Research Article

Evaluation of coagulation abnormalities in acute liver failure

Banwari Agarwal1,�, Gavin Wright2,7,�, Alex Gatt3, Anne Riddell4, Vishwaraj Vemala2,Susan Mallett1, Pratima Chowdary4, Andrew Davenport5, Rajiv Jalan6, Andrew Burroughs2,⇑

1Department of Intensive Care Medicine, Royal Free Hospital, London, United Kingdom; 2Sheila Sherlock Liver Centre, Royal FreeHospital, London, United Kingdom; 3Department of Haematology, Mater Dei Hospital, Tal-Qroqq, Malta; 4KD Haemophilia Centre

and Thrombosis Unit, Royal Free Hospital, London, United Kingdom; 5UCL Centre for Nephrology, Royal Free Hospital, London,United Kingdom; 6Liver Failure Group, Institute of Hepatology, UCL Medical School, Royal Free Hospital,

London, UK; 7Basildon & Thurrock University Hospital, Essex, United Kingdom

Background & Aims: In acute liver failure (ALF), prothrombin (HLE) was also noted in most patients. No significant clinical

time (PT) and its derivative prothrombin time ratio (PTR) are ele-vated, and are considered predictors of increased bleeding risk.We aimed at determining whether increased PT/PTR reflects thehaemostatic potential and bleeding risk in ALF patients.Methods: Twenty consecutive ALF patients were recruited. Sam-ples were analysed on admission for standard laboratory clottingtests (e.g. PT), thromboelastography (TEG), individual pro andanticoagulant factors and thrombin generation (TG) kinetics withand without Protac�, a snake venom protein C activator, andmicroparticle assay. TG was also measured in 20 age and sexmatched healthy volunteers.Results: PT was significantly raised (50.7 s ± 7.2, p = 0.0001) butdid not correlate with TEG parameters. TEG tracings were consis-tent with a hypocoagulable state in 20%, normal in 45%, andhypercoagulable in 35% of the patients. There was a concomitantand proportional reduction in plasma levels of both procoagu-lants and natural anticoagulant proteins, in conjunction with asignificant elevation in plasma levels of factors-VIII (FVIII) andVon Willebrand factor, and microparticles, culminating in anoverall efficient, albeit reduced, thrombin generation capacityin comparison with healthy individuals. A heparin-like effect

Journal of Hepatology 20

Keywords: Acute liver failure; Thromboelastography; Thrombin generation;Coagulopathy.Received 29 March 2012; received in revised form 27 May 2012; accepted 14 June2012; available online 23 June 2012⇑ Corresponding author. Address: Sheila Sherlock Liver Centre, Royal FreeHospital, London United Kingdom. Tel.: +44 (0)2077940500.E-mail address: andrew.burroughs@nhs.net (A. Burroughs).

� These authors contributed equally to this work.Abbreviations: ALF, Acute liver failure; INR, international normalised ratio; TEG,thromboelastogram/thromboelastography; PT, prothrombin time; ICP, intracra-nial pressure; AT, antithrombin; APTT, activated partial thromboplastin time; R-time, reaction time of TEG; K-time, kinetic time of TEG; MA, maximum amplitudeof TEG; SIRS, systemic inflammatory response syndrome; WBC, white blood cellcount; CRRT, continuous renal replacement therapy; OLT, orthotopic liver trans-plantation; MELD, model for end-stage liver disease score; VWF, Von Willebrandfactor Spp. Supplementary; PPP, platelet poor plasma; TG, thrombin generation;LT, lag-time of thrombogram; TP, time to peak of thrombogram; PH, peak heightof thrombogram; slope (velocity index), rate of thrombin generation [PH/(TP–LT)]; ETP, endogenous thrombin potential; ETP%, endogenous thrombinpotential percentage [ETP + protac/ETP-protac] � 100; MP, microparticles; PPL,procoagulant phospholipids; TF, tissue factor.

bleeding complications occurred and no blood transfusions wererequired.Conclusions: In ALF, despite grossly deranged PT in all patients,estimation of bleeding risk suggests that the coagulation distur-bance in ALF patients is complex and heterogeneous for whichan individualised approach is required.� 2012 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Introduction

Coagulopathy and hepatic encephalopathy (HE) are cardinal fea-tures of acute liver failure (ALF). The prothrombin time (PT) andits derivation, the PTR, and the commonly used ‘International nor-malised ratio’ (INR) have become the standard tests to assess coag-ulopathy in liver disorders, as not only do they serve as goodprognostic indicators, but are broadly considered to correlate withbleeding risk and thus guide blood product replacement for inva-sive procedures. However, there is increasing evidence that in liverdisease, the PT/INR as an indicator of coagulopathy may be mis-leading, as bleeding is rare unless when it occurs as a complicationof co-existent portal hypertension and/or sepsis and renal failure.The overall haemostatic potential and the clinical consequencesthereof, in liver disease, seem to represent the sum total of themodulating effect of a multitude of pro and antihaemostatic driv-ers activated simultaneously and in varying degrees. The impor-tant players are clotting factors, both pro and anticoagulants(most of which are synthesized solely by the liver), platelets, andthe endothelium. Correcting coagulopathy delays intervention,introduces transfusion-related complications, and eliminates rele-vance of PT as an indicator of prognosis and need for liver trans-plantation. In patients with stable non-infected compensatedand decompensated cirrhosis and, during liver transplantation,an increasing body of evidence not only shows a state of ‘balanced’haemostasis in chronic liver disease [1–3], but even hypercoagula-bility with thrombotic complications [4,5]. Our group have alreadydocumented increased clotting of continuous renal replacementtherapy (CRRT) circuits in those liver failure patients requiringrenal support [6]. It is not clear whether a similar discrepancy

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exists between the clotting parameters in ALF when patients typ-ically present with much higher PT/INRs and derangement ofother measures of coagulation than patients with cirrhosis.

The aims of this study were to determine whether increasedPT/PTR reflects the haemostatic potential and bleeding risk inALF patients. A comprehensive assessment of plasma levels ofpro and anticoagulants factors, the relative contribution of plate-lets and fibrinogen towards clot strength, the role of endogenousheparinoids and the resultant heparin-like effect (HLE, plasmamicroparticle activity), and the role of procoagulant phospholip-ids on clotting time were undertaken in order to understandcoagulation findings and their relationship with clinical out-comes (bleeding/thrombosis) in patients presenting with ALF.

Patients and methods

Twenty (n = 20) consecutive and unselected adult ALF patients admitted to theliver unit were studied as approved by our local research ethics committee andNational Research Ethics Service guidelines. The diagnosis of ALF was based onclinical and laboratory evidence (defined by de novo liver failure, coagulopathyPT P20 s or INR P1.5, and hepatic encephalopathy (HE)) [7]. Pregnant women,patients on anticoagulants (such as warfarin and anti-platelet agents) and oralcontraceptives, and those with prothrombotic disorders including ALF secondaryto acute Budd–Chiari syndrome, were excluded.

In addition to the ALF patients, after written informed consent, 20 healthyvolunteers were also enrolled for thrombin generation (TG), microparticles, andprocoagulant assay.

Data was collected for patient demographics, aetiology of liver failure, sever-ity of liver failure (Model of End-stage Liver Disease (MELD)) and organ dysfunc-tion (Sequential Organ Failure Assessment (SOFA), and degree of theinflammatory response (Systemic Inflammatory Response Score (SIRS)) on admis-sion. Clinical outcome including bleeding or clotting episodes was followed untilthe time they either improved spontaneously, or were transplanted or died. TheInternational Society for Thrombosis and Haemostasis (ISTH) definition of majorbleeding was used for this study [8].

Blood sampling

Blood sampling via atraumatic venepuncture was performed in patients who hadfulfilled ALF criteria at the time of admission to our hospital, either on the liverward or on the intensive care unit (ICU). In addition to the comprehensive coag-ulation studies, samples were collected for full blood count, liver function tests,urea, creatinine and electrolytes, albumin, lactate, and arterial ammonia.

Coagulation studies

Whole ‘native’ blood was used for immediate TEG analysis within 5 min of sam-pling, while citrated samples, using BD Vacutainer� tubes with a blood to citrateconcentration of 9:1 (Becton Dickinson, Plymouth, Devon, UK), were collected forother laboratory coagulation tests. Platelet poor plasma (PPP) was prepared bydouble centrifugation at 2000g for 12 min at 4 �C. Samples were stored in aliquotsat �85 �C until testing.

Routine coagulation tests

The PT was measured using HemosIL™ PT Fibrinogen HS Plus thromboplastinreagent (Instrumentation Laboratory (IL), Warrington, UK) with an ISI of 1.15,according to the manufacturer’s instructions using an ACL TOP automated coag-ulometer (IL). PT ratios were calculated for each patient from the geometric meanPT (GMPT) calculated from normal individuals. INRs were calculated using theGMPT and the manufacturers ISI. The APTT was obtained using HemosIL™ Syn-thASIL APTT reagent (IL) on an ACL TOP coagulometer.

Additional coagulation tests

We analysed factors VIII, IX, XI, and XII by standard one-stage APTT based assays.Factors II, V, and VII were analysed by a one stage clotting PT-based assay on anACL 3000 (IL, Warrington, UK) [4]. Von Willebrand factor (VWF) antigen was

Journal of Hepatology 201

analysed by an in-house ELISA. We also analysed anticoagulant factors proteinC (PC), protein S (PS), antithrombin (AT), as previously described [4]. Anti-Xaassays were used to determine endogenous heparinoids in patient samples aspreviously described [9]. Fibrinogen was measured using the recommendedClauss method [10]. Microparticles were quantified in the platelet poor plasma(PPP) using Zymuphen MP-Activity assay, Huphen BioMed, Neuville-sur-oise,France; and procoagulant phospholipids (procoagulant PPL) (Stago, Asnieres surSeine, France) on an ACL TOP coagulometer were measured in both ALF patientsand healthy volunteers.

Thromboelastography

Thromboelastography (TEG) (Thromboelastograph� Hemostasis Analyzer 5000(Haemonetics Corporation, Niles IL, USA) measures the dynamics of thrombinproduction and provides a global assessment of coagulation incorporating thecumulative effect of the interactions at various levels between plasma compo-nents (clotting proteins) and cellular components (platelets, red and white bloodcells, and microparticles) of coagulation; allowing for dynamic assessment of dif-ferent stages of clot formation (clot initiation, amplification and propagation tofibrinolysis). All ALF patients’ whole blood samples were analysed by the follow-ing TEG assays: (1) TEG-native, (2) TEG-heparinase (with heparinase embeddedcup and pin) was used to assess the endogenous heparin effect, and (3) TEG-func-tional fibrinogen (using abciximab reagent to inhibit platelet function via glyco-protein IIb/IIIa receptor complex), to assess the relative contribution to clotstrength of platelets and ‘functional’ fibrinogen [11]. Six parameters wereassessed: R-time (minutes): latency of clot formation from beginning of clottingreaction to initial fibrin formation (defined 2-millimetre amplitude), representingthe enzymatic component of coagulation, the point at which standard tests ofcoagulation (e.g. PT/INR) reach their end point, and therefore thought to reflectPT/INR. K-time (minutes): time from initial fibrin formation required to reachspecific clot firmness (defined 20-millimetre amplitude), representing clotdynamics. Alpha-angle (degrees): the kinetics of clot formation, measuring rateof fibrin formation and cross-linkage representing fibrinogen levels. Maximumamplitude (MA) (mm): measures maximal clot strength representing plateletfunction and aggregation as is mainly contributed to by platelets and fibrinogen.Lysis-30 (%): lysis at 30 min representing clot lysis, and it is a measure of fibrino-lysis. Interpretation of TEG data is based on normal ranges provided by the man-ufacturer; R (min) 12–26, K (min) 3–13, Angle (degree) 14–46, MA (mm) 42–63,and Lysis-30 (%) 0–5, and TEG-functional fibrinogen and MA (mm) 9–29.

Thrombin generation assessment

Thrombin generation was assessed in both the patient group and the healthy vol-unteer PPP samples prepared as previously stated, by using the Calibrated Auto-mated Thrombography (CAT) method as published by Hemker et al. [12]. TGassays were triggered with 5 pM tissue factor (TF) reagent (ThrombinoscopeB.V., Maastricht, The Netherlands). The CAT TG parameters of endogenous throm-bin potential (ETP), peak height (PH), time to peak (TP), and lag-time (LT) wereassessed. In addition, we calculated the velocity index (slope) [PH/TP–LT)] inorder to assess the efficiency of the amplification part of coagulation. We alsoused a modified TG assay as previously described [4], in the presence of Protac�

(Pentapharm, Basel, Switzerland) to assess the impact of ALF on the protein Cpathway. The ETP% [(ETP + protac)/(ETP-protac) � 100] was calculated.

Statistics

All groups of results were tested for normality, with continuous data expressed asmean ± standard error of mean (SEM), and further analysed by either t-test orNewman–Keuls multiple comparison test as appropriate. As results displayednormal Gaussian distribution, the Pearson correlation test was used to calculatecorrelation. A p value <0.05 was considered statistically significant. GraphPadPrism version 5 (GraphPad Software Inc., La Jolla, CA, USA) was used for allanalyses.

Results

Clinical characteristics

Twenty consecutive ALF patients admitted to the Royal FreeHospital between the period of November 2009 and December

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Research Article

2010 were recruited into the study. Nine patients were admittedto ICU directly, and a further four patients required admission toICU from the ward within 48 h of admission. All patients wereencephalopathic on enrolment (14 patients’ grade 62 and sixpatients’ grade 3–4). The average age of the patients was42.4 ± 3.8 years; with 65% male and 70% of Caucasian ethnicity.The aetiology of ALF was as follows – 55% paracetamol overdose(n = 11), 20% ischaemic (n = 4), 15% seronegative (n = 3), and 10%idiosyncratic drug reactions (n = 2). The mean SOFA score(6.8 ± 1.0) and the MELD score (27.7 ± 2.2) at admission indicatedpoor prognosis without intervention. Systemic inflammatory

Table 1. Demographic, clinical, and laboratory characteristics and outcome.

ALF (n = 20) Median (IQR)Outcomes

Survivors 12Death 6OLT 3

Bleeding eventsAny bleeding complication 0Blood Tx-units 0

DemographicsAge (yr) 43 (28-81)Male gender (%) 65Female gender (%) 35Caucasian race (%) 70

Clinical characteristicsEtiology of ALF (number of patients)

Seronegative 3Drugs (bodybuilding) 2Ischemic/hypotensive 4POD 11

Hepatic encephalopathy-grade 1.5 (1-3)*Number of patients with SIRS 2 or more 11Number of patients with positive cultures 8

Laboratory dataCreatinine (µmol/L) 108 (70-244)Total bilirubin (µmol/L) 80 (49-129)***Albumin (g/L) 32 (28-37)**Venous ammonia (µmol/L) 115 (95-138)***ALT (IU/L) 3849 (1558-7513)***AST (IU/L) 3108 (1375-8521)***Phosphate (mmol/L) 0.9 (0.7-1.2)CRP (mg/L) 13 (8-42)*Standard coagulation:

INR 4.1 (2.2-6.1)***PTT (sec) 39 (27-67)***APTT (sec) 35 (32-41)*PLT (x109/L) 111 (74-153)*

Fibrinogen (g/L) 1.8 (1.2-2.1)*

AP

T

Data are expressed as mean ± SEM.⁄p <0.05, ⁄⁄p <0.01, and ⁄⁄⁄p <0.001 indicate significant difference from population mean

782 Journal of Hepatology 201

response was observed in most patients with the mean SIRS scorebeing 1.95 ± 0.19 (9/20 patients had P2 SIRS points) and C Reac-tive Protein (CRP) elevated at 39.7 ± 15.3 mg/L. Twelve patientshad variable degrees of renal dysfunction with 9 of these requir-ing renal support. None of the 20 patients received fresh frozenplasma (FFP) or platelets on admission. In the course of disease,however, 7/20 patients received FFP to correct INR to 63 (localICU policy) before insertion of central vascular catheters. Nopatient had intracranial monitoring device inserted. All patientsreceived routine antimicrobial prophylaxis according to localguidelines.

LF (n = 20) Median (IQR)rognostic score

SOFA 6 (3-12)*EG parameters:1) TEG-native

R-time (min) 12.1 (8.9-30.6)K-time (min) 5.2 (2.9-21.4)α-angle (degrees) 40.4 (11.5-55.9)Maximum amplitude (mm) 43.7 (30.0-52.2)Lysis 30 (%) 0.4 (0.1-0.5)

2) TEG-heparinase

3) TEG-functional fibrinogen

R-time (min) 10.8 (7.2-21.5)

K-time (min) 4.6 (2.3-8.4)α-angle (degrees) 37.4 (24.2-49.9)Maximum amplitude (mm) 45.9 (36.5-56.1)Lysis 30 (%) 0.4 (0.1-0.7)

R-time (min) 3.8 (2.8-5.5)K-time (min) 9.5 (3.5-10.1)α-angle (degrees) 24.9 (14.5-29.4)Maximum amplitude (mm) 11.0 (3.3-15)Lysis 30 (%) 0.3 (0.1-0.4)

.

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Twenty healthy volunteers were used for comparative TG

analysis with mean age 42.6 ± 2.5, 25% male without knowncoagulation or other major illnesses (Table 1).

Clinical and coagulation outcome

Eleven patients survived without the need for liver transplanta-tion, four underwent orthotopic liver transplantation and sixpatients died (1 post-orthotopic liver transplantation); equatingto 60% transplant-free survival. No recorded bleeding events(either spontaneous or following intervention) occurred in thesepatients and no transfusion of blood was required. Similarly, noevidence of thrombotic events was observed except frequentclotting of extracorporeal renal replacement therapy circuit intwo out of the nine patients who required renal support.

2 Factor VIII

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1

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Coagulation results

Standard clotting tests

On admission, there was a rise in PT (50.7 s ± 7.2, p = 0.0001), PTR(4.3 ± 0.6), p = 0.0001) and INR (4.3 ± 0.5, p = 0.0001), and APTT(38.1 s ± 2.5, p = 0.7 (ns)), and a reduction in platelet count(140.4 ± 24.5, p = 0.0825); fibrinogen level remained within nor-mal limits (1.8 ± 0.2).

Thromboelastography results

Four out of the 20 patients had a hypocoagulable profile (R-time41.3 ± 7.4; K-time 33.6 ± 10.1, Alpha-angle 9.0 ± 2.6 and MA27.6 ± 5.3), 9 had a normal coagulable state (R-time 11.2 ± 3.8,K-time 7.0 ± 6.6, Alpha-angle 38.7 ± 14.4 and MA 44.2 ± 10.4)

Table 2. Relative changes in pro and anticoagulant factor levels of ALFpatients at admission.

Normal range (NR)

Median (IQR)

% change from NR

Procoagulant activityFactor II 50-150 IU/dl 24 (17-37) -59***Factor VII 50-150 IU/dl 15 (8-29) -66***Factor IX 50-150 IU/dl 41 (30-67) -42**Factor X 50-150 IU/dl 34 (15-46) -51***Factor V 50-150 IU/dl 21 (12-44) -34***Factor XI 70-150 IU/dl 51 (38-60) -50***Factor XII 50-150 IU/dl 49 (32-69) -44***Fibrinogen 1.5-2.5 g/L 1.6 (1.1-2.1) -1

Endothelial factors Factor VIII 70-175 IU/dl 194 (174-248) 94**VWF:Ag 45-175 IU/dl 288 (240-356) 184***

Anticoagulant activityProtein C 70-140 IU/dl 14 (8-28) -70***Protein S 66-126 IU/dl 41 (24-66) -30**Antithrombin III 45-175 IU/dl 39 (28-50) -61***

Data represent mean ± SEM, with negative numbers denoting inverse correlation.⁄⁄p <0.01, and ⁄⁄⁄p <0.001 indicate significant correlation.

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and the remaining 7 were hypercoagulable (R-time 7.3 ± 0.1, K-time 2.6 ± 0.2, Alpha-angle 57.4 ± 1.3 and MA 58.5 ± 5.2). Nofibrinolysis was observed in any patient until 30 min (Supple-mentary Table 1).

Endogenous heparinoids

An endogenous heparin-like effect (HLE) occurred in mostpatients, which was confirmed by the observation of a trendtowards improving R-time (15.46 ± 2.53, p = 0.43), K-time(8.01 ± 1.74, p = 0.41) and a-angle (37.29 ± 4.04, p = 0.56) in theheparinase modified TEG, and the presence of significantly ele-vated plasma anti-Xa concentrations (0.06 ± 0.01, p = 0.0002).

Coagulation factor levels

A significant decline occurred in nearly all factors, both procoag-ulant and anticoagulant factors, except endothelium derived fac-tors (factor VIII and VWF), which displayed a multiple foldincrease in plasma concentration (Table 2 and Fig. 1).

AnticoagulantsPC, PS and ATIII

ProcoagulantsFII, FV, FVII, FIX,

FX, FXI, FXII, Fibrinogen

Fold

c

-1

Fig. 1. Balanced relationship between reduced procoagulants and anticoag-ulants at admission to ICU with ALF. At admission (0 h), both pro andanticoagulants are significantly reduced to the same extent (see Table 2).Endothelial factors VWF and factor VIII are significantly elevated and remainelevated at 48 h post admission.

80

TEG-native

Platelet effect

TEG-Ffib

Fibrinogen (g/L)

MA

(mm

)

60

40

20

00 1 2 3

Fig. 2. Correlation between fibrinogen and TEG native versus functionalfibrinogen. Correlation graphs (with 95% CI) of fibrinogen compared to TEG-native (p = 0.081) and TEG-Ffib (p = 0.123) with functional fibrinogen accountingfor 25%, and platelets (blue line) for 75% of clot strength (MA).

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Research Article

Platelets and fibrinogen contribution to clot dynamics

On admission, patients displayed mild thrombocytopenia(140 ± 24 � 109/L) and an increased mean platelet volume10.9 fl ± 0.3 (normal = 9.8 fl). No direct correlation was observedbetween clot strength (MA) and platelet count. However, patientswith higher MA had higher platelet counts (135.0 ± 17.5 � 109/Lvs. 103.9 ± 15.5 � 109/L). More marked thrombocytopenia (plate-let count 6100) affected other TEG parameters (R-time, K-time,a-angle) also. A greater but statistically non-significant associa-tion was seen between MA and fibrinogen (p = 0.08). While thetotal MA in native TEG reflects contributions from both plateletsand fibrinogen, TEG-functional fibrinogen showed an obvious dif-ference in MA (Table 1 and Fig. 2), with functional fibrinogenaccounting for 25%, and platelets for 75% of clot strength; derivedby subtracting MA of TEG-Ffib from MA TEG-native.

Thrombin generation data

There was no significant correlation between PT/PTR and throm-bin generation parameters. When compared to healthy controls,the endogenous thrombin potential (ETP, the total amount ofthrombin that the test plasma can generate using the conditionsspecified) and the peak height (PH) to generate thrombin weresignificantly lower in ALF patients (p <0.0001 for both parame-ters), but in the presence of Protac� (a protein C activator), theETP and ETP% (representing percentage inhibition of ETP in thepresence of Protac�) were significantly higher than the corre-sponding healthy controls (p <0.05 and p <0.001, respectively);this reflects the relative protein C deficiency in ALF patients.Lag-time (LT, time to first thrombin formation) was longer inALF patients (p <0.01), with no correction following addition ofProtac�. The ALF group generated thrombin faster than the nor-mal cohort, as evidenced by the increase in the velocity indexof thrombin generation (p = 0.048) whilst the time to peak (TP)thrombin generation was similar for both groups (p = 0.068)(Table 3; Fig. 3).

Microparticle data

Microparticle activity as assessed using a functional MP-activityassay was clearly significantly increased in ALF patients when

Table 3. Relative changes in thrombin generation parameters of ALF patients comp

TG parameters Healthy cETP (nM.min) 1602 (12ETP + P (nM.min) 488 (300ETP% 29 (22-3Lag-time (sec) 2.5 (2.3-Time-to-peak (min) 5.8 (5.2-Peak height (nM) 278 (221Slope (velocity index) nM.min 91 (69-1Microparticles (PPL assay) sec 68 (55-7Microparticles (activity assay) nM 4.0 (3.2-

Data represent Mean ± SEM of thrombin generation parameters between plasma of heSymbols represent: ⁄p <0.05, ⁄⁄p <0.01, ⁄⁄⁄p <0.001 compared to data from healthy contr

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compared to the normal cohort (p <0.0001). This was confirmedby using the procoagulant phospholipid assay (PPL), whichshowed significantly shorter clotting times in the ALF group thanin normal volunteers (p <0.05) (Fig. 3).

Discussion

The liver synthesizes almost all clotting factors and their inhibi-tors, though the endothelium also acts as an additional source ofFVIII while also synthesizing VWF. PT and its derivative PTR havebeen used to assess coagulability of plasma in liver disease sincethe time of the test’s inception by Quick [13]. As PT is onlyaffected by five clotting factors (factors VII, FX, FV, FII and fibrin-ogen), it does not take into account anticoagulant pathways andeffects of platelet and endothelial contributions. In the last dec-ade, global assays of coagulation (utilising TEG and TG) havereceived much attention as they, at least in theory, represent animprovement over standard plasmatic coagulation tests, byincorporating the effects of blood cells (in TEG assays) and coag-ulation proteins present in platelet poor plasma for TG assess-ment. Our group and others [4,1] have shown thrombingeneration capacity to more accurately reflect the in vivo milieuof liver disease, leading to the observation that patients with cir-rhosis exhibit normal to even exaggerated coagulation. The cur-rent study shows that this observation also holds true forpatients with ALF and confirms that although traditional coagula-tion tests such as PT may reflect the degree of liver failure, theydo not provide a true reflection of the coagulation balance inthese patients, an observation that is in keeping with data fromthe US liver failure group. Our study in addition, and for the firsttime, explored other potential haemostatic mechanisms bydescribing thrombin generation, role of endogenous heparinoids,microparticles and the relative role of platelets and fibrinogen inmodulating the coagulation disturbances in patients with ALF. Asin cirrhosis, patients with ALF exhibit a similar but much magni-fied reduction in pro- and anticoagulant proteins, and elevationof factors VIII and VWF levels. Although the overall thrombingeneration potential is reduced in ALF, these patients, like thosewith cirrhosis, demonstrate brisker thrombin production oncethe small initial amount of thrombin required to activate factorsVIII, IX, and XI is formed, which then leads to rapid thrombin

ared to healthy controls at admission.

Median + IQRontrols ALF patients89-1891) 932 (608-1211)***-572) 724 (461-996)**6) 84 (65-97)***2.8) 3.4 (2.8-4.2)**6.1) 4.6 (4.0-5.7)*-319) 132 (98-198)***10) 122 (39-195)*3) 56 (45-70)*5.3) 16 (11-26)***althy controls versus ALF patients. Statistical significance is defined by p <0.05.ols.

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150

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*** ***

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A B

C D

E F

G H

Fig. 3. Thrombin generation and microparticle studies. Graphs depicting TGparameter differences between ALF patients and healthy controls. (A) Endoge-nous thrombin potential percentage (ETP), (B) ETP percentage (ETP%) =[(ETP + protac/ETP-protac) � 100], (C) Lag phase (LT), (D) peak height (peak), (E)time-to-peak (TP), (F) slope (velocity index) [slope = PH/(TP – Lag-time)] andmicroparticles using both (G) PPL (secs) and (H) TG-activity assays (nM)).Statistical significance is defined by p <0.05. Symbols represent: ⁄p <0.05,⁄⁄p <0.01, and ⁄⁄⁄p <0.001 compared to data from healthy controls.

JOURNAL OF HEPATOLOGY

production due to increased availability of factor VIII. Rapidthrombin generation, along with reduced thrombin inactivationsecondary to activated protein C resistance, shown by the mini-mal change in ETP in presence of Protac�, enhances clot exten-sion and thrombotic potential.

The microparticles (MP) are small cell fragments present inthe blood, derived mostly from platelets and monocytes, but alsofrom other cells also including erythrocytes and even tumourcells [14]. Since they are blebs arising from the cell surface, theypossess a procoagulant activity due to the active phospholipidslike phosphatidylserine that they carry. Microparticles derived

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from monocytes are also known to carry tissue factor and are ableto initiate and support coagulation [15]. An increase in micropar-ticles has been demonstrated in various thrombotic scenariossuch as acute coronary syndrome and cancer [16,17]. However,here, we show for the first time that this is true in ALF as well,thus contributing to thrombotic potential in these patients.

Increased platelet size in acute liver failure, as evidenced byan elevated mean platelet volume, suggests an increased turn-over of platelets with ‘younger’ and more reactive plateletsreleased in the circulation acting to preserve platelet biomass,and, in conjunction with increased microparticles, probably cir-cumvent the effects of thrombocytopenia. This hypothesis is sup-ported by our TEG data on whole blood (containing platelets)showing a large majority of patients as hypercoagulable. Largerplatelets have also been shown to be linked with other throm-botic states such as venous thromboembolism, acute pulmonaryembolism, and stroke patients [18–20].

Platelets have to be supported by fibrinogen, and these aretwo key players in determining clot strength. Fibrinogen bindsto the platelet surface via GPIIb/IIIa receptors to induce plateletaggregation, and acts as a precursor to fibrin, which increasesthe clot meshwork tensile strength. While in health, plateletsand fibrinogen contribute almost equally to clot strength (55%vs. 45% respectively [11]), the TEG-functional fibrinogen analysisrevealed that, in ALF, functional fibrinogen accounted for only25%, and platelets 75% of clot strength, despite a normal serumfibrinogen level and a general thrombocytopenic state, indicatinga state of relative ‘functional’ hypofibrinogenaemia and adisproportionately stronger platelet-effect on clot strength inALF patients, independent of platelet count.

In contrast to cirrhosis [4], there is significant reduction in thefibrinolysis potential in ALF, probably related to elevated levels ofplasminogen activator inhibitor-1 (PAI-1), as previously shown[21]. Reduced fibrinolysis and lack of hyperfibrinogenaemiaexpected in acute inflammation and reduced functional fibrino-gen in ALF explain some of the underlying mechanisms involvedin our observation of a state of preserved haemostasis.

A heparin-like effect in ALF, also shown previously in ALFpatients undergoing liver transplantation [22], and confirmedagain in this study, possibly reflects release of heparan sulphatefrom the damaged vascular endothelium, heparin from the dam-aged liver, and reduced renal clearance of endogenous hepari-noids. Heparinoids through the binding of antithrombin inhibitthrombin and factor X activity, limiting cleavage of fibrinogento form fibrin, thus delaying clot formation and leading to bleed-ing risk. Heparinase-modified TEG therefore can be a valuableadjunct in the assessment of coagulopathy related to ALF [23].

In conclusion, the commonly held clinical perception of ableeding diathesis with progressive ALF, as indicated by standardclotting tests (especially the PT), is not substantiated by the morecomprehensive assessment of clot dynamics using TEG andthrombin generation. These assays do not correlate with PT anddemonstrate a more balanced coagulation state in ALF. The under-lying mechanisms responsible for this seemingly paradoxical andbalanced coagulation state in patients with ALF are likely to reflectinterplay of various simultaneously activated pro haemostatic(reduced natural anticoagulants – proteins C and S, and anti-thrombin, elevated factors VIII and VWF, lack of fibrinolysis,increased microparticles, and larger platelets) and anti haemo-static (reduced production of liver derived procoagulant factors,thrombocytopaenia, reduced fibrinogen activity and endogenous

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Research Article

heparin like effect) pathways. This does not, however, apply to allpatients and whilst our data suggests a more judicious rather thana ‘blanket’ approach to the use of blood products to correct ‘appar-ent’ coagulopathy in ALF, it underscores the importance of furtherstudies in replacing PT with a combination of global assays ofcoagulation to assess bleeding risk in these patients.

Author’s contribution

BA, project conception and supervision, data collection, manu-script preparation and revision; AKB, project conception andsupervision, manuscript revision; GW, data analysis, manuscriptpreparation and revision; AG, project conception, data analysis,manuscript revision; AR, laboratory analyses (assays and meth-ods), statistical analysis, manuscript revision; AD, data analysis,manuscript revision, renal aspects; SM, thromboelastographyanalysis; PC, project conception, manuscript revision; RJ, manu-script revision; and VV, patient recruitment, data collection. Allauthors contributed to manuscript review and revision.

Conflict of interest

The authors who have taken part in this study declared that theydo not have anything to disclose regarding funding or conflict ofinterest with respect to this manuscript.

Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.jhep.2012.06.020.

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