further decrease in arterial Controls was not seen. In arterial APAP samples, these factors continued to decrease: at ' ALF' mean arterial activities of PC, PS and AT were 8%, 1% and 7% that at 0 h respectively (Fig. 5). Low levels of anti-Xa activity were only detected at time 0 h in two arterial APAP samples (0.07 and 0.24 units/ ml) and one arterial Control sample (0.19 units/ml), consistent with TEG results and probably a reflection of exogenous heparin at this time point.
D-dimer concentrations in arterial and portal vein samples did not differ significantly between Controls and the APAP group. Mean fibrinogen concentrations were significantly lower in arterial APAP samples at 12 h compared to Controls (p = 0.015) and all times thereafter to ' ALF' (p < 0.001, Fig. 3).
Portal vein platelet aggregates are observed during hypercoagulable phase of ALI. At 0 h, there was no significant difference in mean number of portal vein platelet aggregates detected per ×10 microscope field in APAP animals (0.81 ± 0.18 per field) compared to Controls (1.14 ± 0.18 per field). However, at 12 h a significant difference was detected with a greater number of portal vein platelet aggregates in the APAP group (1.3 ± 0.5 per field) compared to Controls (0.3 ± 0.2 per field) (Fig. 6). Platelet aggregates were also detected in hepatic sinusoids, but there was no significant effect of time, zone of the hepatic lobule or experimental group on abundance of sinusoidal platelet aggregates.

Plasma endotoxin increases with progression of ALI.
In APAP pigs, plasma endotoxin concentration at ' ALF' was significantly higher compared to 0 h in both arterial (p = 0.004) and portal (p = 0.010) blood, but there was no significant difference in endotoxin concentration between portal and arterial blood at any time point. Endotoxin concentrations in APAP pigs were 2.6 ± 1.6 endotoxin units (EU)/ml and 14.3 ± 1.6 EU/ml in portal blood at 0 h and ' ALF' respectively and 2.9 ± 1.5 EU/ml and 13.4 ± 1.5 EU/ml in arterial blood at 0 h and ' ALF' respectively. In Controls plasma endotoxin concentration was unaffected by time and independent of blood vessel sampled. Plasma endotoxin concentrations in Control pigs were 4.3 ± 2.0 EU/ml and 8.2 ± 2.0 EU/ml in portal blood at 0 h and 20 h respectively and 5.5 ± 2.1 EU/ml and 6.8 ± 2.1 EU/ml in arterial blood at 0 h and 20 h respectively.
Portal vein flow is unaffected by ALI. Portal vein flow (PVF) in APAP and Control groups did not change significantly with time and no significant difference was seen between these groups. PVF in Controls was Platelet count, ADAMTS-13, endotoxin, fibrinogen and PC are independently associated with TEG in ALI. There were significant strong correlations between the activities/concentrations of all of the procoagulants and anticoagulants measured, platelet count, neutrophil count and endotoxin concentration. Platelet count, as expected, had a significant independent effect on α (estimate = 0.05 ± 0.02, p = 0.006), MA (estimate = 0.03 ± 0.01, p = 0.022) and K (estimate = −0.02 ± 0.01, p = 0.039) with a fall in platelet count being associated with trend to hypocoagulability. ADAMTS-13 activity had a significant independent effect on α (estimate = −0.50 ± 0.17, p = 0.006) and K (estimate = 0.44 ± 0.17, p < 0.001) with a fall in ADAMTS-13 activity being associated with increased speed of clot formation, in agreement with the hypercoagulability and fall in ADAMTS-13 activity seen at 12 h. Endotoxin concentration had a significant independent effect on MA (estimate = −1.21 ± 0.50, p = 0.024) and R (estimate = 3.19 ± 1.13, p = 0.008), emphasising the role of endotoxin in progression of hypocoagulability. Finally, fibrinogen (estimate = 12.10 ± 3.23, p = 0.001) and protein C (estimate = 0.26 ± 0.08, p = 0.004) were independently associated with MA, emphasising the role of both procoagulants and anticoagulants in determination of coagulation status.

Discussion
We describe for the first time the evolution of coagulation disturbance from health through ALI to established ALF, prior to the onset of multi-organ failure, in a pig model of APAP-induced ALF. TEG analysis clearly demonstrated early hypercoagulability at 12 h, progressing to hypocoagulability. In this study, a key independent event implicated in early hypercoagulability was a reduction in ADAMTS-13, which preceded the fall in the pro-coagulants (FV, FVIII) synthesised by the liver and occurred in the face of normal vWF Ag concentrations. There is disproportionate decline in platelet count suggesting potential consumption and this is associated with presence of platelet aggregates in small intrahepatic portal vessels at 12 h. Notably, portal blood flow was unchanged and there was no evidence of enhanced fibrin breakdown, distinguishing the hypercoagulability of ALF from the syndromes of reduced blood flow and disseminated intravascular coagulation (DIC) respectively. The progression of hypercoagulability to hypocoagulability was demonstrated first by changes in TEG and later by changes in thrombin generation, PT, PTT and INR. Key independent events associated with hypocoagulability in this study were increase in endotoxin and decline in platelet count, PC and fibrinogen, demonstrating again the importance of concurrent decline in procoagulants and anticoagulants. These key events were also correlated with neutrophilia with left shift and presence of a heparin-like effect, which may result from endothelial injury due to primary APAP-related liver injury, response to systemic inflammation or local hypoxic response due to platelet aggregates. This study highlights ADAMTS-13 reduction, endotoxemia and platelet aggregates in the liver as key events in the evolution of the coagulopathy in ALI. In this study we show for the first time an in vivo association between hypercoagulability and an increased vWF antigen to ADAMTS13 ratio, platelet aggregates in intrahepatic portal vessels and decline in natural anticoagulants. This occurred at the 12 h time point. At this time, the greater number of intrahepatic portal platelet aggregates in the APAP pigs compared to Controls was not associated with differences in portal flow. Inflammation and endotoxemia may have been contributory factors, as they are known to activate coagulation [13][14][15][16] . However, in this study increases in neutrophil count and endotoxin concentration were not yet apparent at 12 h. Endothelial changes secondary to ALI may also have contributed to platelet aggregation 17,18 . ADAMTS-13 is synthesised primarily by hepatic stellate cells and cleaves hyperactive multimeric or ultra-long forms of vWF into less active vWF fragments. This reduction in vWF activity appears to be greater in the presence of FVIII and platelet glycoprotein 1bα. It has previously been reported that ADAMTS-13 activity is reduced in ALF/ALI patients and plasma taken from these patients resulted in increased aggregation of normal platelets ex vivo compared to plasma from normal controls 22 . We report now in vivo evidence of this observation, which was previously made ex vivo. This study does not allow us to determine whether platelet microthrombi were present in other organs apart from the liver, as no other tissues were biopsied at 12 h. However, a previous clinical study, which reviewed peroperative deaths, identified pulmonary platelet aggregates as a finding particular to liver transplant surgeries, despite the absence of other risk factors for thrombosis 23 . We now report a possible mechanism for this post-mortem finding.
The importance of other anticoagulants in ALF, including PC, has also been reported previously. PC is synthesized by the liver and secreted into the circulation. It is localised to endothelial surfaces by the endothelial PC receptor. PC is activated by endothelium bound thrombomodulin (TM)-thrombin complexes and binds PS to inactivate activated FV and FVIII 24 . Reduction in hepatic PC synthesis combined with PC consumption and/or endothelial injury therefore results in a prothrombotic state. In ALF, Yamaguchi et al. (2006) showed increased plasma thrombin-AT complexes and free TM, decreased activated PC-PC inhibitor complexes and perhaps most importantly decreased ratio of activated PC-PC inhibitor complexes to PC 25 . They concluded that in ALF, endothelial cell injury results in decreased PC activation and a prothrombotic state. Warkentin and Pai also suggest a central role for PC deficiency in acute hypoxic liver injury, which may predispose to microthrombi and ischaemic limb necrosis 26 . Recombinant human PC is available as a therapeutic agent 24 . However, it is unlicensed in patients with liver failure due to potential risk of bleeding. Moreover, its major reported therapeutic effects Figure 3. Change in (a) endogenous thrombin potential (ETP) and (b) peak height for thrombin generation and (c) concentrations of fibrinogen in arterial plasma with progression of acute liver injury to acute liver failure (ALF), which was defined as international normalised ratio (INR) of 3 or more. Mean ( ± se) for each parameter are plotted for acetaminophen (APAP) treated pigs (solid lines) and control pigs (broken lines). P values are given above significant differences between the APAP and control groups.
SCIeNTIFIC RepoRts | 7: 9347 | DOI:10.1038/s41598-017-09508-3 appear not to be due to its anti-coagulant activity, but rather its anti-inflammatory, anti-apoptotic and endothelial stabilisation effect 24 . Therefore, consideration for use in early ALF/ALI with appropriate monitoring of coagulation status may be beneficial in selected cases 27,28 .
Endotoxin is known to activate the coagulation cascade via a pathway which is dependent on increased tissue factor expression on circulating monocytes and release of tissue factor positive microparticles into the circulation. Experimental treatment with endotoxin results in hypercoagulability followed by hypocoagulability in man, rodents and pigs 14,29,30 . However contrary to our findings, the hypocoagulability of experimental endotoxemia is a consumptive coagulopathy, associated with elevation in d-dimers and DIC 14,29,30 .
In the current study a heparin-like effect (HLE) was observed, supporting a role for endothelial injury in the coagulopathy of ALI. We previously defined a heparin-like effect in nine out of 10 human ALF patients using TEG analysis, as a correction of R plus K of more than 50% in the presence of heparinase 18 . Anti-Xa assays can also be used to measure the effect of endogenous and exogenous heparins in plasma 31 and the results of the chromogenic anti-Xa assay used in this study has previously been shown to be correlated with the R TEG parameter when using heparinase cups in infected cirrhotic patients 17 . In the current study, anti-Xa activity was only detected in 3 samples at time 0 h: this may have been due to contamination with unfractionated heparin sodium used to flush catheters prior to insertion. Reason for failure to detect anti-Xa activity when INR exceeded 3 in presence of significant HLE is unclear and warrants further investigation of the assay used with porcine samples.
The pig model of APAP-induced ALF used in this study has been previously shown to mimic the clinical time course of hyperacute liver failure in man 32 . In this study we confirm hepatocyte necrosis at the time INR exceeded 3, defined as ' ALF' , by demonstrating significant elevations in HMGB1, a biomarker of hepatocyte necrosis in Figure 4. Change in arterial plasma activities/concentrations of (a) factor V (FV), (b) factor VIII (FVIII), (c) von Willebrand factor (vWF Ag) and (d) A disintegrin and metalloprotease with thrombospondin type 1 repeats-13 (ADAMTS-13) with progression of acute liver injury to acute liver failure (ALF) which was defined as international normalised ratio (INR) of 3 or more. Mean ( ± se) for each parameter are plotted for acetaminophen (APAP) treated pigs (solid lines) and control pigs (broken lines). P values are given above significant differences between the APAP and control groups.
SCIeNTIFIC RepoRts | 7: 9347 | DOI:10.1038/s41598-017-09508-3 APAP-induced ALF in man 21 . Studies using biomarkers to map the time course of liver injury to clinical liver failure in man and pigs are yet to be published despite considerable progress in this field 21,33 .
In conclusion, the results of this longitudinal study shows for the first time that APAP related ALI is associated with the initial development of hypercoagulability, consequent upon reduction in levels of ADAMTS-13 and natural anticoagulants, which may contribute to intrahepatic portal venous platelet aggregation and is a potential therapeutic target. Hypercoagulability progresses to hypocoagulability due to a combination of reduction in platelets, reduction in circulating procoagulants and anticoagulants and endotoxemia. The results of this study highlight reduction in ADAMTS-13, platelet aggregation and decline and endotoxemia as key events in the evolution of the coagulopathy of ALI and emphasises the importance of global assessments of coagulation status in ALI/ALF and the use of blood products on an "on-demand" basis.

Methods
Porcine model of acute liver failure. A previously described pig model of APAP-induced ALF was used in this study 32 . Ten ALF pigs (' APAP pigs') and three control pigs ('Controls') from a previously published study 34 , from which appropriate blood samples had been taken, were included in this study. All animal procedures were approved by the Animal Welfare and Ethical Review Board of the Royal Veterinary College, University of London and were carried out in accordance with the Animals (Scientific Procedures) Act 1986.
Female, 26-36 kg, Landrace cross Large White pigs were induced to and maintained under general anaesthesia until study end. At the beginning of the study all pigs were instrumented for repeat blood sampling including a 5Fr, 20 cm catheter in a femoral artery, a 7.5Fr, 16 cm catheter in the right external jugular vein and a 5Fr, 20 cm catheter in the portal vein via an ileal vein.
Time 0 h was defined as the time immediately following instrumentation of the pigs for experimental procedures and was just prior to onset of induction to ALF. Acute liver failure was induced in ' APAP pigs' with an aqueous APAP suspension, given via an oroduodenal tube as described previously 32 . A loading dose of 0.25 g/ kg APAP was followed by an hourly maintenance APAP dose, adjusted between 0.5 and 4 g to achieve toxic serum APAP concentrations of greater than 300 mg/L. Progression of ALI to ALF was monitored by measuring prothrombin time (PT), initially every 4 h and then every 1 h when prolongation of PT was observed. PT was measured using venous sodium citrate whole blood samples and a bedside analyser, CoagDx (Idexx Laboratories Ltd, West Yorkshire, UK): this PT will hereafter be referred to as PT CoagDx . ' ALF' was defined as the time point at which PT CoagDx exceeded 3 times that at time 0 h, which was equivalent to an INR of 3. This was based upon previous studies, where INR of 3 was associated with subsequent progression to death in 100% of pigs 32 . 'Control Figure 5. Change in arterial plasma activities of (a) protein C (PC), (b) protein S (PS) and (c) antithrombin (AT) with progression of acute liver injury to acute liver failure (ALF) which was defined as international normalised ratio (INR) of 3 or more. Mean ( ± se) for each parameter are plotted for acetaminophen (APAP) treated pigs (solid lines) and control pigs (broken lines). P values are given above significant differences between the APAP and control groups.
SCIeNTIFIC RepoRts | 7: 9347 | DOI:10.1038/s41598-017-09508-3 pigs' were managed using the same protocols as APAP pigs, except that they received water without APAP for the 20 h required for ' ALF' induction in APAP pigs.
All pigs included in these studies were ultimately sacrificed or died within 20 h of ' ALF' for APAP pigs and at 40 h for Control pigs. Histological examination of paraffin embedded post-mortem liver specimens was used to confirm acute APAP-induced hepatocyte necrosis in APAP pigs.
In the following studies progression of coagulopathy was assessed between Time 0 h and ' ALF' unless otherwise stated. During this time all pigs received intensive supportive care according to defined protocols 32 , which did not include treatments for coagulopathy nor any blood products.

Haematology.
Femoral artery blood samples were collected in Ethylenediaminetetraacetic acid (EDTA) tubes (Becton Dickinson UK Ltd, Plymouth, UK) and fresh blood smears were made for complete blood cell counts at a commercial laboratory (Pathology and Diagnostic Laboratories, RVC, UK) at 0 h, 4 h, 8 h, 12 h and then every 2 h until ' ALF' . EDTA blood was stored at 4 °C prior to analysis within 24 h. Thromboelastography (TEG). TEG was performed on the TEG analyser 5000 (Haemonetics Ltd, Coventry, UK). Baseline TEG was performed after intramuscular premedication with atropine, azaperone, midazolam and ketamine, from a blood sample obtained directly from an ear vein to achieve normal TEG values for this study. Subsequent blood samples were obtained from femoral artery and portal vein catheters at 0 h, 12 h, then every 2 h until ' ALF' . One ml of native whole blood collected in a clean 2.5 ml plastic syringe was analysed within 4 minutes of collection. TEG analysis was carried out with (heparinase cup) and without (natural cup) addition of heparinase simultaneously. Two TEG analysers (Analyzer 5000) permitted simultaneous analysis of portal vein and arterial samples. All TEG traces were reviewed graphically and those with errors due to technical problems, e.g. bubbles in the sample, calibration error, discontinuation of analysis prior to achievement of all data points, were discarded. The following machine generated parameters were analysed: R, reaction time, representing time to initial fibrin formation; K, representing time from beginning of clot formation until Statistical analysis. IBM SPSS Statistics V22 (IBM Corporation, New York, USA) was used for all data analysis and significance was set at the 5% level. The Linear Mixed Models procedure was employed to analyse each recorded parameter in turn and to consider the effect of time, group (APAP or Control) and blood vessel (artery or portal vein) on each parameter. The ' ALF' time point for the APAP group was compared to the 20 h time point for the Control group. The Linear Mixed Models procedure was used to determine which of these parameters may act as independent determinants of the four TEG parameters recorded (α, K, R, MA) for native whole blood without heparinase. Parameters considered for inclusion in this model were platelet count, neutrophil count, endotoxin concentrations and all procoagulants and anticoagulants assayed. A backward elimination method was used for parameter selection. Data Availability. The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.