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In vitro efficacy of pro‐ and anticoagulant strategies in compensated and acutely ill patients with cirrhosis

In vitro efficacy of pro‐ and anticoagulant strategies in compensated and acutely ill patients... Section of Hepatobiliary Surgery and Liver Transplantation, Department of Background & Aims: A simultaneous decline in pro- and anticoagulant drivers in pa- Surgery, University of Groningen, University tients with liver diseases results in a “rebalanced” haemostatic system, even in acutely Medical Center Groningen, Groningen, The Netherlands ill patients. Nevertheless, both bleeding and thrombotic events are common. Here, we Liver Intensive Care Unit, Institute of Liver explored efficacy of pro- and antihaemostatic strategies in compensated and acutely Studies, King College Hospital, London, UK ill cirrhotics which may be unpredictable given the profound haemostatic changes. Institute of Liver Studies, King College Hospital, London, UK Methods: We tested the effects in vitro of the addition of clinically relevant doses of commonly used pro- and antihaemostatic strategies in plasma from healthy individu- Correspondence Ton Lisman, Department of Surgery, als (n = 30) and patients with compensated (n = 18) and acutely decompensated cir- University Medical Center Groningen, rhosis (n = 18), and acute- on- chronic liver failure (n = 10). We used thrombin generation Groningen, The Netherlands. Email: j.a.lisman@umcg.nl tests and fibrin clot permeability assays to assess potency of various approaches. Results: Fresh frozen plasma and recombinant factor VIIa modestly increased throm- Funding information This study was funded in part by the Tekke bin generation (10%- 20%). Prothrombin complex concentrate increased thrombin Huizinga Foundation (The Netherlands). generation two- fold in controls and 2- 4- fold in patients. Clot permeability decreased Handling Editor: Juan Abraldes after addition of fibrinogen concentrate by 51% in controls and by 50%- 60% in pa- tients. Low molecular weight heparin decreased thrombin generation by 18% in con- trols and by 23%- 54% in patients. Similarly, dabigatran decreased thrombin generation by 33% in controls and by 47%- 100% in patients. In contrast, rivaroxaban decreased thrombin generation by 55% in controls, but only by 11%- 38% in patients. Conclusions: These in vitro data suggest little prohaemostatic effect of fresh frozen plasma and recombinant factor VIIa in acutely ill cirrhotics, whereas prothrombin complex concentrate and fibrinogen concentrate clearly improved haemostasis. Furthermore, our data suggest the requirement for dose adjustments of commonly used anticoagulants in these patients. K E Y WO R D S bleeding, cirrhosis, haemostasis, thrombosis Abbreviations: ACLF, acute of chronic liver failure; AD, acute decompensation; ETP, endogenous thrombin potential; FFP, fresh frozen plasma; LMWH, low molecular weight hepari; PCC, prothrombin complex concentrate; TM, thrombomodulin This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2018 The Authors. Liver International Published by John Wiley & Sons Ltd 1988     wileyonlinelibrary.com/journal/liv Liver International. 2018;38:1988–1996. LISMAN et AL.       1989 1  |   I NTR O D U C TI O N Key points Patients with liver diseases frequently acquire substantial alterations • Bleeding and thrombotic events are common in patients in their haemostatic system. A simultaneous decline in pro- and an- 2-4 with liver disease, but there is uncertainty on optimal ticoagulant drivers results in a “rebalanced” haemostatic system. management strategies External factors may tip the balance towards hypo- or hypercoagu- 5 6-10 11-14 • Efficacy of pro- and anticoagulant agents are likely al- lability, and distinct hypo- and hypercoagulable features in tered in patients with liver disease as a result of pro- patients with liver disease which may predispose them to bleeding or found changes in their haemostatic system thrombotic complications. • Using plasma samples from patients with compensated Bleeding and thrombosis are not uncommon in patients with cirrhosis, acute decompensation of cirrhosis or acute- liver disease, in particular in those with advancing and decompen- on-chronic liver failure we demonstrate little prohaemo - sated illness. However, little evidence- based treatment strategies static effect of fresh frozen plasma and recombinant for prevention or treatment of bleeding or thrombosis are available. factor VIIa, whereas prothrombin complex concentrate Current expert recommendations propose a very restrictive prophy - and fibrinogen concentrate clearly improved lactic prohaemostatic management, and a more active anticoagulant 5,16,17 haemostasis approach. Importantly, anticoagulant therapy in patients with • Commonly used anticoagulants have profoundly altered cirrhosis is challenging, and new generation anticoagulant drugs anticoagulant potency in critically ill cirrhotics have not been extensively studied in patients with cirrhosis, al- 19,20 though the clinical use in these patients is increasing. Decompensation may tip the haemostatic balance of patients with liver disease towards a bleeding phenotype. A recent study With the aim to provide a more rational approach to pro- and has identified thrombocytopenia (with a platelet count <30 000 μ L) antihaemostatic treatment of acutely ill patients with cirrhosis, and and hypofibrinogenemia (<0.6 g/L) as independent risk factors for to facilitate design of future clinical studies, we tested the in vitro bleeding in acutely ill patients with cirrhosis. It is, however, un - effects of commonly used pro- and antihaemostatic strategies in known whether there is a causal link between bleeding risk and plasma from patients with acutely decompensated cirrhosis and these laboratory abnormalities, and studies assessing whether re - acute- on- chronic liver failure. versal of thrombocytopenia and/or hypofibrinogenemia decrease the bleeding risk in these patients will be required to ascertain this. 2  |  M AT E R I A L S A N D M E T H O DS It is unknown whether acutely ill patients with cirrhosis are at risk for development of venous thrombosis similar to the increased 2.1 | Patients risk in well compensated patients. Nevertheless, prophylactic or therapeutic antithrombotic strategies may be required in acutely The study was performed at King’s College Hospital, a 950- bed ter- ill patients with cirrhosis, in the context of venous thrombosis, tiary hospital in London, United Kingdom, between August 2013 and portal vein thrombosis, and thrombosis of extracorporeal assist August 2015. The study was approved by NRES Committee London- 15,23 devices. Westminster, Study Number 12/LO/1417. Informed consent or assent We have recently studied the haemostatic status of patients with was obtained from participants or their personal consultees. Details acutely decompensated or acute- on- chronic liver failure and found on patient recruitment and blood sampling have been published pre- 24 24 a remarkably preserved haemostatic system. The relatively well- viously. In short, patients were sampled on admission and patients preserved haemostatic balance therefore suggests that a defensive were only excluded when currently using antihaemostatic agents. prohaemostatic and a proactive antihaemostatic approach may be From the published cohort we studied 30 healthy volunteers, 18 pa- warranted in these patients. tients with acute decompensation (AD) of cirrhosis and 10 patients Given the major alterations in the haemostatic system of pa- with acute- on- chronic liver failure (ACLF). Eighteen patients with well tients with cirrhosis, the efficacy of pro- and antihaemostatic strat- compensated cirrhosis were newly recruited in the outpatient clinic, egies may be unpredictable. We have previously demonstrated that and were not using antihaemostatic drugs at the time of sampling. in patients with compensated cirrhosis the in vitro anticoagulant ef - Acute decompensation of chronic liver disease and ACLF were de- fects of some of the commonly used drugs was decreased, whereas fined and graded according to number of organ failures in concord- the anticoagulant effects of others were increased as compared to ance with criteria reported in the CANONIC study. From the 10 25,26 anticoagulant effects in healthy individuals. Another study has patients with ACLF, 1 was classified as grade 1 and 9 were grade 3. shown a lack of prohaemostatic effect of in vitro addition of fresh frozen plasma to plasma from patients with compensated cirrho- 2.2 | Routine laboratory tests sis, despite improvements in plasma levels of coagulation factors. Similarly, transfusion of platelets to patients with cirrhosis did in - Haemoglobin, white blood cell count, albumin, creatinine, bilirubin, crease the platelet count, but did not improve global haemostasis. aspartate transaminase, alanine transaminase and gamma- glutamyl LISMAN et AL. 1990       transpeptidase were measured in the diagnostic laboratory of King’s Thrombinoscope BV, Maastricht, the Netherlands) was dispensed in College Hospital for routine clinical care. International normalized each well to allow a continuous registration of thrombin generation. ratios, and plasma levels of fibrinogen, antithrombin, factor II, fac- Fluorescence was read in time by a fluorometer, Fluoroskan Ascent tor VIII and factor X were measured in stored frozen samples on an (ThermoFisher Scientific, Helsinki, Finland). All procedures were un- automated coagulation analyzer (ACL 300 TOP) with reagents and dertaken according to the protocol suggested by Thrombinoscope B.V. protocols from the manufacturer (Werfen, Breda, The Netherlands). The pro- or anticoagulant potency of the different agents was expressed as the percentual change in endogenous thrombin po - tential (ETP), lag time, peak or velocity index after addition of the 2.3 | In vitro addition of pro- and anticoagulants study agent. These percentages were compared between patients We added the following agents to plasma samples of each patient and controls. and control: 2.5 | Fibrin concentration and fibrin permeability • Recombinant factor VIIa (Novo Nordisk, Bagsvaerd, Denmark)- final concentration 50 nmol/L Fibrinogen levels in plasma of patients and healthy volunteers • Cofact (a 4-factor prothrombin complex concentrate (PCC), were determined on an ACL TOP 300 analyzer using reagents from Sanquin, Amsterdam, Netherlands)-final concentration 0.5 U/mL Instrumentation Laboratory (Breda, the Netherlands) according to • Pooled normal plasma (to mimick fresh frozen plasma [FFP] trans- the manufacturer’s instructions. fusion-obtained by combining plasma from >200 healthy volun- The average pore size of the fibrin clot (expressed as the Darcy teers, a generous gift from Dr. J.C. Meijers, Academic Medical constant, Ks) was determined in permeation studies as previously Center Amsterdam, the Netherlands)-final concentration 20% (v/v) described. In short, plasma samples (100 μL) were incubated with • Fibrinogen concentrate (CSL Behring, Marburg, Germany)-final 10 μL of activation buffer (final concentration of 1 IU/mL thrombin, concentration 1 g/L 20 mmol/L CaCl , in tris- buffered saline, pH 7.5) to generate clots. After • Rivaroxaban, a direct factor Xa inhibtor (Alsachim, Illkirch mixing, 100 μL was immediately transferred to a 4.5- cm plastic tip with Graffenstaden, France)-final concentration 25 ng/mL a roughened interior surface, which was cut off from a 1 mL serological • Dabigatran, a direct thrombin inhibitor (Alsachim, Illkirch pipette (Corning Costar Stripette; Sigma- Aldrich, St Louis, MO, USA), Graffenstaden, France)-final concentration 300 ng/mL and left for 2 hours in a moist chamber at room temperature to conso- l • The low molecular weight heparin (LMWH) Clexane (Sanofi- idate. The plastic tip was then connected through a flexible silicon tube Aventis BV, Gouda, the Netherlands)-final concentration 0.2 U/mL to a syringe containing TBS with a 4- cm pressure drop. After a wash- out period of 90 minutes with tris- buffered saline, preweighed tubes Plasma levels of procoagulant drugs were chosen to represent clin - were attached to the clotting tip, and tris- buffered saline drops passing ically relevant (peak) levels observed in clinical use in the general pop- through the clot were collected every 30 minutes for 2 hours. The total ulation. The final concentrations of the anticoagulant drugs were also volume of liquid passing through the clot was weighed after collection. chosen to represent clinically relevant plasmas levels and were identi- Permeation of tris- buffered saline through the clot was quantified ac- cal to levels used in previously published experiments. Importantly, cording to the flow rate and the following equation, Ks = (Q × L × η)/T drug concentrations which gave appreciable (but not maximal) inhibi- × A × P, where Ks = Darcy’s constant, Q = volume of liquid (mL), L = clot tion of thrombin generation in pooled normal plasma were selected length (cm), η = viscosity (poise), T = time (s), A = cross- sectional area of so it would be possible to detect both increased and decreased drug the clot (cm ), and P = pressure drop (dyne/cm). effects in patients compared to controls. 2.6 | Statistical analyses 2.4 | Thrombin generation Data are expressed as means (with standard deviations (SDs)), me- The thrombin generation test was performed using platelet- poor dians (with interquartile ranges), or numbers (with percentages) plasma with the fluorimetric method described by Hemker, as appropriate. Multiple groups were compared using One- way Calibrated Automated Thrombography in absence or presence of ANOVA (with the Tukey’s post- test) or Kruskal- Wallis H test (with the above- mentioned agents, except for fibrinogen concentrate. Dunn’s post- test) as appropriate. P values of .05 or less were consid- Coagulation was activated using commercially available reagents ered statistically significant. GraphPad Prism (San Diego, USA) and containing recombinant tissue factor (final concentration 5 pmol/L), GraphPad Instat (San Diego, USA) were used for analyses. phospholipids (final concentration 4 μmol/L), in the presence of soluble thrombomodulin (TM, the concentration of which is not revealed by the manufacturer). These reagents were purchased 3  |   RE S U LT S from Thrombinoscope BV, Maastricht, the Netherlands. Thrombin Calibrator (Thrombinoscope BV) was added to calibrate the throm- We studied the effects of ex vivo addition of commonly used bin generation curves. A fluorogenic substrate with CaCl (FluCa-k it, pro- and anticoagulant drugs in patients with compensated and 2 LISMAN et AL.       1991 decompensated cirrhosis, and in patients with acute- on- chronic liver velocity index increased substantially more in patients compared to failure and compared results with those obtained in healthy controls. controls. The lag time did not appreciably change in either controls Table 1 summarises baseline characteristics of patients and controls. or patients (Table 3, Figure 1). Without the addition of pro- or anticoagulant agents, patients Addition of pooled normal plasma (20% v/v) increased total generated more thrombin as compared to controls, using TM- thrombin generation in controls by 16%, with very similar changes in modified thrombin generation tests, which is in line with our previ- compensated and AD patients (9%- 19% increase). Addition of pooled ously published data (Table 2). normal plasma to samples of patients with ACLF led to a more pro- When recombinant factor VIIa (50 nmol/L) was added to plasma found increase in thrombin generation (38% increase), but given the of healthy individuals, an increase of ~20% in ETP, peak, and velocity lower baseline thrombin generation levels in the ACLF group, total index was observed, with an ~20% decrease in lag time. In patients, thrombin generation after addition of pooled normal plasma was however, addition of recombinant factor VIIa did not appreciably comparable between AD and ACLF patients (913 [841- 980] vs 883 change ETP, peak, and velocity index, whereas the lag time was [757- 993] nmol/L IIa × min) - Table 3, Figure 1. shortened to a similar extent as in controls (Table 3, Figure 1). The permeability of clots generated from plasma from healthy Addition of the PCC Cofact (0.5 U/mL) to plasma of healthy controls was remarkably similar to that of the permeability of clots individuals resulted in an approximate doubling of total thrombin from patients, despite the lower fibrinogen levels in patient plasma. generation (to 829 [697- 1141] nmol/L IIa × min). An exaggerated re- When fibrinogen concentrate (1 g/L) was added to control samples, sponse was observed in plasma from patients with a 110% increase a 51% reduction in permeability was observed. A similar effect of in the compensated cirrhosis group (to 1525 [1052- 1697] nmol/L IIa fibrinogen concentrate was observed in patients compensated cir- × min), a 150% increase in thrombin generation in the AD group (to rhosis, whereas a slightly more robust effect was observed in plasma 1869 [1734- 2210] nmol/L IIa × min) and a 270% increase in the ACLF from AD and ACLF patients with a 61% reduction in permeability in group (to 2383 [1731- 3538] nmol/L IIa × min). Similarly, the peak and the AD group and a 63% reduction in the ACLF group (Table 4). TABLE 1  Patient characteristics Compensated Controls (n = 30) (n = 18) AD (n = 18) ACLF (n = 10) Age 37 ± 7 60 ± 13 54 ± 14 56 ± 10 Gender (male, %) 15 (50%) 12 (67%) 5 (50%) 13 (72%) CLIF- SOFA n/a n/a 4 ± 2 10 ± 4 MELD n/a 9 ± 2 16 ± 9 31 ± 9 Reason for n/a n/a Ascites n = 4 Sepsis n = 4 decompensation Variceal bleeding n = 4 Variceal bleeding n = 4 Encephalopathy n = 10 SBP n = 2 Haemoglobin (g/L) n/d 145 (113- 145) 111 (93- 128) 95 (77- 116) WBC (× 10 /L) n/d 5 (4- 6) 6 (3- 9) 10 (8- 19) Platelets (× 10 /L) n/d 119 (87- 150) 95 (65- 132) 89 (65- 111) Albumin (g/L) n/d 41 (39- 45) 33 (30- 37) 28 (26- 33) Creatinin (μmol/L) n/d 82 (70- 89) 67 (56- 93) 135 (94- 224) Bilirubin (μmol/L) n/d 19 (11- 25) 44 (30- 71) 362 (116- 493) AST (U/L) n/d 42 (37- 45) 47 (38- 56) 79 (50- 86) ALT (U/L) n/d 36 (24- 43) 25 (18- 30) 46 (24- 61) GGT (U/L) n/d 60 (33- 143) 77 (47- 135) 37 (30- 91) , , INR 1.0 ± 0.1 1.1 ± 0.1 1.6 ± 0.4** *** 2.1 ± 0.4** **** , , Fibrinogen (g/L) 2.8 ± 0.4 3.3 ± 0.7 2.2 ± 0.9* *** 1.3 ± 0.3** **** , , Antithrombin (%) 107 ± 11 88 ± 26* 46 ± 18** *** 28 ± 14** **** FII (%) 101 ± 18 78 ± 17** 48 ± 16** *** 31 ± 10** FVIII (%) 99 ± 36 157 ± 42** 146 ± 27* 212 ± 93** **** FX (%) 105 ± 23 85 ± 21* 57 ± 20** *** 41 ± 15** ALT, alanine transaminase; AST, aspartate transaminase; CLIF- SOFA, Chronic Liver Failure- Sequential Organ Failure Assessment; F, factor; GGT, gamma- glutamyl transpeptidase; INR, international normalised ratio; MELD, model for end- stage liver disease; n/a, not applicable; n/d, not determined; SBP, spontaneous bacterial peritonitis; WBC, white blood cell. Shown are means ± standard deviation or medians (interquartile range). *P < .05 vs control, **P < .01 vs control, ***P < .01 vs compensated, ****P < .01 vs AD. LISMAN et AL. 1992       TABLE 2  Thrombin generation ETP (nmol/L IIa Vel index (nmol/L parameters of thrombomodulin- modified × min) Peak (nmol/L IIa) Lag time (min) IIa × min) thrombin generation testing in controls Controls 440.0 122.8 (93.9- 180.3) 1.67 (1.46- 1.98) 61.9 (46.3- 96.5) and patients with compensated (CC) or (343.4- 639.8) acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) CC 699.5 184.5 (152.0- 211.3) 1.67 (1.33- 1.67) 99.0 (75.8- 111.0) (538.5- 819.3) AD 736.3 182.3 (173.2- 215.6)** 1.33 (1.33- 1.63)* 102.4 (696.2- 882.3)**  (90.3- 112.1)** ACLF 648.1 124.5 (86.5- 144.6) 1.92 (1.67- 2.00) 60.3 (43.4- 75.0) (473.3- 743.7) Shown are medians (interquartile ranges). *P < .05, **P < .01 vs controls. TABLE 3  Percentual changes in ETP Peak Lag time Vel index thrombin generation parameters of rFVIIa thrombomodulin- modified thrombin generation testing in controls and patients Controls 21 (8- 32) 20 (8- 32) 28 (20- 34) 20 (11- 42) with compensated (CC) or acutely CC 3 (0- 16)* 2 (−3 to 9)** 21 (20- 30) 1 (−6 to 2 0)* decompensated cirrhosis (AD), or AD 2 (−1 to 5 )*** −1 (−3 to 3 )*** 24 (17- 37) 0 (−10 to 5 )*** acute- on- chronic liver failure (ACLF) after in vitro addition of prohaemostatic agents ACLF 3 (−3 to 27)* −1 (−4 to 19)* 20 (15- 25)* 3 (−6 to 2 5) Cofact Controls 92 (788- 101) 72 (46- 89) 0 (0- 17) 72 (38- 93) CC 109 (82- 149) 89 (55- 102) 0 (0- 0) 78 (55- 104) AD 151 (135- 197)*** 109 (87- 150)** 0 (−13- 6) 97 (75- 136)* ACLF 273 (212- 393)*** 176 (141- 219)*** 10 (0- 17) 178 (156- 265)*** Pooled normal plasma Controls 16 (2- 21) 15 (−3- 19) 0 (−10- 0) 15 (−4- 19) CC 7 (−1- 21) 7 (0- 18) 20 (0- 26) 6 (−4- 18) AD 19 (8- 29) 23 (10- 33) −11 (−25- 0) 22 (12- 31) ACLF 38 (27- 69) *** 56 (47- 124) *** 0 (−9- 9) 59 (45- 117) *** Shown are the percentual increase of the ETP, peak, or velocity index, and the percentual decrease in the lag time. Data are expressed as median percentages with interquartile rangeP .* < .05, **P < .01, ***P < .001 vs controls. Addition of rivaroxaban (25 ng/mL) to plasma from healthy con- generated any thrombin in the presence of dabigatran (Table 5, trols resulted in a 55% decrease in the ETP (to 178 [152- 339] nmol/L Figure 2). IIa × min), with similar changes in peak and velocity index. The re- LMWH (0.2 U/mL) decreased the ETP by 18% in controls (to 374 duction in total thrombin generation was less profound in patients, [291- 471] nmol/L IIa × min), with similar changes in peak and velocity particularly in the AD group in which the ETP was only reduced by index. The decrease in the ETP was more pronounced in patients 11% (to 629 [527- 692] nmol/L IIa × min) with a 25% reduction in the with a 23% decrease in the compensated group (to 526 [425- 632]), ACLF group (to 465 [367- 570] nmol/L IIa × min), and a 38% reduc- a 41% decrease in the AD group (to 463 [390- 594] nmol/L IIa × min) tion in the compensated group (to 403 [271- 510] nmol/L IIa × min). and a 54% decrease (to 311 [213- 403] nmol/L IIa × min) in the ACLF Although the reduction in thrombin generation was substantially group (Table 5, Figure 2). However, given the higher baseline ETP in decreased in patients, the lag time was substantially more prolonged patients, the ETP in the presence of LMWH were similar between in patients compared to controls, particularly in the ACLF group patients and controls. (Table 5, Figure 2). Addition of dabigatran (300 ng/mL) decreased total thrombin generation in controls by 33% (to 333 [221- 539] nmol/L IIa × min), 4  |  D I S CU S S I O N and prolonged the lag time more than 6- fold. In patients, total throm- bin generation was decreased by 47% in the compensated group (to Here we have studied the in vitro effects of commonly used pro- and 308 [256- 401] nmol/L IIa × min), by 75% in the AD group (to 171 [63- anticoagulant strategies in compensated and acutely ill patients with 229] nmol/L IIa × min), and none of the patients in the ACLF group cirrhosis in comparison to healthy individuals and found remarkable LISMAN et AL.       1993 FIGURE 1  Absolute ETP values from thrombomodulin- modified thrombin generation testing in controls and patients with compensated or acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) prior to and after in vitro addition of prohaemostatic agents. Indicated are percentual changes in the ETP after addition of procoagulant agents. Shown are medians with interquartile ranges . PNP, pooled normal plasma TABLE 4  Fibrinogen levels and fibrinogen permeability in controls and patients with compensated (CC) or acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) in the absence and presence of fibrinogen concentrate Permeability + 1 g/L fibrinogen Percentual decrease Fibrinogen (g/L) Permeability (Ks) concentrate (Ks) in permeability −9 −9 −9 −9 Controls 2.8 ± 0.4 9.2 × 10 ± 3.2 × 10 4.2 × 10 ± 1.0 × 10 51 ± 11 −9 −9 −9 −9 CC 3.3 ± 0.7 9.0 × 10 ± 4.0 × 10 4.1 × 10 ± 2.0 × 10 52 ± 11 −9 −9 −9 −9 AD 2.2 ± 0.9* 8.8 × 10 ± 3.6 × 10 3.7 × 10 ± 1.1 × 10 61 ± 15 * −9 −9 −9 −9 ACLF 1.3 ± 0.3** 10.9 × 10 ± 5.1 × 10 4.0 × 10 ± 1.8 × 10 63 ± 17* Data are expressed as means ± standard deviation. *P < .05, **P < .001 vs controls. differences in the potency of these strategies between patients and bleeding during liver transplantation. However, based on our data, controls. Notably, the in vitro efficacy of the procoagulant PCC and dosing of PCCs should be performed conservatively in acutely ill pa- the anticoagulants dabigatran and LMWH increased with increas - tients with cirrhosis given the profoundly exaggerated procoagulant ing severity of disease, and the efficacy of rivaroxaban was lower response in patients compared to controls. Similarly, our data sug- in patients although not proportional to the severity of disease. The gest that substantial dose- adjustments may be required when low increase in efficacy of PCC, dabigatran, and LMWH with increasing molecular weight heparin or direct oral anticoagulants are consid - severity of disease mirror the haemostatic changes that are more ered for prophylaxis or treatment of thrombotic complications in profound in the sicker patients. The results of this study may have acutely ill patients with cirrhosis. clinical relevance as it suggests a requirement for dose- adjustments The lack of a procoagulant effect of recombinant factor VIIa for a number of agents tested, and suggests some agents to be clini- in patients with cirrhosis suggests competent tissue factor- cally ineffective in these patients. Specifically, the lack of a procoag- mediated activation of coagulation in these patients, and is also ulant effect of recombinant factor VIIa in patients, and the minimal in line with a ver y modest increase in thrombin generation follow- effect of FFP in the AD group are in line with the lack of evidence ing administration of recombinant factor VIIa to healthy volun - supporting the use of recombinant factor VIIa in patients with liver teers. However, as recombinant factor VIIa also has important 31-33 37 diseases, and with the doubts on the use of FFP in patients with tissue factor- independent procoagulant activity, it cannot be liver disease in general. Our data suggest a procoagulant approach presumed that this drug does not have some prohaemostatic ef - with PCCs and or fibrinogen concentrate to substantially improve fect in acutely ill patients with cirrhosis in vivo. It has been pre - haemostatic status in acutely ill patients with liver disease given the viously demonstrated that FFP has no appreciable effects on in profound procoagulant effects of PCC in thrombin generation tests vitro thrombin generation in plasma from patients with compen - and the improvement of fibrin clot structure by fibrinogen concen- sated cirrhosis, despite clear changes in plasma levels of coagu - trate. This strategy has been reported to be effective in managing lation factors and a decrease in the INR. Here we demonstrate LISMAN et AL. 1994       TABLE 5  Percentual decrease in thrombin generation parameters of thrombomodulin- modified thrombin generation testing in controls and patients with compensated (CC) or acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) after addition of anticoagulants ETP Peak Lag time Vel index Rivaroxaban Controls 55 (50- 63) 62 (56- 69) 34 (25- 46) 70 (62- 75) CC 42 (32- 50)* 53 (44- 57)* 60 (50- 100)*** 60 (44- 68)* AD 11 (10- 30)*** 19 (18- 35)*** 52 (41- 63)* 33 (27- 47)*** ACLF 25 (11- 35)** 43 (27- 52)** 70 (56- 87)*** 54 (43- 64)* Dabigatran Controls 33 (16- 48) 33 (20- 65) 571 (314- 711) 30 (5- 76) CC 50 (35- 63) 56 (41- 73) 455 (365- 617) 53 (29- 75) AD 75 (93- 73)*** 92 (84- 98)*** 483 (298- 614) 95 (87- 99)** ACLF No thrombin formed*** No thrombin formed*** No thrombin formed*** No thrombin formed*** LMWH Controls 18 (9- 28) 15 (6- 26) 0 (0- 10) 14 (−2- 32) CC 25 (16- 30) 15 (5- 27) 26 (17- 54) 8 (−4- 20) AD 41 (26- 48)** 26 (13- 30) 17 (7- 29)** 16 (1- 29) ACLF 54 (39- 61)*** 29 (24- 42)** 9 (0- 28) 25 (14- 41) Shown are the percentual decrease of the ETP, peak, or velocity index, and the percentual increase in the lag time. Data are expressed as median per- centages with interquartile range.*P < .05, **P < .01, ***P < .001 vs controls. FIGURE 2  Absolute ETP values from thrombomodulin- modified thrombin generation testing in controls and patients with compensated or acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) prior to and following addition of anticoagulant agents. Indicated are percentual changes in the ETP after addition of anticoagulant agents. Shown are medians with interquartile ranges comparable effects of pooled normal plasma (mimicking FFP individuals (from London, UK) and our pooled normal plasma transfusion) in plasma from healthy individuals and patients with (which was generated in Amsterdam, the Netherlands). Our data acutely decompensated cirrhosis, suggesting FFP to be ineffec - suggests FFP does have some haemostatic effect in patients with tive in AD patients. The slight increase in thrombin generation in ACLF. However, the drawback of FFP administration in these our control group is likely clinically insignificant and may reflect acutely ill patients is the risk of volume overload and exacerbation minor differences in coagulation profiles between our healthy of portal hypertension. LISMAN et AL.       1995 The substantially increased procoagulant capacity of PCCs in C O N FL I C T O F I N T E R E S T plasma from acutely ill patients with cirrhosis likely relates to the The authors do not have any disclosures to report. low baseline plasma levels of procoagulant proteins. The same dose of PCC thus results in a much larger relative increase in levels of the vitamin K- dependent factors in patients compared to controls. O RC I D Baseline thrombin generating capacity is elevated in patients com- Ton Lisman http://orcid.org/0000-0002-3503-7140 pared to controls, despite the much lower levels of procoagulants, Caleb Fisher http://orcid.org/0000-0003-3336-3898 as the levels of anticoagulant proteins are also low. Therefore, the William Bernal http://orcid.org/0000-0002-6508-3287 procoagulant effects of PCCs are much more prominent in patients compared to controls. The preserved fibrin clot structure in pa- tients is in line with our previous results showing preserved fibrin clot structure in patients with compensated cirrhosis to be related R EF ER EN C E S to oxidative modifications to the fibrinogen molecule. Fibrinogen 1. Lisman T, Leebeek FW, de Groot PG. Haemostatic abnormalities in concentrate profoundly decreases fibrin clot permeability in acutely patients with liver disease. J Hepatol. 2002;37:280-287. ill patients with cirrhosis, which is in line with our previous study 2. Lisman T, Porte RJ. Rebalanced hemostasis in patients with liver disease: evidence and clinical consequences. Blood. showing that fibrinogen concentrate normalizes fibrin clot permea- 2010;116:878-885. bility in samples taken from patients during liver transplantation. 3. Lisman T, Stravitz RT. Rebalanced hemostasis in patients with acute The increased anticoagulant potency of LMWH and pro - liver failure. Semin Thromb Hemost. 2015;41:468-473. foundly increased anticoagulant potency of dabigatran are in line 4. Tripodi A, Mannucci PM. The coagulopathy of chronic liver disease. 25,39 N Engl J Med. 2011;365:147-156. with previous studies in patients with compensated cirrhosis, 5. Lisman T, Caldwell SH, Burroughs AK, et al. Hemostasis and throm- and suggest a requirement for conservative dosing particularly bosis in patients with liver disease: the ups and downs. J Hepatol. of dabigatran in acutely ill patients with cirrhosis. Importantly, 2010;53:362-371. although it remains to be established whether dose- adjustments 6. Bedreli S, Sowa JP, Malek S, et al. Rotational thromboelastometry can detect factor XIII deficiency and bleeding diathesis in patients and/or monitoring of drug levels improves the anticoagulant with cirrhosis. Liver Int. 2017;37:562-569. management of acutely ill patients with cirrhosis, monitoring of 7. Kleinegris MC, Bos MH, Roest M, et al. Cirrhosis patients have a LMWH by anti- Xa testing is unreliable in patients with cirrhosis. coagulopathy that is associated with decreased clot formation ca- Thrombin generation tests are currently not ready for clinical use, pacity. J Thromb Haemost. 2014;12:1647-1657. 8. Narvaiza MJ, Fernandez J, Cuesta B, Paramo JA, Rocha E. Role of but the development of whole blood generation tests may result sialic acid in acquired dysfibrinogenemia associated with liver cir - in a point- of- care thrombin generation test which may be suitable rhosis. Ric Clin Lab. 1986;16:563-568. for anticoagulant monitoring, which might be used to monitor 9. Leebeek FW, Kluft C, Knot EA, de Maat MP, Wilson JH. A shift anticoagulant treatment in these difficult patients. Rivaroxaban in balance between profibrinolytic and antifibrinolytic fac- appears less effective in patients compared to controls which is tors causes enhanced fibrinolysis in cirrhosis. Gastroenterology. 1991;101:1382-1390. in line with studies in patients with compensated cirrhosis, and 10. Laffi G, Marra F, Gresele P, et al. Evidence for a storage pool de- dose increases may be required. However, importantly, although fect in platelets from cirrhotic patients with defective aggregation. rivaroxaban is less effective in patients as assessed by total throm - Gastroenterology. 1992;103:641-646. bin generation, it appears more effective compared to controls in 11. Hugenholtz GC, Mccrae FL, Adelmeijer J, et al. Procoagulant changes in fibrin clot structure in patients with cirrhosis are associ- prolonging the lag time of thrombin generation which was also ob- ated with oxidative modifications of fibrinogen. J Thromb Haemost. served in our previous study in compensated cirrhosis. 2016;15:1054-1066. Taken together, our results suggest a profound difference in 12. Raparelli V, Basili S, Carnevale R, et al. Low- grade endotoxemia and clinical efficacy of some of the commonly used pro- and antico- platelet activation in cirrhosis. Hepatology. 2017;65:571-581. 13. Saliola M, Lorenzet R, Ferro D, et al. Enhanced expression of agulant strategies in compensated and acutely ill patients with monocyte tissue factor in patients with liver cirrhosis. Gut. cirrhosis. Dose adjustments of some of these agents may be re - 1998;43:428-432. quired, either to improve efficacy or to decrease the risk of side 14. Lisman T, Bongers TN, Adelmeijer J, et al. Elevated levels of von effects. Although our studies have been performed using in vitro, willebrand factor in cirrhosis support platelet adhesion despite re - duced functional capacity. Hepatology. 2006;44:53-61. plasma- based systems, which do not take the role of blood cells 15. Roberts LN, Bernal W. Management of bleeding and thrombosis in haemostasis into account, these results may still be clinically in critically ill patients with liver disease. Semin Thromb Hemost. relevant. We propose that our study is the starting point for future 2015;41:520-526. preclinical and clinical studies that will further explore the need 16. Saner FH, Kirchner C. Monitoring and treatment of coagulation dis- orders in end- stage liver disease. Visc Med. 2016;32:241-248. for alternative dosing of pro- and anticoagulants in patients with 17. Northup PG, Caldwell SH. Coagulation in liver disease: a guide for cirrhosis. Our data may assist in development of urgently needed the clinician. Clin Gastroenterol Hepatol. 2013;11:1064-1074. clinical studies to assess safety and efficacy of strategies to pre - 18. Lisman T, Kamphuisen PW, Northup PG, Porte RJ. Established and vent or treat bleeding and thrombotic complications in these com - new- generation antithrombotic drugs in patients with cirrhosis - plex patients. possibilities and caveats. J Hepatol. 2013;59:358-366. LISMAN et AL. 1996       19. De Gottardi A, Trebicka J, Klinger C, et al. Antithrombotic treatment liver transplantation due to chronic liver disease. Liver Transpl. with direct- acting oral anticoagulants in patients with splanchnic 2005;11:895-900. vein thrombosis and cirrhosis. Liver Int. 2017;37:694-699. 33. Bosch J, Thabut D, Albillos A, et al. Recombinant factor VIIa for 20. Intagliata NM, Henry ZH, Maitland H, et al. Direct oral anti- variceal bleeding in patients with advanced cirrhosis: a randomized, coagulants in cirrhosis patients pose similar risks of bleed - controlled trial. Hepatology. 2008;47:1604-1614. ing when compared to traditional anticoagulation. Dig Dis Sci. 34. Shah NL, Intagliata NM, Northup PG, Argo CK, Caldwell SH. 2016;61:1721-1727. Procoagulant therapeutics in liver disease: a critique and clinical 21. Drolz A, Horvatits T, Roedl K, et al. Coagulation parameters and rationale. Nat Rev Gastroenterol Hepatol. 2014;11:675-682. major bleeding in critically ill patients with cirrhosis. Hepatology. 35. Kirchner C, Dirkmann D, Treckmann JW, et al. Coagulation man- 2016;64:556-568. agement with factor concentrates in liver transplantation: a single- 22. Ambrosino P, Tarantino L, Di Minno G, et al. The risk of venous center experience. Transfusion. 2014;54:2760 -2768. thromboembolism in patients with cirrhosis. A systematic review 36. Bijsterveld NR, Moons AH, Boekholdt SM, et al. Ability of re- and meta- analysis. Thromb Haemost. 2017;117:139-148. combinant factor VIIa to reverse the anticoagulant effect of the 23. Northup PG, Sundaram V, Fallon MB, et al. Hypercoagulation and pentasaccharide fondaparinux in healthy volunteers. Circulation. thrombophilia in liver disease. J Thromb Haemost. 2008;6:2-9. 2002;106:2550-2554. 24. Fisher C, Patel VC, Stoy SH, et al. Balanced haemostasis with both 37. Lisman T, de Groot PG. The role of cell surfaces and cellular recep - hypo- and hyper- coagulable features in critically ill patients with tors in the mode of action of recombinant factor VIIa. Blood Rev. acute- on- chronic- liver failure. J Crit Care. 2017;43:54-60. 2015;29:223-229. 25. Potze W, Arshad F, Adelmeijer J, et al. Differential in vitro inhibition 38. Groeneveld DJ, Adelmeijer J, Hugenholtz GC, Ariens RA, Porte RJ, of thrombin generation by anticoagulant drugs in plasma from pa- Lisman T. Ex vivo addition of fibrinogen concentrate improves fibrin tients with cirrhosis. PLoS ONE. 2014;9:e88390. network structure in plasma samples taken during liver transplanta - 26. Potze W, Adelmeijer J, Lisman T. Decreased in vitro anticoagulant tion. J Thromb Haemost. 2015;13:2192-2201. potency of rivaroxaban and apixaban in plasma from patients with 39. Senzolo M, Rodriguez-Castro KI, Rossetto V, et al. Increased an- cirrhosis. Hepatology. 2015;61:1435-1436. ticoagulant response to low- molecular- weight heparin in plasma 27. Tripodi A, Chantarangkul V, Primignani M, et al. Thrombin gener- from patients with advanced cirrhosis. J Thromb Haemost. ation in plasma from patients with cirrhosis supplemented with 2012;10:1823-1829. normal plasma: considerations on the efficacy of treatment with 40. Potze W, Arshad F, Adelmeijer J, et al. Routine coagulation assays fresh- frozen plasma. Intern Emerg Med. 2012;7:139-144. underestimate levels of antithrombin- dependent drugs but not of 28. Tripodi A, Primignani M, Chantarangkul V, et al. Global hemostasis direct anticoagulant drugs in plasma from patients with cirrhosis. Br tests in patients with cirrhosis before and after prophylactic plate- J Haematol. 2013;163:666-673. let transfusion. Liver Int. 2013;33:362-367. 41. Ninivaggi M, Apitz-Castro R, Dargaud Y, de Laat B, Hemker HC, 29. Moreau R, Jalan R, Gines P, et al. Acute- on- chronic liver failure is a Lindhout T. Whole- blood thrombin generation monitored with distinct syndrome that develops in patients with acute decompen - a calibrated automated thrombogram- based assay. Clin Chem. sation of cirrhosis. Gastroenterology. 2013;144:1426-1437.e1-9. 2012;58:1252-1259. 30. Hemker HC, Giesen P, Al Dieri R, et al. Calibrated automated thrombin generation measurement in clotting plasma. Pathophysiol Haemost Thromb. 2003;33:4-15. How to cite this article: Lisman T, Kleiss S, Patel VC, et al. In 31. Lodge JP, Jonas S, Jones RM, et al. Efficacy and safety of repeated vitro efficacy of pro- a nd anticoagulant strategies in perioperative doses of recombinant factor VIIa in liver transplanta - compensated and acutely ill patients with cirrhosis. Liver Int. tion. Liver Transpl. 2005;11:973-979. 32. Planinsic RM, van der Meer J, Testa G, et al. Safety and efficacy 2018;38:1988–1996. https://doi.org/10.1111/liv.13882 of a single bolus administration of recombinant factor VIIa in http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Liver International Pubmed Central

In vitro efficacy of pro‐ and anticoagulant strategies in compensated and acutely ill patients with cirrhosis

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Abstract

Section of Hepatobiliary Surgery and Liver Transplantation, Department of Background & Aims: A simultaneous decline in pro- and anticoagulant drivers in pa- Surgery, University of Groningen, University tients with liver diseases results in a “rebalanced” haemostatic system, even in acutely Medical Center Groningen, Groningen, The Netherlands ill patients. Nevertheless, both bleeding and thrombotic events are common. Here, we Liver Intensive Care Unit, Institute of Liver explored efficacy of pro- and antihaemostatic strategies in compensated and acutely Studies, King College Hospital, London, UK ill cirrhotics which may be unpredictable given the profound haemostatic changes. Institute of Liver Studies, King College Hospital, London, UK Methods: We tested the effects in vitro of the addition of clinically relevant doses of commonly used pro- and antihaemostatic strategies in plasma from healthy individu- Correspondence Ton Lisman, Department of Surgery, als (n = 30) and patients with compensated (n = 18) and acutely decompensated cir- University Medical Center Groningen, rhosis (n = 18), and acute- on- chronic liver failure (n = 10). We used thrombin generation Groningen, The Netherlands. Email: j.a.lisman@umcg.nl tests and fibrin clot permeability assays to assess potency of various approaches. Results: Fresh frozen plasma and recombinant factor VIIa modestly increased throm- Funding information This study was funded in part by the Tekke bin generation (10%- 20%). Prothrombin complex concentrate increased thrombin Huizinga Foundation (The Netherlands). generation two- fold in controls and 2- 4- fold in patients. Clot permeability decreased Handling Editor: Juan Abraldes after addition of fibrinogen concentrate by 51% in controls and by 50%- 60% in pa- tients. Low molecular weight heparin decreased thrombin generation by 18% in con- trols and by 23%- 54% in patients. Similarly, dabigatran decreased thrombin generation by 33% in controls and by 47%- 100% in patients. In contrast, rivaroxaban decreased thrombin generation by 55% in controls, but only by 11%- 38% in patients. Conclusions: These in vitro data suggest little prohaemostatic effect of fresh frozen plasma and recombinant factor VIIa in acutely ill cirrhotics, whereas prothrombin complex concentrate and fibrinogen concentrate clearly improved haemostasis. Furthermore, our data suggest the requirement for dose adjustments of commonly used anticoagulants in these patients. K E Y WO R D S bleeding, cirrhosis, haemostasis, thrombosis Abbreviations: ACLF, acute of chronic liver failure; AD, acute decompensation; ETP, endogenous thrombin potential; FFP, fresh frozen plasma; LMWH, low molecular weight hepari; PCC, prothrombin complex concentrate; TM, thrombomodulin This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2018 The Authors. Liver International Published by John Wiley & Sons Ltd 1988     wileyonlinelibrary.com/journal/liv Liver International. 2018;38:1988–1996. LISMAN et AL.       1989 1  |   I NTR O D U C TI O N Key points Patients with liver diseases frequently acquire substantial alterations • Bleeding and thrombotic events are common in patients in their haemostatic system. A simultaneous decline in pro- and an- 2-4 with liver disease, but there is uncertainty on optimal ticoagulant drivers results in a “rebalanced” haemostatic system. management strategies External factors may tip the balance towards hypo- or hypercoagu- 5 6-10 11-14 • Efficacy of pro- and anticoagulant agents are likely al- lability, and distinct hypo- and hypercoagulable features in tered in patients with liver disease as a result of pro- patients with liver disease which may predispose them to bleeding or found changes in their haemostatic system thrombotic complications. • Using plasma samples from patients with compensated Bleeding and thrombosis are not uncommon in patients with cirrhosis, acute decompensation of cirrhosis or acute- liver disease, in particular in those with advancing and decompen- on-chronic liver failure we demonstrate little prohaemo - sated illness. However, little evidence- based treatment strategies static effect of fresh frozen plasma and recombinant for prevention or treatment of bleeding or thrombosis are available. factor VIIa, whereas prothrombin complex concentrate Current expert recommendations propose a very restrictive prophy - and fibrinogen concentrate clearly improved lactic prohaemostatic management, and a more active anticoagulant 5,16,17 haemostasis approach. Importantly, anticoagulant therapy in patients with • Commonly used anticoagulants have profoundly altered cirrhosis is challenging, and new generation anticoagulant drugs anticoagulant potency in critically ill cirrhotics have not been extensively studied in patients with cirrhosis, al- 19,20 though the clinical use in these patients is increasing. Decompensation may tip the haemostatic balance of patients with liver disease towards a bleeding phenotype. A recent study With the aim to provide a more rational approach to pro- and has identified thrombocytopenia (with a platelet count <30 000 μ L) antihaemostatic treatment of acutely ill patients with cirrhosis, and and hypofibrinogenemia (<0.6 g/L) as independent risk factors for to facilitate design of future clinical studies, we tested the in vitro bleeding in acutely ill patients with cirrhosis. It is, however, un - effects of commonly used pro- and antihaemostatic strategies in known whether there is a causal link between bleeding risk and plasma from patients with acutely decompensated cirrhosis and these laboratory abnormalities, and studies assessing whether re - acute- on- chronic liver failure. versal of thrombocytopenia and/or hypofibrinogenemia decrease the bleeding risk in these patients will be required to ascertain this. 2  |  M AT E R I A L S A N D M E T H O DS It is unknown whether acutely ill patients with cirrhosis are at risk for development of venous thrombosis similar to the increased 2.1 | Patients risk in well compensated patients. Nevertheless, prophylactic or therapeutic antithrombotic strategies may be required in acutely The study was performed at King’s College Hospital, a 950- bed ter- ill patients with cirrhosis, in the context of venous thrombosis, tiary hospital in London, United Kingdom, between August 2013 and portal vein thrombosis, and thrombosis of extracorporeal assist August 2015. The study was approved by NRES Committee London- 15,23 devices. Westminster, Study Number 12/LO/1417. Informed consent or assent We have recently studied the haemostatic status of patients with was obtained from participants or their personal consultees. Details acutely decompensated or acute- on- chronic liver failure and found on patient recruitment and blood sampling have been published pre- 24 24 a remarkably preserved haemostatic system. The relatively well- viously. In short, patients were sampled on admission and patients preserved haemostatic balance therefore suggests that a defensive were only excluded when currently using antihaemostatic agents. prohaemostatic and a proactive antihaemostatic approach may be From the published cohort we studied 30 healthy volunteers, 18 pa- warranted in these patients. tients with acute decompensation (AD) of cirrhosis and 10 patients Given the major alterations in the haemostatic system of pa- with acute- on- chronic liver failure (ACLF). Eighteen patients with well tients with cirrhosis, the efficacy of pro- and antihaemostatic strat- compensated cirrhosis were newly recruited in the outpatient clinic, egies may be unpredictable. We have previously demonstrated that and were not using antihaemostatic drugs at the time of sampling. in patients with compensated cirrhosis the in vitro anticoagulant ef - Acute decompensation of chronic liver disease and ACLF were de- fects of some of the commonly used drugs was decreased, whereas fined and graded according to number of organ failures in concord- the anticoagulant effects of others were increased as compared to ance with criteria reported in the CANONIC study. From the 10 25,26 anticoagulant effects in healthy individuals. Another study has patients with ACLF, 1 was classified as grade 1 and 9 were grade 3. shown a lack of prohaemostatic effect of in vitro addition of fresh frozen plasma to plasma from patients with compensated cirrho- 2.2 | Routine laboratory tests sis, despite improvements in plasma levels of coagulation factors. Similarly, transfusion of platelets to patients with cirrhosis did in - Haemoglobin, white blood cell count, albumin, creatinine, bilirubin, crease the platelet count, but did not improve global haemostasis. aspartate transaminase, alanine transaminase and gamma- glutamyl LISMAN et AL. 1990       transpeptidase were measured in the diagnostic laboratory of King’s Thrombinoscope BV, Maastricht, the Netherlands) was dispensed in College Hospital for routine clinical care. International normalized each well to allow a continuous registration of thrombin generation. ratios, and plasma levels of fibrinogen, antithrombin, factor II, fac- Fluorescence was read in time by a fluorometer, Fluoroskan Ascent tor VIII and factor X were measured in stored frozen samples on an (ThermoFisher Scientific, Helsinki, Finland). All procedures were un- automated coagulation analyzer (ACL 300 TOP) with reagents and dertaken according to the protocol suggested by Thrombinoscope B.V. protocols from the manufacturer (Werfen, Breda, The Netherlands). The pro- or anticoagulant potency of the different agents was expressed as the percentual change in endogenous thrombin po - tential (ETP), lag time, peak or velocity index after addition of the 2.3 | In vitro addition of pro- and anticoagulants study agent. These percentages were compared between patients We added the following agents to plasma samples of each patient and controls. and control: 2.5 | Fibrin concentration and fibrin permeability • Recombinant factor VIIa (Novo Nordisk, Bagsvaerd, Denmark)- final concentration 50 nmol/L Fibrinogen levels in plasma of patients and healthy volunteers • Cofact (a 4-factor prothrombin complex concentrate (PCC), were determined on an ACL TOP 300 analyzer using reagents from Sanquin, Amsterdam, Netherlands)-final concentration 0.5 U/mL Instrumentation Laboratory (Breda, the Netherlands) according to • Pooled normal plasma (to mimick fresh frozen plasma [FFP] trans- the manufacturer’s instructions. fusion-obtained by combining plasma from >200 healthy volun- The average pore size of the fibrin clot (expressed as the Darcy teers, a generous gift from Dr. J.C. Meijers, Academic Medical constant, Ks) was determined in permeation studies as previously Center Amsterdam, the Netherlands)-final concentration 20% (v/v) described. In short, plasma samples (100 μL) were incubated with • Fibrinogen concentrate (CSL Behring, Marburg, Germany)-final 10 μL of activation buffer (final concentration of 1 IU/mL thrombin, concentration 1 g/L 20 mmol/L CaCl , in tris- buffered saline, pH 7.5) to generate clots. After • Rivaroxaban, a direct factor Xa inhibtor (Alsachim, Illkirch mixing, 100 μL was immediately transferred to a 4.5- cm plastic tip with Graffenstaden, France)-final concentration 25 ng/mL a roughened interior surface, which was cut off from a 1 mL serological • Dabigatran, a direct thrombin inhibitor (Alsachim, Illkirch pipette (Corning Costar Stripette; Sigma- Aldrich, St Louis, MO, USA), Graffenstaden, France)-final concentration 300 ng/mL and left for 2 hours in a moist chamber at room temperature to conso- l • The low molecular weight heparin (LMWH) Clexane (Sanofi- idate. The plastic tip was then connected through a flexible silicon tube Aventis BV, Gouda, the Netherlands)-final concentration 0.2 U/mL to a syringe containing TBS with a 4- cm pressure drop. After a wash- out period of 90 minutes with tris- buffered saline, preweighed tubes Plasma levels of procoagulant drugs were chosen to represent clin - were attached to the clotting tip, and tris- buffered saline drops passing ically relevant (peak) levels observed in clinical use in the general pop- through the clot were collected every 30 minutes for 2 hours. The total ulation. The final concentrations of the anticoagulant drugs were also volume of liquid passing through the clot was weighed after collection. chosen to represent clinically relevant plasmas levels and were identi- Permeation of tris- buffered saline through the clot was quantified ac- cal to levels used in previously published experiments. Importantly, cording to the flow rate and the following equation, Ks = (Q × L × η)/T drug concentrations which gave appreciable (but not maximal) inhibi- × A × P, where Ks = Darcy’s constant, Q = volume of liquid (mL), L = clot tion of thrombin generation in pooled normal plasma were selected length (cm), η = viscosity (poise), T = time (s), A = cross- sectional area of so it would be possible to detect both increased and decreased drug the clot (cm ), and P = pressure drop (dyne/cm). effects in patients compared to controls. 2.6 | Statistical analyses 2.4 | Thrombin generation Data are expressed as means (with standard deviations (SDs)), me- The thrombin generation test was performed using platelet- poor dians (with interquartile ranges), or numbers (with percentages) plasma with the fluorimetric method described by Hemker, as appropriate. Multiple groups were compared using One- way Calibrated Automated Thrombography in absence or presence of ANOVA (with the Tukey’s post- test) or Kruskal- Wallis H test (with the above- mentioned agents, except for fibrinogen concentrate. Dunn’s post- test) as appropriate. P values of .05 or less were consid- Coagulation was activated using commercially available reagents ered statistically significant. GraphPad Prism (San Diego, USA) and containing recombinant tissue factor (final concentration 5 pmol/L), GraphPad Instat (San Diego, USA) were used for analyses. phospholipids (final concentration 4 μmol/L), in the presence of soluble thrombomodulin (TM, the concentration of which is not revealed by the manufacturer). These reagents were purchased 3  |   RE S U LT S from Thrombinoscope BV, Maastricht, the Netherlands. Thrombin Calibrator (Thrombinoscope BV) was added to calibrate the throm- We studied the effects of ex vivo addition of commonly used bin generation curves. A fluorogenic substrate with CaCl (FluCa-k it, pro- and anticoagulant drugs in patients with compensated and 2 LISMAN et AL.       1991 decompensated cirrhosis, and in patients with acute- on- chronic liver velocity index increased substantially more in patients compared to failure and compared results with those obtained in healthy controls. controls. The lag time did not appreciably change in either controls Table 1 summarises baseline characteristics of patients and controls. or patients (Table 3, Figure 1). Without the addition of pro- or anticoagulant agents, patients Addition of pooled normal plasma (20% v/v) increased total generated more thrombin as compared to controls, using TM- thrombin generation in controls by 16%, with very similar changes in modified thrombin generation tests, which is in line with our previ- compensated and AD patients (9%- 19% increase). Addition of pooled ously published data (Table 2). normal plasma to samples of patients with ACLF led to a more pro- When recombinant factor VIIa (50 nmol/L) was added to plasma found increase in thrombin generation (38% increase), but given the of healthy individuals, an increase of ~20% in ETP, peak, and velocity lower baseline thrombin generation levels in the ACLF group, total index was observed, with an ~20% decrease in lag time. In patients, thrombin generation after addition of pooled normal plasma was however, addition of recombinant factor VIIa did not appreciably comparable between AD and ACLF patients (913 [841- 980] vs 883 change ETP, peak, and velocity index, whereas the lag time was [757- 993] nmol/L IIa × min) - Table 3, Figure 1. shortened to a similar extent as in controls (Table 3, Figure 1). The permeability of clots generated from plasma from healthy Addition of the PCC Cofact (0.5 U/mL) to plasma of healthy controls was remarkably similar to that of the permeability of clots individuals resulted in an approximate doubling of total thrombin from patients, despite the lower fibrinogen levels in patient plasma. generation (to 829 [697- 1141] nmol/L IIa × min). An exaggerated re- When fibrinogen concentrate (1 g/L) was added to control samples, sponse was observed in plasma from patients with a 110% increase a 51% reduction in permeability was observed. A similar effect of in the compensated cirrhosis group (to 1525 [1052- 1697] nmol/L IIa fibrinogen concentrate was observed in patients compensated cir- × min), a 150% increase in thrombin generation in the AD group (to rhosis, whereas a slightly more robust effect was observed in plasma 1869 [1734- 2210] nmol/L IIa × min) and a 270% increase in the ACLF from AD and ACLF patients with a 61% reduction in permeability in group (to 2383 [1731- 3538] nmol/L IIa × min). Similarly, the peak and the AD group and a 63% reduction in the ACLF group (Table 4). TABLE 1  Patient characteristics Compensated Controls (n = 30) (n = 18) AD (n = 18) ACLF (n = 10) Age 37 ± 7 60 ± 13 54 ± 14 56 ± 10 Gender (male, %) 15 (50%) 12 (67%) 5 (50%) 13 (72%) CLIF- SOFA n/a n/a 4 ± 2 10 ± 4 MELD n/a 9 ± 2 16 ± 9 31 ± 9 Reason for n/a n/a Ascites n = 4 Sepsis n = 4 decompensation Variceal bleeding n = 4 Variceal bleeding n = 4 Encephalopathy n = 10 SBP n = 2 Haemoglobin (g/L) n/d 145 (113- 145) 111 (93- 128) 95 (77- 116) WBC (× 10 /L) n/d 5 (4- 6) 6 (3- 9) 10 (8- 19) Platelets (× 10 /L) n/d 119 (87- 150) 95 (65- 132) 89 (65- 111) Albumin (g/L) n/d 41 (39- 45) 33 (30- 37) 28 (26- 33) Creatinin (μmol/L) n/d 82 (70- 89) 67 (56- 93) 135 (94- 224) Bilirubin (μmol/L) n/d 19 (11- 25) 44 (30- 71) 362 (116- 493) AST (U/L) n/d 42 (37- 45) 47 (38- 56) 79 (50- 86) ALT (U/L) n/d 36 (24- 43) 25 (18- 30) 46 (24- 61) GGT (U/L) n/d 60 (33- 143) 77 (47- 135) 37 (30- 91) , , INR 1.0 ± 0.1 1.1 ± 0.1 1.6 ± 0.4** *** 2.1 ± 0.4** **** , , Fibrinogen (g/L) 2.8 ± 0.4 3.3 ± 0.7 2.2 ± 0.9* *** 1.3 ± 0.3** **** , , Antithrombin (%) 107 ± 11 88 ± 26* 46 ± 18** *** 28 ± 14** **** FII (%) 101 ± 18 78 ± 17** 48 ± 16** *** 31 ± 10** FVIII (%) 99 ± 36 157 ± 42** 146 ± 27* 212 ± 93** **** FX (%) 105 ± 23 85 ± 21* 57 ± 20** *** 41 ± 15** ALT, alanine transaminase; AST, aspartate transaminase; CLIF- SOFA, Chronic Liver Failure- Sequential Organ Failure Assessment; F, factor; GGT, gamma- glutamyl transpeptidase; INR, international normalised ratio; MELD, model for end- stage liver disease; n/a, not applicable; n/d, not determined; SBP, spontaneous bacterial peritonitis; WBC, white blood cell. Shown are means ± standard deviation or medians (interquartile range). *P < .05 vs control, **P < .01 vs control, ***P < .01 vs compensated, ****P < .01 vs AD. LISMAN et AL. 1992       TABLE 2  Thrombin generation ETP (nmol/L IIa Vel index (nmol/L parameters of thrombomodulin- modified × min) Peak (nmol/L IIa) Lag time (min) IIa × min) thrombin generation testing in controls Controls 440.0 122.8 (93.9- 180.3) 1.67 (1.46- 1.98) 61.9 (46.3- 96.5) and patients with compensated (CC) or (343.4- 639.8) acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) CC 699.5 184.5 (152.0- 211.3) 1.67 (1.33- 1.67) 99.0 (75.8- 111.0) (538.5- 819.3) AD 736.3 182.3 (173.2- 215.6)** 1.33 (1.33- 1.63)* 102.4 (696.2- 882.3)**  (90.3- 112.1)** ACLF 648.1 124.5 (86.5- 144.6) 1.92 (1.67- 2.00) 60.3 (43.4- 75.0) (473.3- 743.7) Shown are medians (interquartile ranges). *P < .05, **P < .01 vs controls. TABLE 3  Percentual changes in ETP Peak Lag time Vel index thrombin generation parameters of rFVIIa thrombomodulin- modified thrombin generation testing in controls and patients Controls 21 (8- 32) 20 (8- 32) 28 (20- 34) 20 (11- 42) with compensated (CC) or acutely CC 3 (0- 16)* 2 (−3 to 9)** 21 (20- 30) 1 (−6 to 2 0)* decompensated cirrhosis (AD), or AD 2 (−1 to 5 )*** −1 (−3 to 3 )*** 24 (17- 37) 0 (−10 to 5 )*** acute- on- chronic liver failure (ACLF) after in vitro addition of prohaemostatic agents ACLF 3 (−3 to 27)* −1 (−4 to 19)* 20 (15- 25)* 3 (−6 to 2 5) Cofact Controls 92 (788- 101) 72 (46- 89) 0 (0- 17) 72 (38- 93) CC 109 (82- 149) 89 (55- 102) 0 (0- 0) 78 (55- 104) AD 151 (135- 197)*** 109 (87- 150)** 0 (−13- 6) 97 (75- 136)* ACLF 273 (212- 393)*** 176 (141- 219)*** 10 (0- 17) 178 (156- 265)*** Pooled normal plasma Controls 16 (2- 21) 15 (−3- 19) 0 (−10- 0) 15 (−4- 19) CC 7 (−1- 21) 7 (0- 18) 20 (0- 26) 6 (−4- 18) AD 19 (8- 29) 23 (10- 33) −11 (−25- 0) 22 (12- 31) ACLF 38 (27- 69) *** 56 (47- 124) *** 0 (−9- 9) 59 (45- 117) *** Shown are the percentual increase of the ETP, peak, or velocity index, and the percentual decrease in the lag time. Data are expressed as median percentages with interquartile rangeP .* < .05, **P < .01, ***P < .001 vs controls. Addition of rivaroxaban (25 ng/mL) to plasma from healthy con- generated any thrombin in the presence of dabigatran (Table 5, trols resulted in a 55% decrease in the ETP (to 178 [152- 339] nmol/L Figure 2). IIa × min), with similar changes in peak and velocity index. The re- LMWH (0.2 U/mL) decreased the ETP by 18% in controls (to 374 duction in total thrombin generation was less profound in patients, [291- 471] nmol/L IIa × min), with similar changes in peak and velocity particularly in the AD group in which the ETP was only reduced by index. The decrease in the ETP was more pronounced in patients 11% (to 629 [527- 692] nmol/L IIa × min) with a 25% reduction in the with a 23% decrease in the compensated group (to 526 [425- 632]), ACLF group (to 465 [367- 570] nmol/L IIa × min), and a 38% reduc- a 41% decrease in the AD group (to 463 [390- 594] nmol/L IIa × min) tion in the compensated group (to 403 [271- 510] nmol/L IIa × min). and a 54% decrease (to 311 [213- 403] nmol/L IIa × min) in the ACLF Although the reduction in thrombin generation was substantially group (Table 5, Figure 2). However, given the higher baseline ETP in decreased in patients, the lag time was substantially more prolonged patients, the ETP in the presence of LMWH were similar between in patients compared to controls, particularly in the ACLF group patients and controls. (Table 5, Figure 2). Addition of dabigatran (300 ng/mL) decreased total thrombin generation in controls by 33% (to 333 [221- 539] nmol/L IIa × min), 4  |  D I S CU S S I O N and prolonged the lag time more than 6- fold. In patients, total throm- bin generation was decreased by 47% in the compensated group (to Here we have studied the in vitro effects of commonly used pro- and 308 [256- 401] nmol/L IIa × min), by 75% in the AD group (to 171 [63- anticoagulant strategies in compensated and acutely ill patients with 229] nmol/L IIa × min), and none of the patients in the ACLF group cirrhosis in comparison to healthy individuals and found remarkable LISMAN et AL.       1993 FIGURE 1  Absolute ETP values from thrombomodulin- modified thrombin generation testing in controls and patients with compensated or acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) prior to and after in vitro addition of prohaemostatic agents. Indicated are percentual changes in the ETP after addition of procoagulant agents. Shown are medians with interquartile ranges . PNP, pooled normal plasma TABLE 4  Fibrinogen levels and fibrinogen permeability in controls and patients with compensated (CC) or acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) in the absence and presence of fibrinogen concentrate Permeability + 1 g/L fibrinogen Percentual decrease Fibrinogen (g/L) Permeability (Ks) concentrate (Ks) in permeability −9 −9 −9 −9 Controls 2.8 ± 0.4 9.2 × 10 ± 3.2 × 10 4.2 × 10 ± 1.0 × 10 51 ± 11 −9 −9 −9 −9 CC 3.3 ± 0.7 9.0 × 10 ± 4.0 × 10 4.1 × 10 ± 2.0 × 10 52 ± 11 −9 −9 −9 −9 AD 2.2 ± 0.9* 8.8 × 10 ± 3.6 × 10 3.7 × 10 ± 1.1 × 10 61 ± 15 * −9 −9 −9 −9 ACLF 1.3 ± 0.3** 10.9 × 10 ± 5.1 × 10 4.0 × 10 ± 1.8 × 10 63 ± 17* Data are expressed as means ± standard deviation. *P < .05, **P < .001 vs controls. differences in the potency of these strategies between patients and bleeding during liver transplantation. However, based on our data, controls. Notably, the in vitro efficacy of the procoagulant PCC and dosing of PCCs should be performed conservatively in acutely ill pa- the anticoagulants dabigatran and LMWH increased with increas - tients with cirrhosis given the profoundly exaggerated procoagulant ing severity of disease, and the efficacy of rivaroxaban was lower response in patients compared to controls. Similarly, our data sug- in patients although not proportional to the severity of disease. The gest that substantial dose- adjustments may be required when low increase in efficacy of PCC, dabigatran, and LMWH with increasing molecular weight heparin or direct oral anticoagulants are consid - severity of disease mirror the haemostatic changes that are more ered for prophylaxis or treatment of thrombotic complications in profound in the sicker patients. The results of this study may have acutely ill patients with cirrhosis. clinical relevance as it suggests a requirement for dose- adjustments The lack of a procoagulant effect of recombinant factor VIIa for a number of agents tested, and suggests some agents to be clini- in patients with cirrhosis suggests competent tissue factor- cally ineffective in these patients. Specifically, the lack of a procoag- mediated activation of coagulation in these patients, and is also ulant effect of recombinant factor VIIa in patients, and the minimal in line with a ver y modest increase in thrombin generation follow- effect of FFP in the AD group are in line with the lack of evidence ing administration of recombinant factor VIIa to healthy volun - supporting the use of recombinant factor VIIa in patients with liver teers. However, as recombinant factor VIIa also has important 31-33 37 diseases, and with the doubts on the use of FFP in patients with tissue factor- independent procoagulant activity, it cannot be liver disease in general. Our data suggest a procoagulant approach presumed that this drug does not have some prohaemostatic ef - with PCCs and or fibrinogen concentrate to substantially improve fect in acutely ill patients with cirrhosis in vivo. It has been pre - haemostatic status in acutely ill patients with liver disease given the viously demonstrated that FFP has no appreciable effects on in profound procoagulant effects of PCC in thrombin generation tests vitro thrombin generation in plasma from patients with compen - and the improvement of fibrin clot structure by fibrinogen concen- sated cirrhosis, despite clear changes in plasma levels of coagu - trate. This strategy has been reported to be effective in managing lation factors and a decrease in the INR. Here we demonstrate LISMAN et AL. 1994       TABLE 5  Percentual decrease in thrombin generation parameters of thrombomodulin- modified thrombin generation testing in controls and patients with compensated (CC) or acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) after addition of anticoagulants ETP Peak Lag time Vel index Rivaroxaban Controls 55 (50- 63) 62 (56- 69) 34 (25- 46) 70 (62- 75) CC 42 (32- 50)* 53 (44- 57)* 60 (50- 100)*** 60 (44- 68)* AD 11 (10- 30)*** 19 (18- 35)*** 52 (41- 63)* 33 (27- 47)*** ACLF 25 (11- 35)** 43 (27- 52)** 70 (56- 87)*** 54 (43- 64)* Dabigatran Controls 33 (16- 48) 33 (20- 65) 571 (314- 711) 30 (5- 76) CC 50 (35- 63) 56 (41- 73) 455 (365- 617) 53 (29- 75) AD 75 (93- 73)*** 92 (84- 98)*** 483 (298- 614) 95 (87- 99)** ACLF No thrombin formed*** No thrombin formed*** No thrombin formed*** No thrombin formed*** LMWH Controls 18 (9- 28) 15 (6- 26) 0 (0- 10) 14 (−2- 32) CC 25 (16- 30) 15 (5- 27) 26 (17- 54) 8 (−4- 20) AD 41 (26- 48)** 26 (13- 30) 17 (7- 29)** 16 (1- 29) ACLF 54 (39- 61)*** 29 (24- 42)** 9 (0- 28) 25 (14- 41) Shown are the percentual decrease of the ETP, peak, or velocity index, and the percentual increase in the lag time. Data are expressed as median per- centages with interquartile range.*P < .05, **P < .01, ***P < .001 vs controls. FIGURE 2  Absolute ETP values from thrombomodulin- modified thrombin generation testing in controls and patients with compensated or acutely decompensated cirrhosis (AD), or acute- on- chronic liver failure (ACLF) prior to and following addition of anticoagulant agents. Indicated are percentual changes in the ETP after addition of anticoagulant agents. Shown are medians with interquartile ranges comparable effects of pooled normal plasma (mimicking FFP individuals (from London, UK) and our pooled normal plasma transfusion) in plasma from healthy individuals and patients with (which was generated in Amsterdam, the Netherlands). Our data acutely decompensated cirrhosis, suggesting FFP to be ineffec - suggests FFP does have some haemostatic effect in patients with tive in AD patients. The slight increase in thrombin generation in ACLF. However, the drawback of FFP administration in these our control group is likely clinically insignificant and may reflect acutely ill patients is the risk of volume overload and exacerbation minor differences in coagulation profiles between our healthy of portal hypertension. LISMAN et AL.       1995 The substantially increased procoagulant capacity of PCCs in C O N FL I C T O F I N T E R E S T plasma from acutely ill patients with cirrhosis likely relates to the The authors do not have any disclosures to report. low baseline plasma levels of procoagulant proteins. The same dose of PCC thus results in a much larger relative increase in levels of the vitamin K- dependent factors in patients compared to controls. O RC I D Baseline thrombin generating capacity is elevated in patients com- Ton Lisman http://orcid.org/0000-0002-3503-7140 pared to controls, despite the much lower levels of procoagulants, Caleb Fisher http://orcid.org/0000-0003-3336-3898 as the levels of anticoagulant proteins are also low. Therefore, the William Bernal http://orcid.org/0000-0002-6508-3287 procoagulant effects of PCCs are much more prominent in patients compared to controls. The preserved fibrin clot structure in pa- tients is in line with our previous results showing preserved fibrin clot structure in patients with compensated cirrhosis to be related R EF ER EN C E S to oxidative modifications to the fibrinogen molecule. Fibrinogen 1. Lisman T, Leebeek FW, de Groot PG. Haemostatic abnormalities in concentrate profoundly decreases fibrin clot permeability in acutely patients with liver disease. J Hepatol. 2002;37:280-287. ill patients with cirrhosis, which is in line with our previous study 2. Lisman T, Porte RJ. 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Published: May 30, 2018

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