Haemostasis Patterns in Patients With Acute-on-chronic Liver Failure and Acute Decompensation of Cirrhosis Including Thromboelastometric Tests With and Without the Addition of Protac: a Pilot Study

doi:10.21203/rs.3.rs-1739516/v1

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Haemostasis Patterns in Patients With Acute-on-chronic Liver Failure and Acute Decompensation of Cirrhosis Including Thromboelastometric Tests With and Without the Addition of Protac: a Pilot Study

https://doi.org/10.21203/rs.3.rs-1739516/v1

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Background: Thromboelastometry is considered as the best method to assesses hemostasis in liver disease, diagnostic performance could be improved by adding protein C activators such as thrombomodulin or Protac®.

We assess the changes in ROTEM parameters after the addition of Protac® in patients with acute-on-chronic liver failure (ACLF), acute decompensation (AD), and healthy individuals (HI), to define a different hemostasis pattern, considering standard and velocity ROTEM parameters, and to assess whether the Protac® can improve this pattern definition.

Methods: In a pre-test, we investigated whether diluted EXTEM reagent improves the effect of Protac® on clotting time (CT)-ratio with and without Protac®. Ten ACLF, 20 AD patients and 21 HI were included in the main study.

Results: Standard EXTEM was used in the main study. INTEM CFT, INTEM A5 (inverse), and INTEM TPI (inverse) were the best parameters to differentiate liver disease from HI (ROC AUC, 0.921, 0.906, and 0.928, respectively; all P-values < 0.001). Combining INTEM CFT with EXTEM LI60-ratio only slightly improved the diagnostic performance (ROC AUC, 0.948; P<0.001). EXTEM LI60 and INTEM maxV-t were the best parameters to differentiate between ACLF and AD patients (ROC AUC, 0.743, P=0.033; and 0.723, P=0.050; respectively). Combining EXTEM LI60 + INTEM maxV-t moderately improved the diagnostic performance, (ROC AUC, 0.81, P<0.001).

Conclusions: ROTEM velocity, fibrinolysis parameters and calculated indices improve the diagnostic in combination with standard parameters (e.g., CFT and A5). Ratios calculated with and without Protac (e.g., EXTEM LI60-ratio) only slightly increased the diagnostic performance to discriminate hemostasis patterns.

Cirrhosis

Haemostasis

Thromboelastometry

Thrombomodulin

Protein C

Protac

The preservation of the haemostatic capacity in patients with liver disease relies on the balanced reduction in pro- and anticoagulant drivers.(1)(2) For comprehension of the concept of rebalanced haemostasis, knowledge about methods and limits of the coagulation tests employed in these patients is essential.(3)(4)(5) Standard coagulation tests only mirror the amount of procoagulant factors in plasma, and they are not capable of detecting the contribution of cells in the whole blood; although the anticoagulant pathway (protein C and S) is not properly explored in any coagulation assay currently used in the clinical practice, but rotational thromboelastometry (ROTEM) is considered as the best method to assesses haemostasis patterns in patients with liver disease.(6)(7)(8)(9)(10)(11)(12)

Protein C levels are decreased in patients with cirrhosis.(13) Therefore, global test per definition underestimates the coagulation capacity of the blood sample from cirrhotic patients. The protein C system requires the endothelial cell transmembrane protein thrombomodulin (TM) to become activated.(14) The lack of endothelial cells or any protein C activators results in a lack of protein C activation during clot formation.(15) This concept has been extensively investigated using thrombin generation tests, which have been executed in the presence and absence of a soluble form of TM to allow for protein C activation.(16) Due to the low protein C levels, TM-modified thrombin generation does not result in decreased thrombin generation in patients with cirrhosis (TM-resistance), whereas adding thrombomodulin to blood samples from healthy individuals result in significant decrease in thrombin generation.(17)(18) Similar results can be achieved by adding Protac® (Pentapharm, Basel, Switzerland), a snake venom which activates protein C, too.(19)(20)(21)(22)(23) Accordingly, Protac® results in a significant decrease in the endogenous thrombin potential (ETP) of healthy individuals but not in patients with cirrhosis. Similarly, in the presence of Protac®, ETP was significantly higher in patients with acute liver failure than in healthy controls.(2)(24)(16)(22)

ROTEM assesses global coagulation and clot dynamics in real time through the natural phases of clotting initiation, clot kinetics, and stability until clot breakdown. It is performed on whole blood. ROTEM is considered as the mainstay of coagulation monitoring during liver transplantation. Anyway, similar limitations may apply for ROTEM regarding the protein C system, which is profoundly altered in patients with liver disease.(25) Thromboelastometry with the addition of thrombomodulin or Protac® has not been reported, yet.

Therefore, the primary end point of this study is to assess the changes in ROTEM parameters after the addition of Protac® in patients with acute-on-chronic liver failure (ACLF) and acute decompensation (AD) of cirrhosis and comparing these results with healthy individuals.

Secondary end point was to define a different haemostasis pattern, considering both standard and velocity ROTEM parameters, for patients with acute-on-chronic liver failure, acute decompensation of cirrhosis and healthy individuals; and to assess whether the assays including Protac® can improve this pattern definition.

Study Design

This is a single centre prospective observational study, approved by the Ethical Committee of the Hospital Clinic of Barcelona (approval number HCB/2017/0948) on April 4th, 2018. Healthy individuals and patients were enrolled after giving their written informed consent. All study procedures were performed following the ethical standards of the Declaration of Helsinki. This study followed the “Strengthening the Reporting of Observational Studies in Epidemiology (STROBE)” statement guidelines for observational cohort studies.(26)

Study Population and Data Collection

Adult patients admitted at Hospital Clinic of Barcelona Spain who were diagnosed with ACLF or AD according to diagnostic criteria of Consortium definition (27) (28) and healthy individuals were enrolled between December 1st, 2020, and June 18th, 2021. Patients with previous coagulation disorders or thrombotic events, those with transfusions of blood products (plasma, platelets, or fibrinogen) within the last 7 days before admission were excluded, as well as those taking anticoagulants or platelet aggregation inhibitors.

Outcome Measurements

Demographic data, the severity of liver disease evaluated with Child-Pugh, Model for End-Stage Liver Disease (MELD), Sequential Organ Failure Assessment (SOFA), and chronic liver failure (CLIF)–SOFA scores were collected. Data on haemorrhagic or thrombotic events and on transfusion requirements were collected throughout hospitalization. Bleeding events were defined according to the following criteria: fatal bleeding, symptomatic bleeding in a critical area or organ, and/or bleeding causing a fall in haemoglobin level of ≥ 2 g/dL or leading to transfusion ≥ 2 units of whole blood or packed red cells.(29)

Standard Coagulation Test (international normalized ratio [INR], aPTT, platelet count, and fibrinogen) and routine laboratory tests also were collected.

Pre-test Study

Initially we analysed blood samples from 10 patients with ACLF, 10 patients with AD, and 10 healthy individuals to assess whether diluted EXTEM reagent (tissue factor activation) improves the effect of Protac® on clotting time (CT)-ratio with and without Protac®. The pre-test study was based on the hypothesis that ROTEM tests with reduced tissue factor concentration might be more sensitive to decreased thrombin generation as expected in advanced stages of liver disease.(20)

Sample Preparation

ROTEM was performed with EXTEM reagent diluted to different levels by mixing with HEPES buffer: 1:1 (undiluted), 1:10, 1:100, and 1:500 diluted. Protac® vials (3 U) were reconstituted with 200 µl saline (15 U/ml corresponding to 0.3 U /20 µl), and then 20 µl of reconstituted Protac® were added to 300 ml of citrated blood and incubated for 5 min at 37 ºC (on the ROTEM preheating platform) before tissue factor activated test were started with different EXTEM reagent dilutions.

The CT-ratio with and without Protac® found with the 1:1 undiluted EXTEM reagent was 1.22 ± 0.35 for healthy individuals and 0.99 ± 0.23 for cirrhotic patients. With the dilution of the EXTEM reagent 1:10, the CT-ratio increases from 1.38 ± 0.29 for healthy volunteers to 1.17 ± 0.15 for cirrhotic patients. Accordingly, no advantage in performing the test with diluted EXTEM reagent could be demonstrated. With dilutions 1:100 and 1:500 the results did not provide any advantage, too. Therefore, the main study was carried out with the undiluted EXTEM reagent according to the instructions of the manufacturer.

Main Study

Twenty consecutive AD, 10 ACLF patients, and 21 healthy individuals were included in the main study. Here, standard, velocity, fibrinolysis and calculated thromboelastometric indices were used to discriminate between patients with liver disease from healthy individuals and to discriminate between ACLF and AD patients, as well.

Thromboelastometry (ROTEM) Assays and Parameter

ROTEM was performed within one hour after blood sampling. Blood samples were collected in a BD Vacutainer (9NC 0.129M) through a peripheral venous catheter during the first 24 hours of admission. ROTEM was performed with a point-of-care rotational thromboelastometry (ROTEM) delta device (TEM Innovations, Munich Germany). Measurements were carried out by extrinsic activation (tissue factor at different concentrations; EXTEM and diluted EXTEM), extrinsic activation with platelet inhibition (tissue factor plus cytochalasin D; FIBTEM), intrinsic activation (ellagic acid without and with heparinase; INTEM and HEPTEM) and running tests with and without Protac® (Pentapharm, Basel, Switzerland), a snake venom extract that activates protein C (PC).(30)

Standard ROTEM parameters

clotting time (CT in s), reflecting the initiation of the clotting process; clot formation time (CFT in s), reflecting the propagation of clot formation; time-to-20 mm clot firmness amplitude (TT20 = CT + CFT in s) (31); alpha angle (ALPHA in °), reflecting clot kinetics; clot firmness amplitude 5 min after CT (A5 in mm), clot firmness amplitude 10 min after CT (A10), and maximum clot firmness (MCF in mm).

Calculated thrombosis indices

shear elastic modulus strength (G = 5000 x MCF / (100 – MCF)) and thrombodynamic potential index (TPI = [(100 x MCF) / (100 – MCF)] / CFT in s^− 1).(32)

Velocity parameters for the first derivate curve

area under the first derivative curve until MCF (AUC in mm x 100), maximum velocity (maxV in mm/min), and time to maximum velocity (maxV-T in s).

Fibrinolysis parameters

lysis index 30, 45, and 60 min after CT (LI30, LI45 and LI60, reflecting residual clot firmness amplitude in % of MCF at 30, 45, and 60 min after CT), and maximum lysis (ML reflecting the decrease in clot firmness amplitude in % of MCF during run time).

The first derivative of the thromboelastometric curve (velocity curve) described by Sorensen et al. shows a profile similar to the thrombin generation curve.(33) Here, the ROTEM velocity parameter maxV-t corresponds to the thrombin burst rate (time to peak (ttP)), maxV to the thrombin generation peak (TPi), and AUC to the total amount of thrombin generated (endogenous thrombin potential (ETP)). CalcuRo software (Tem Innovations, Munich, Germany) was used to evaluate the rate of tensile strength exerted during clot formation.

Definition of a Hypocoagulable Profile

Presence of three or more parameters outside the reference range in patients with both EXTEM and INTEM tests (prolonged CT or CFT, decreased alpha angle, and decreased MCF) and two or more parameters in those with just the EXTEM test.(19)

Definition of a Hypercoagulable profile

Presence of at least two of the following: shortened CT or CFT, increased alpha angle, or increased MCF.(19)

Statistical Analysis

No sample size calculation was carried out for this pilot study. Continuous variables were expressed as median (interquartile range), and categorical variables were presented as numbers (percentages). Missing data was minimal (below 0.5%) and missing observations were not imputed. Comparisons between groups were conducted with either the Fisher Exact test or the Mann-Whitney test. Receiver operating characteristic (ROC) curve analyses were performed to differentiate patients with liver disease from healthy subjects, using ROTEM parameters which were significantly different between both groups.

A threshold of 0.05 was used for statistical significance. All analyses were performed using the Statistical Package for Social Sciences, version 22.0(SPSS, Chicago, IL); and the level of significance was established at the two-sided 5% level.

Demographic data of healthy controls and patients with liver disease are presented in Table 1. Alcohol was the most frequent etiology in all patients. Sixty percent of patients with acute-on-chronic liver failure (ACLF) were grade 3, at expenses of haemodynamics, renal and liver failure, predominantly. Four patients with acute decompensation of cirrhosis (AD) and 3 with ACLF had a bleeding event portal hypertension related during hospitalization. No patient had thrombotic events. One patient with ACLF died after 2 weeks of admission due to sepsis complicated with multiorgan failure.

Table 1

DEMOGRAFIC DATA OF LIVER PATIENT POPULATIONS AND HEALTHY CONTROLS (MEDIANS AND INTERQUARTILE RANGES)
DEMOGRAPHIC DATA	HEALTHY CONTROLS (CON; N = 21)	ACUTE DECOMPENSATION (AD; N = 20)	ACUTE-ON-CHRONIC LIVER FAILURE (ACLF; N = 10)
Age [years]	53 (48–70)	62 (51–68)	56 (52–67)
Sex [male/female]	10/12	14/6	7/3
Child [A/B/C]	-	1/15/4	1/1/8
Etiology of the liver disease [OH/HCV/NASH/others]	-	8/4/6/2	7/1/2/0
MELD Score	-	18 (14–24)	26 (19–30)
Hemoglobin [g/dL]	13.1 (12.3–14.0)	9.6 (8.8–9.7)	9.1 (7.3–10.0)
ACLF grade* [1/2/3]	-	-	2/2/6
Creatinin [mg/dL]	-	1.1 (0.7–2.7)	2.2 (1.0-4.2)
Na [mmol/L]	-	136 (134–139)	137 (134–140)
K [mmol/L]	-	3.8 (3.5–4.1)	4 (3.6–4.4)
Albumin [g/L]	-	32 (23–33)	24 (23–29)
Bilirubin [mg/dL]	-	2.3 (1.5–3.4)	9 (2–22)
PT [Quick; %]	-	49 (46–58)	37 (33–46)
APTT [sec]	-	32 (29–35)	39 (31–50)
Platelet count [nL^− 1]	-	65 (55–101)	46 (33–98)
Fibrinogen [g/L]	-	2.3 (1.9–2.6)	1.9 (1.4–2.9)

* at the time of blood sampling.

1. Pre-test study: Diluted EXTEM reagent did not improve the diagnostic performance of the effect of Protac® on clotting time (CT)-ratio (with and without Protac®).

Accordingly, the main study was carried out with the undiluted EXTEM reagent according to the instructions of the manufacturer (see methods section).

2. Samples from patients with cirrhosis show a hypocoagulable profile in standard thromboelastometry when compared with healthy individuals.

Thromboelastometry performed in standard conditions showed an hypocoagulable profile in 55% of the patients whit chronic liver disease compared to healthy controls who no one showed hypocoagulability. Kinetic parameters, such as CT and CFT, were significantly prolonged in the former. Additionally, maximum clot firmness (MCF) was significantly lower in patients compared to controls. Overall, there were no significant differences in thromboelastometry parameters across the severity of liver disease. LI60 in EXTEM was significantly higher in patients with ACLF vs. controls: 99 (96–100) vs. 93 (90–96), P < 0.01. Velocity parameters from the first derivative curve were in agreement with standard thromboelastometric parameters, with lower area under the curve (AUC) and maximum velocity (maxV) in liver patients compared to controls, whereas the time to reach the maximum velocity (maxV-t) showed more variability (Table 2.1).

Table 2.1

STANDARD THROMBOELASTOMETRIC TESTS IN LIVER PATIENT POPULATIONS AND HEALTHY CONTROLS (ANOVA WITH BONFERRONI ADJUSTMENT)
	HEALTHY CONTROLS (CON; a; N = 21	ACUTE DECOMPENSATION (AD; b; N = 20	ACUTE-ON-CHRONIC LIVER DISEASE (ACLF; c; N = 10	P-VALUES
EXTEM Reference Ranges)
CT (38–79 sec)	55 (51–60)	62 (56–74)	71 (64–77)	< 0.05, a vs. b and c
CFT (34–159 sec)	80 (72–92)	146 (100–181)	139 (94–214)	< 0.05, a vs. b and c
TT20 (CT + CFT)	141 (126–149)	221 (159–255)	205 (168–281)	< 0.01, a vs. b and c
ALPHA (63–83˚)	74 (71–75)	67 (59–74)	65 (56–71)	< 0.05, a vs. b and c
A5	48 (43–52)	32 (28–38)	30 (19–39)	< 0.01, a vs. b and c
A10 (43–65 mm)	58 (54–60)	43 (37–48)	44 (35–54)	< 0.01, a vs. b and c
MCF (50–72 mm)	65 (63–67)	52 (48–57)	53 (46–57)	< 0.01, a vs. b and c
G	9108 (8534–10267)	5402 (4661–6759)	5923 (4476–7880)	< 0.01, a vs. b and c
TPI	2.33 (1.82–2.81)	0.69 (0.53–1.30)	0.84 (0.42–1.37)	< 0.01, a vs. b and c
INTEM (Reference Ranges)
CT (100–240 sec)	182 (167–184)	190 (163–218)	215 (194–247)	ns
CFT (30–110 sec)	67 (61–83)	138 (103–172)	136 (93–213)	< 0.01, a vs. b and c
TT20 (CT + CFT)	255 (239–275)	326 (306–349)	369 (312–442)	< 0.01, a vs. b and c
ALPHA (70–83˚)	76 (73–77)	67 (60–71)	62 (55–70)	< 0.01, a vs. b and c
A5	46 (42–49)	33 (28–37)	32 (24–38)	< 0.01, a vs. b and c
A10 (44–66 mm)	57 (54–59)	43 (37–47)	41 (33–47)	< 0.01, a vs. b and c
MCF (50–72 mm)	62 (61–65)	50 (45–56)	51 (43–55)	< 0.01, a vs. b and c
G	8322 (7988–9442)	5278 (4313–6558)	5192 (3918–6360)	ns
TPI	2.43 (1.79–3.04)	0.72 (0.51–1.11)	0.80 (0.35–1.30)	< 0.01, a vs. b and c
CT-Ratio (INTEM/HEPTEM)	1.04 (0.95–1.08)	0.96 (0.89–1.07)	1.03(1.01–1.25)	ns
FIBTEM (Reference Ranges)
CT	52 (49–57)	61 (50–67)	61 (51–77)	ns
A5	16 (12–17)	14 (10–16)	10 (8–14)	ns
A10 (7–23 mm)	16 (13–19)	14 (11–16)	10 (8–16)	ns
MCF (9–25 mm)	17 (14–19)	15 (12–18)	11 (9–17)	ns
VELOCITY PARAMETER
EXTEM AUC	6446 (6263–6731)	5152 (4777–5692)	5405 (4705–6049)	< 0.01, a vs. b and c
EXTEM maxV	15 (13–17)	11 (8–14)	10 (8–14)	< 0.01, a vs. b and c
EXTEM maxV-t	103 (73–118)	92 (63–145)	105 (85–129)	ns
FIBTEM AUC	1699 (1409–2161)	1532 (1206–1817)	1095 (965–1601)	ns
FIBTEM maxV	14 (11–16)	10 (8–16)	8 (6–11)	ns
FIBTEM maxV-t	58 (53–69)	66 (61–77)	72 (60–80)	ns
INTEM AUC	6257 (6082–6427)	5178 (4662–5687)	5241 (4395–5618)	< 0.01, a vs. b and c
INTEM maxV	18 (15–20)	11 (9–13)	10 (7–13)	< 0.01, a vs. b and c
INTEM maxV-t	205 (193–224)	205 (180–243)	254 (214–288)	0.05, b vs. c
FIBRINOLIYSIS PARAMETER
EXTEM LI30 (94–100%)	100 (100–100)	100 (100–100)	100 (100–100)	ns
EXTEM LI45	97 (96–98)	99 (96–100)	100 (99–100)	< 0.05 a vs. c
EXTEMLI60 (86–100%)	93 (90–96)	96 (91–98)	99 (96–100)	< 0.05 a vs. c
EXTEM ML (< 15% at 60 min after CT)	7.0 (4.0–9.0)	4.0 (2.0-8.5)	1.0 (0.0-3.2)	< 0.05 a vs. c
FIBTEM LI30	100 (100–100)	100 (99–100)	100 (97–100)	ns
FIBTEM LI45	100 (100–100)	100 (98–100)	100 (97–100)	ns
FIBTEM LI60	100 (99–100)	100 (97–100)	100 (96–100)	ns
FIBTEM ML	0 (0-0.5)	0 (0-2.5)	0 (0-3.5)	ns
ΔLI60 (FIBTEM – EXTEM)	6(4–9)	2.5(1-6.7)	0.5(-2.7-3.2)	< 0.05 a vs. c

3. Healthy individuals, but not patients with cirrhosis, show a pattern of hypocoagulability in thromboelastometry performed after the addition of Protac® to the blood sample.

ROTEM kinetic parameters were prolonged when Protac® was added to blood samples from healthy subjects: TT20 EXTEM, 141 (126–149) vs. 163 (140–183) s, P = 0.003 and INTEM CFT, 67 (61–83) vs. 81 (72–97) s, P = 0.009. This effect was less pronounced in patients with AD cirrhosis (only INTEM CT increased from (190 (163–218) to 212 (166–237) s, P = 0.02) after adding Protac®, whereas in patients with ACLF opposite changes were observed: CFT, 136 (93–213) vs. 119 (76–207) s, P = 0.02, and TPI, 0.80 (0.35–1.30) vs. 1.01 (0.34–1.50) s^− 1, P = 0.01, without and with Protac®, respectively (Table 3.1). Few variations were seen on velocity parameters in healthy controls and in patients after the Protac® challenge. EXTEM LI60 significantly increased with compared to without Protac® in healthy subjects and in patients with AD. No changes were observed in FIBTEM LI60 run in parallel. (Table 3.1).

Table 3.1

THROMBOELASTOMETRIC TESTS WITHOUT and WITH PROTAC CHALLENGE IN LIVER PATIENT POPULATIONS AND HEALTHY CONTROLS
	WITHOUT PROTAC	WITH PROTAC	P-VALUE			RATIO W/WHO PROTAC
HEALTHY CONTROLS (N = 21)
EXTEM
CT	55 (51–60)	66 (53–77)	0.02			1.2
CFT	80 (72–92)	92 (80–108)	0.01			1.1
TT20 (CT + CFT)	141 (126–149)	163 (140–183)	0.003			1.1
ALPHA	74 (71–75)	72 (69–74)	0.03			0.9
A5	48 (43–52)	44 (41–48)	0.01			0.9
A10	58 (54–60)	55 (53–59)	0.02			0.9
MCF	65 (63–67)	65 (62–68)	0.59			1.0
G	9108 (8534–10267)	9511 (8507–10735)	0.25			1.0
TPI	2.33 (1.82–2.81)	2.04 (1.54–2.61)	0.17			0.9
INTEM
CT	182 (167–194)	180 (157–199)	0.66			0.9
CFT	67 (61–83)	81 (72–97)	0.009			1.1
CT + CFT (TT20)	255 (239–275)	255 (247–288)	0.38			1.0
ALPHA	76 (73–77)	73 (71–75)	0.03			0.9
A5	46 (42–49)	45 (43–48)	0.36			0.9
A10	57 (54–59)	56 (53–58)	0.43			0.9
MCF	62 (61–65)	62 (60–66)	0.57			1.0
G	8322 (7988–9442)	9210 (8193–9797)	0.28			1.0
TPI	2.43 (1.79–3.04)	2.02 (1.68–2.34)	0.10			0.9
FIBTEM
CT	52 (49–57)	59 (54–67)	0.01			1.1
A5	16 (12–17)	14 (13–16)	0.22			0.9
A10	16 (13–19)	15 (14–18)	0.42			0.9
MCF	17 (14–19)	16 (15–19)	0.57			0.9
VELOCITY PARAMETER
EXTEM AUC	6446 (6263–6731)	6518 (6262–6819)	0.54			1.0
EXTEM maxV	15 (13–17)	14 (11–17)	0.25			0.9
EXTEM maxV-t	103 (73–118)	127 (87–146)	0.03			1.2
FIBTEM AUC	1699 (1409–2161)	1601 (1463–1798)	0.09			0.9
FIBTEM maxV	14 (11–16)	12 (9–15)	0.19			0.9
FIBTEM maxV-t	58 (53–69)	74 (59–85)	0.06			1.3
INTEM AUC	6257 (6082–6427)	6454 (6278–6657)	0.02			1.0
INTEM maxV	18 (15–20)	17 (15–18)	0.21			1.3
INTEM maxV-t	205 (193–224)	229 (199–250)	0.23			1.0
FIBRINOLYSIS PARAMETER
EXTEM LI30	100 (100–100)	100 (100–100)	0.10			1.0
EXTEM LI45	97 (96–98)	100 (98–100)	< 0.001			1.0
EXTEM LI60	93 (90–96)	98 (95–99)	< 0.001			1.0
EXTEM ML	7 (4–9)	2 (1–5)	< 0.001			0.3
FIBTEM LI30	100 (100–100)	98 (100–100)	0.12			0.9
FIBTEM LI45	100 (100–100)	97 (100–100)	0.10			0.9
FIBTEM LI60	100 (99–100)	100 (100–100)	0.53			0.9
FIBTEM ML	0 (0-0.5)	0 (0-3.5)	0.53			0.8
ACUTE DECOMPENSATED PATIENTS (AD; N = 20)
EXTEM
CT	62 (56–74)	63 (55–75)	0.67	1.0
CFT	146 (100–181)	136 (113–169)	0.49	1.0
TT20 (CT + CFT)	221 (159–255)	198 (173–248)	0.77	1.0
ALPHA	67 (59–74)	67 (63–70)	0.54	1.0
A5	32 (28–38)	33 (29–38)	0.60	1.0
A10	43 (37–48)	44 (39–47)	0.46	0.9
MCF	52 (48–57)	52 (48–56)	0.61	0.9
G	5402 (4661–6759)	5624 (4738–6519)	0.50	1.0
TPI	0.69 (0.53–1.30)	0.80 (0.61–1.04)	0.27	1.0
INTEM
CT	190 (163–218)	212 (166–237)	0.02	1.1
CFT	138 (103–172)	106 (88–152)	0.067	0.9
TT20 (CT + CFT)	326 (306–349)	312 (278–348)	0.73	1.0
ALPHA	67 (60–71)	70 (62–72)	0.43	1.0
A5	33 (28–37)	35 (29–38)	0.79	1.0
A10	43 (37–47)	44 (39–47)	0.74	1.0
MCF	50 (45–56)	50 (47–56)	0.72	1.0
G	5278 (4313–6558)	5595 (4445–6604)	0.61	1.0
TPI	0.72 (0.51–1.11)	1.02 (0.59–1.33)	0.48	1.5
FIBTEM
CT	61 (50–67)	65 (58–72)	0.03	1.0
A5	14 (10–16)	12 (8–16)	0.06	0.8
A10	14 (11–16)	13 (9–17)	0.07	0.9
MCF	15 (12–18)	13 (9–17)	0.11	0.9
VELOCITY PARAMETER
EXTEM AUC	5152 (4777–5692)	5191 (4851–5590)	0.47	0.9
EXTEM maxV	11 (8–14)	10 (9–12)	0.35	0.9
EXTEM maxV-t	92 (63–145)	85 (68–155)	0.38	1.2
FIBTEM AUC	1532 (1206–1817)	1340 (841–1652)	0.01	0.8
FIBTEM maxV	10 (8–16)	10 (8–14)	0.08	0.8
FIBTEM maxV-t	66 (61–77)	69 (64–79)	0.40	1.0
INTEM AUC	5178 (4662–5687)	5239 (4976–5670)	0.57	0.9
INTEM maxV	11 (9–13)	11 (10–13)	0.51	1.0
INTEM maxV-t	205 (180–243)	215 (196–256)	0.05	1.0
FIBRINOLYSIS PARAMETER
EXTEM LI30	100 (100–100)	100 (100–100)	0.31	1.0
EXTEM LI45	99 (96–100)	100 (99–100)	0.06	1.0
EXTEM LI60	96 (91–98)	97 (95–98)	0.02	1.0
ML EXTEM	4.0 (2.0-8.5)	2.5 (2.0-4.7)	0.02	0.7
FIBTEM LI30	100 (99–100)	100 (100–100)	0.49	0.9
FIBTEM LI45	100 (98–100)	100 (100–100)	0.48	0.9
FIBTEM LI60	100 (97–100)	100 (98–100)	0.48	0.9
ML FIBTEM	0 (0-2.5)	0 (0-1.5)	0.48	0.5
ACUTE-ON-CHRONIC LIVER FAILURE PATIENTS (ACLF, N = 10)
EXTEM
CT	71 (64–77)	64 (54–76)	0.28		0.9
CFT	139 (94–214)	159 (98–251)	0.95		1.0
TT20 (CT + CFT)	205 (168–281)	221 (160–334)	0.50		1.0
ALPHA	65 (56–71)	62 (57–72)	0.85		1.0
A5	30 (19–39)	35 (22–47)	0.41		0.9
A10	44 (35–54)	41 (31–52)	0.72		0.9
MCF	53 (46–57)	51 (41–60)	0.85		1.0
G	5923 (4476–7880)	5380 (3617–7748)	0.50		0.9
TPI	0.84 (0.42–1.37)	0.69 (0.29–1.64)	0.87		1.1
INTEM
CT	215 (194–247)	212 (187–238)	0.61		0.9
CFT	136 (93–213)	119 (76–207)	0.02		0.9
TT20 (CT + CFT)	369 (312–442)	331 (260–458)	0.16		0.9
ALFA	62 (55–70)	67 (61–72)	0.04		1.0
A5	32 (24–38)	33 (24–40)	0.59		1.0
A10	41 (33–47)	41 (32–49)	0.71		0.9
MCF	51 (43–55)	51 (41–56)	0.71		0.9
G	5192 (3918–6360)	5144 (3541–6784)	0.72		1.0
TPI	0.80 (0.35–1.30)	1.01 (0.34–1.50)	0.01		1.1
FIBTEM
CT	61 (51–77)	65 (54–85)	0.30		1.1
A5	10 (8–14)	10 (6–15)	0.36		0.9
A10	10 (8–16)	11 (6–16)	0.57		0.9
MCF	11 (9–17)	12 (9–18)	0.59		1.9
VELOCITY PARAMETER
EXTEM AUC	5405 (4705–6049)	5145 (4194–6064)	0.57		0.9
EXTEM maxV	10 (8–14)	9 (7–13)	0.85		1.0
EXTEM maxV-t	105 (85–129)	103 (57–144)	0.95		1.1
FIBTEM AUC	1095 (965–1601)	1244 (712–1776)	0.87		1.0
FIBTEM maxV	8 (6–11)	10 (4–15)	1.00		1.0
FIBTEM maxV-t	72 (60–80)	71 (63–89)	0.33		1.1
INTEM AUC	5241 (4395–5618)	5028 (4140–5719)	0.50		0.9
INTEM maxV	10 (7–13)	9 (7–15)	0.30		1.0
INTEM maxV-t	254 (214–288)	265 (222–286)	0.67		0.9
FIBRINOLYSIS PARAMETER
EXTEM LI30	100 (100–100)	100 (100–100)	1.00		1.0
EXTEM LI45	100 (99–100)	100 (99–100)	0.10		1.0
EXTEM LI60	99 (96–100)	99 (97–100)	0.04		1.0
EXTEM ML	1.0 (0.0-3.2)	0.5 (0.0-2.2)	0.02		0.4
FIBTEM LI30	100 (97–100)	100 (94–100)	0.71		0.9
FIBTEM LI45	100 (97–100)	100 (90–100)	0.46		0.9
FIBTEM LI60	100 (96–100)	100 (96–100)	0.71		0.9
FIBTEM ML	0.0 (0.0-3.5)	0.0 (0.0–14)	0.71		0.5

4. Differences in standard, velocity, and fibrinolysis thromboelastometric parameters were observed between the liver patient population and healthy controls.

ROC curve analyses, using ROTEM parameters were significantly different between both groups (Table 2.2). INTEM TPI (inverse), INTEM CFT and INTEM A5 (inverse) showed the highest ROC AUC: 0.928, 0.921, and 0.906, respectively (all P-values < 0.001). INTEM CFT provided the highest sensitivity (90%) and INTEM A5 (inverse) the highest specificity (100%). Overall, INTEM TPI (inverse) performed at best as it includes CFT and A5 (sensitivity 80%, specificity 100%, PPV 100%, NPV 78%).

Additionally, EXTEM LI60 and INTEM maxV-t were different between patients with AD and ACLF (ROC AUC of 0.743, P = 0.033; and 0.723, P = 0.050; respectively). Discrimination improved moderately by combining both parameters, INTEM maxV-t + EXTEM LI60 (ROC AUC = 0.81, P < 0.001).

Table 2.2

ROC CURVE ANALYZES FOR STANDARD THROMBOELASTOMETRIC TESTS TO IDENTIFY COAGULATION PATTERN OF LIVER PATIENT POPULATIONS AND HEALTHY CONTROLS
ROTEM PARAMETER	ROC AUC	95% CI	P-VALUE	OPTIMUM CUT-OFF	SENS	SPEC	PPV	NPV
HEALTHY CONTROLS vs. LIVER DISEASE PATIENTS (AD + ACLF)
EXTEM CT	0.788	0.658–0.915	0.001	56	76%	60%	77%	62%
EXTEM CFT	0.863	0.759–0.968	< 0.001	96	83%	81%	83%	81%
EXTEM TT20	0.901	0.811–0.991	< 0.001	149	90%	76%	90%	76%
EXTEM A5	0.100	0.012–0.188	< 0.001	43	20	23	27	17
1 / EXTEM A5	0.900	0.812–0.988	< 0.001	0.025	80	100	100	78
EXTEM TPI	0.110	0.014–0.205	< 0.001	1.17	33	5	33	4.8
1 / EXTEM TPI	0.890	0.795–0.986	< 0.001	0.683	80	95	96	77
EXTEM maxV	0.171	0.057–0.285	< 0.001	12	36	19	39	17
EXTEM LI60	0.741	0.606–0.877	0.004	92	80	47	69	62
EXTEM ML (at 60 min after CT)	0.259	0.123–0.394	0.004	3.5	40	15	40	14
ΔLI60 (FIB-EX)	0.263	0.120–0.405	0.004	-0.9	93	5	56	20
INTEM CFT	0.921	0.845–0.997	< 0.001	82	90	77	84	84
INTEM TT20	0.882	0.776–0.987	< 0.001	304	80	96	96	77
INTEM A5	0.094	0.003–0.185	< 0.001	39	20	10	24	8
1 / INTEM A5	0.906	0.815–0.997	< 0.001	0.026	80	100	96	77
INTEM TPI	0.072	0.000-0.147	< 0.001	1.3	20	15	24	8
1 / INTEM TPI	0.928	0.853-1.000	< 0.001	0.842	80	100	100	78
INTEM maxV	0.138	0.033–0.243	< 0.001	10	53	5	44	7
AD (0) vs. ACLF (1)
EXTEM CFT	0.640	0.414–0.866	0.218	66	80	65	80	65
EXTEM L60	0.743	0.553–0.932	0.033	96	80	50	80	50
EXTEM ML	0.258	0.068–0.447	0.033	0.5	70	5	70	5
1 / EXTEM ML	0.677	0.450–0.904	0.174	0.225	85	52	40	90
ΔLI60 (FIB-EX)	0.295	0.096–0.494	0.071	-0.5	80	10	80	10
FIBTEM AUC	0.370	0.158–0.582	0.253	866	90	20	90	20
INTEM maxV-t	0.723	0.523–0.922	0.050	238	70	75	70	75

5. The addition of Protac® to the blood samples did not improve the ability of standard, velocity, and fibrinolysis thromboelastometric tests performed without Protac® to identify the haemostasis pattern of liver patients from the haemostasis pattern of healthy controls.

To investigate the potential benefit of Protac® in identifying the haemostasis pattern of patients with liver disease, we repeated the ROC curve analysis including thromboelastometry tests performed with and without Protac®. None of the thromboelastometric parameters with Protac® discriminated better between patients and controls compared to the same assay and parameter performed without Protac® (data non shown). To further investigate the potential ability of Protac® to identify the haemostasis pattern of patients with liver disease, we calculated the ROC curves of the ratio of all thromboelastometry parameters performed without and with Protac® (Table 3.2). Overall, no significant improvement in the discrimination between patients with liver disease and healthy controls was observed after addition of Protac® to the standard thromboelastometry tests.

EXTEM LI60-ratio (inverse) and INTEM CFT-ratio (inverse), both with an optimal cut-off of 0.95, showed the best ROC AUC (0.824 and 0.817, respectively; both P-values < 0.001). However, they do not increase the ability to define the hemostatic pattern of the same test performed without Protac® (Table 3.2). EXTEM LI60-ratio showed a sensitivity of 96% and INTEM CFT-ratio a sensitivity of 90% for differentiating healthy controls from patients with liver disease, but specificity was very low (5%) in both cases (Table 3.2, 4.1, 4.2)

Table 3.2

ROC CURVE ANALYZES FOR RATIOS WITHOUT AND WITH PROTAC CHALLENGE TO IDENTIFY COAGULATION PATTERN OF PATIENTS WITH LIVER DISEASE AND HEALTHY CONTROLS
ROTEM PARAMETER	ROC AUC	95% CI	P-VALUE	OPTIMUM CUT-OFF	SENS	SPEC	PPV	NPV
HEALTHY CONTROLS vs. LIVER DISEASE PATIENTS (AD + ACLF)
EXTEM CT-ratio	0.325	0.162–0.487	0.034	0.86	76	29	61	46
EXTEM CFT-ratio	0.420	0.262–0.578	0.334	0.92	73	15	55	27
EXTEM TT20-ratio	0.294	0.150–0.439	0.013	0.92	63	24	54	31
EXTEM A5-ratio	0.613	0.460–0.767	0.171	0.91	70	43	61	47
EXTEM maxV-t-ratio	0.416	0.258–0.573	0.130	0.88	66	24	56	33
EXTEM LI60-ratio	0.176	0.052-0.300	< 0.001	0.99	96	5	59	50
1 / EXTEM LI60-ratio	0.824	0.700-0.948	< 0.001	0.95	86	53	71	71
EXTEM ML-ratio	0.703	0.550–0.857	0.018	0.36	73	58	59	50
FIBTEM CT-ratio	0.452	0.291–0.612	0.559	1.01	70	24	57	36
FIBTEM ML-ratio	0.611	0.298–0.925	0.505	0.23	55	80	83	50
FIBTEM AUC-ratio	0.454	0.291–0.616	0.579	0.73	80	24	60	45
INTEM CT-ratio	0.624	0.465–0.783	0.135	0.86	86	44	63	60
INTEM CFT-ratio	0.183	0.058–0.307	< 0.001	0.56	90	5	57	25
1 / INTEM CFT-ratio	0.817	0.693–0.942	< 0.001	0.95	80	71	80	71
INTEM TPI-ratio	0.659	0.504–0.813	0.056	0.78	86	43	67	67
INTEM AUC-ratio	0.297	0.150–0.443	0.014	0.94	73	10	54	20
AD vs. ACLF
EXTEM LI60-ratio	0.445	0.234–0.656	0.628	1.00	50	45	33	66
1 / EXTEM LI60-ratio	0.500	0.275–0.725	1.00	0.00	100	0	35	65
EXTEM ML-ratio	0.350	0.102–0.597	0.248	0.43	57	32	24	67
INTEM CFT-ratio	0.512	0.307–0.718	0.912	0.88	70	50	39	75
INTEM TPI-ratio	0.555	0.350–0.670	0.628	1.00	90	50	47	90

Table 4.1

EXTEM AND INTEM KINETIC PARAMETER (CT AND CFT) DIFFERENCES WITHOUT AND WITH PROTAC CHALLENGE
DIFFERENCES IN EXTEM AND INTEM KINETIC PARAMETERS	CONTROLS (N = 21)	AD + ACLF (N = 30)	P-VALUE (t-test)
EXTEM ΔCT	16 (-8-21)	0 (-9-6)	0.01
EXTEM ΔCFT	7 (-1-22)	6 (-17-24)	0.33
INTEM ΔCT	-2 (-31-17)	15 (-18-28)	0.20
INTEM ΔCFT	12 (-0.5-24)	-12 (-34-2)	< 0.001

Table 4.2

ROC CURVE ANALYSES REGARDING EXTEM AND INTEM KINETIC PARAMETER (CT AND CFT) DIFFERENCES WITHOUT AND WITH PROTAC CHALLENGE
DIFFERENCES IN EXTEM AND INTEM KINETIC PARAMETER	ROC AUC	95% CI	P-VALUE	OPTIMUM CUT-OFF	SENS	SPEC	PPV	NPV
HEALTHY CONTROLS vs. LIVER DISEASE PATIENTS (AD + ACLD)
EXTEM ΔCT	0.337	0.175–0.498	0.049	-8.5	76	24	46	30
EXTEM ΔCFT	0.437	0.278–0.596	0.450	-18	76	5	53	12
INTEM ΔCT	0.609	0.448–0.769	0.190	-24.5	86	34	65	63
INTEM ΔCFT	0.199	0.069–0.329	< 0.001	-14.5	56	10	70	71
-INTEM Δ CFT	0.81	0.671–0.931	< 0.001	-4.5	90	74	81	75
HEALTHY CONTROLS vs. AD
EXTEM ΔCT	0.362	0.185–0.539	0.130	-6.5	80	29	52	60
EXTEM ΔCFT	0.452	0.265–0.640	0.602	-1	60	24	43	38
INTEM ΔCT	0.676	0.509–0.844	0.054	-1	75	53	60	69
INTEM ΔCFT	0.238	0.074–0.403	0.004	-49	70	5	41	14
-INTEM ΔCFT	0.762	0.597–0.926	0.004	-4.5	90	70	73	79
HEALTHY CONTROLS vs. ACLF
EXTEM ΔCT	0.286	0.088–0.483	0.057	-7	60	29	29	60
EXTEM ΔCFT	0.407	0.149–0.665	0.410	-18	80	5	29	33
INTEM ΔCT	0.474	0.246–0.701	0.816	-24	70	44	33	70
INTEM ΔCFT	0.121	0.000-0.255	0.001	-28	90	5	31	50
-INTEM ΔCFT	0.879	0.745-1.000	0.001	-6	90	80	56	93
AD vs. ACLF
EXTEM ΔCT	0.363	0.140–0.585	0.226	-17	80	10	31	50
EXTEM ΔCFT	0.488	0.253–0.722	0.912	-18	80	25	35	71
INTEM ΔCT	0.350	0.122–0.578	0.187	-21.5	70	10	28	40
INTEM ΔCFT	0.520	0.316–0.724	0.860	-22.5	80	40	40	80
-INTEM ΔCFT	0.480	0.276–0.684	0.860	-20.5	60	30	36	72

6. Combination of thromboelastometric parameters for ROC curve analysis increases the ability to define the hemostatic pattern of patients with liver disease.

Finally, we combined data from standard ROTEM tests and ROTEM test modified by the addition of Protac® in order to improve the haemostasis pattern definition of patients with liver disease. The quotient INTEM CFT/EXTEM LI60-ratio showed the highest ROC AUC 0.948 (P < 0.001). Similarly, INTEM CFT/EXTEM A5 x EXTEM LI60-ratio and INTEM CFT + EXTEM ML-ratio showed both a ROC AUC of 0.947 (both P-values < 0.001). However, they only marginally increased the ability to identify haemostasis pattern of patients with liver disease observed in EXTEM TT20 + INTEM CFT (AUC of 0.944; P < 0.001) (Table 5). Again, the best combination to define the haemostasis pattern of patients with AD from patients with ACLF was the combination of INTEM maxV-t + EXTEM LI60, which showed a ROC AUC of 0.81 (P < 0.001) (Table 5).

Table 5

ROC CURVE ANALYZES FOR COMBINED ROTEM PARAMETERS TO IDENTIFY COAGULATION PATTERN OF LIVER PATIENT POPULATIONS AND HEALTHY CONTROLS
ROTEM PARAMETER	ROC AUC	95% CI	P-VALUE	OPTIMUM CUT-OFF	SENS	SPEC	PPV	NPV
HEALTHY CONTROLS (CON) vs. LIVER DISEASE PATIENTS (AD + ACLF)
EXTEM TT20 + INTEM CFT	0.944	0.884-1.000	< 0.001	0.254	96%	72%	83%	94%
EXTEM TT20 + EXTEM LI60	0.913	0.827–0.998	< 0.001	0.319	90	66	79	82
INTEM CFT + EXTEM LI60	0.927	0.852-1.00	< 0.001	0.329	90	89	87	85
EXTEM TT20 + INTEM CFT + EXTEM LI60	0.943	0.876-1.00	< 0.001	0.678	83	100	100	81
EXTEM TT20 – EXTEM A5	0.902	0.812–0.991	< 0.001	106	86	81	87	81
EXTEM TT20 / EXTEM A5	0.896	0.825–0.987	< 0.001	4.11	80	100	100	78
EXTEM TT20 / (EXTEM A5 x EXTEM ML)	0.881	0.775–0.987	< 0.001	0.780	80	85	87	78
INTEM CFT – INTEM A5	0.929	0.853-1.000	< 0.001	45.5	90	85	90	86
INTEM CFT / INTEM A5	0.929	0.855-1.000	< 0.001	2.571	80	95	96	77
INTEM CFT – 10 x INTEM TPI	0.926	0.850-1.000	< 0.001	67.6	90	85	87	85
INTEM CFT / INTEM TPI	0.925	0.849-1.000	< 0.001	50.1	90	81	87	85
EXTEM TT20 + EXTEM ML-ratio	0.929	0.852-1.000	< 0.001	0.571	80	90	91	79
EXTEM TT20 x EXTEM ML-ratio	0.778	0.639–0.918	0.001	53.21	80	58	94	67
INTEM CFT – INTEM ΔCFT	0.895	0.802–0.988	< 0.001	87.5	86	85	90	82
INTEM CFT / EXTEM LI60-ratio	0.948	0.893-1.000	< 0.001	89	90	85	90	82
INTEM CFT / EXTEM A5 x EXTEM LI60-ratio	0.947	0.885-1.000	< 0.001	2	90	85	87	89
INTEM CFT + EXTEM ML-ratio	0.947	0.887-1.000	< 0.001	0.587	80	95	95	80
INTEM CFT x EXTEM ML-ratio	0.919	0.841–0.998	< 0.001	97.39	80	90	100	63
INTEM CFT-ratio + EXTEM LI60-ratio	0.879	0.779–0.979	< 0.001	0.711	80	95	96	77
INTEM CFT-ratio x EXTEM LI60-ratio	0.170	0.045–0.295	< 0.001	0.662	80	0.04	56	17
1 / (INTEM CFT-ratio + EXTEM LI60-ratio)	0.121	0.021–0.221	< 0.001	1.211	43	0.04	39	6
1 / (INTEM CFT-ratio x EXTEM LI60-ratio)	0.838	0.720–0.956	< 0.001	0.973	80	85	59	41
AD vs. ACLF
INTEM maxV-t – EXTEM ML	0.745	0.555–0.935	0.031	219	80	65	53	87
INTEM maxV-t / EXTEM ML	0.744	0.508–0.981	0.060	49	85	58	43	92
INTEM maxV-t + EXTEM LI60	0.81	0.636–0.984	0.006	0.324	80	70	57	87
INTEM maxV-t x EXTEM LI60	0.750	0.564–0.936	0.028	21481	80	65	53	87
INTEM maxV-t – INTEM ΔCFT	0.713	0.531–0.894	0.062	191	90	55	37	83

The present study assesses for the first time, the effect of adding Protac® to blood samples from healthy individuals and patients with liver disease, on thromboelastometric parameters. Significant impairment, mainly in kinetic parameters, was observed after adding Protac® to samples from healthy subjects, whereas this effect was negligible in patients with cirrhosis. This study also demonstrated that specific combinations of standard, velocity, and fibrinolysis ROTEM parameters, better define the haemostasis profile of patients with liver disease than thromboelastometric assays performed with Protac® do.

The anticoagulant pathway is underexplored in global test currently used in clinical practice (ROTEM®, TEG®), and it constitutes their main limitations. In-vivo, thrombomodulin, located on the cell membrane of endothelial cells, activates Protein C (the main anticoagulant driver). Accordingly, this important biological step should be reflected by assays including thrombomodulin to properly assess the overall hemostatic performance of the subjects. Protein C activation can be replicated in-vitro by the snake venom Protac®. In the present study, Protein C was activated with Protac® leading to hypocoagulability as reflected by prolonged kinetic and velocity ROTEM parameters in blood samples from healthy individuals treated with Protac® compared to blood samples not treated with Protac®. However, this effect was not seen in samples from patients with liver disease. This finding confirms reports on thrombin generation tests performed with and without thrombomodulin, another way to activate protein C in-vitro.(24) As protein C levels are lower in patients with liver disease compared to healthy individuals, the addition of thrombomodulin obviously has much less impact in the liver disease population. However, when thrombomodulin is added to thrombin generation assays, no differences in thrombin generation between patients and controls have been reported.(23) However, samples from patients with liver disease remained hypocoagulable compared to those from healthy controls when we added Protac® to the thromboelastometric assay in our study. Differences in the inherent nature of both tests (e.g., plasma vs. whole blood), and differences in the protein C activation (thrombomodulin vs. Protac®) may be responsible for these differences.(34)(21). At the same time, we must not lose sight that thromboelastometry assess fibrin formation, not thrombin generation, and deficiencies in fibrin polymerization (otherwise reported in patients with liver disease) could result in an output different from the expected.

Activation of the anticoagulant pathway would be particularly interesting in patients with liver disease given the deficiency in both pro and anticoagulant factors seen in this population: This was the rational to include Protac® in the global test. Surprisingly, none of the thromboelastometric parameters performed with Protac® provided better definition of the haemostasis profile of patients with liver disease than the same parameter performed without Protac®. Again, issues related to the in-vitro activation of the anticoagulant pathway in the absence of endothelial cells may account for this finding. Furthermore, CFT and A5 in the INTEM test showed already an excellent diagnostic performance (ROC AUC, 0.921 and 0.906 respectively; both P-values < 0.001) to identify haemostasis patterns patients with cirrhosis (Table 2.2) which is not easy to improve. Accordingly, TPI, a ROTEM index calculated using MCF and CFT, was the parameter that better defines the haemostasis profile of patients with liver disease revealing that these patients show slower clot formation (CFT) and, to a lesser extent, reduced clot firmness amplitudes (A). Interestingly, these changes are not proportional to the severity of the liver disease, as it could be assumed a priori, pointing to a different haemostasis re-balance in AD than in ACLF patients. TPI could be assessed as a new predictor for bleeding or thrombotic complications, as well as for guiding therapy in actively bleeding patients, in future clinical studies.

The combination of EXTEM TT20 + INTEM CFT (AUC 0.944; P < 0.001) slightly increased the diagnostic performance of the TPI. EXTEM TT20 + INTEM CFT values over 0.254 identified patients with liver disease with a sensitivity of 96% and a specificity of 72% compared to healthy individuals. The addition of Protac® to the standard thromboelastometric parameters (e.g., EXTEM LI60-ratio with and without Protac®), as used in the ROTEM index INTEM CFT / EXTEM LI60-ratio, marginally increased the discriminative performance (ROC AUC 0.948; P < 0.001) of the standard parameters but does actually not justify the inclusion of Protac® in thromboelastometric reagents.

Protac® decreased fibrinolysis when added to samples from healthy subjects (Table 3.1), and EXTEM LI60 values over 96% identified patients with ACLF with sensitivity of 80% and specificity of 50%, vs. patients with AD. (Table 2.2). We interpret this finding with caution because this may be due to a decrease in platelet-mediated clot retraction, rather than an actual decrease in fibrinolytic activity, as Hartmann reported in 2020.(35), since FIBTEM ML does not change. On the other hand, hypofibrinolysis/fibrinolysis shutdown is suggested in patients with ACLF compared to patients with AD. It has been shown that higher values of EXTEM LI60, increased the risk of thrombosis and were associated to disease severity in critically ill COVID-19 patients.(36)(37) Protac® can activate platelet factor 4 which regulates whether the thrombin-thrombomodulin complex results in protein C or thrombin-activatable fibrinolysis inhibitor (TAFI) activation. High platelet factor 4 levels can result in decreased TAFI activation and hyperfibrinolysis.(38) Furthermore, the higher TPI values observed in samples from ACLF patients points to microthrombosis as a potential mechanism of organ failure in this population, as recently suggested. (39) However, we cannot exclude that the differences observed in EXTEM ML or LI60 after Protac® may be due to changes in platelet-mediated clot retraction rather than changes in the fibrinolytic activity, since FIBTEM ML remained unchanged.(35)

Besides the novelty, the main strength of this study is that samples from the same individual, drawn at the same time, were run in parallel with ROTEM assays with and without Protac®. The in-vitro setting always precludes definitive clinical conclusions and it should be realized as a limitation of the study. Due to the small sample size, the fibrinolysis parameter approach to discriminate patients with AD from those with ACLF is more hypothesis generating and has to be confirmed in a bigger cohort. Anyway, standard, velocity, and fibrinolysis ROTEM parameter, particularly if used in combination, provide already excellent useful definition of the haemostasis profile of patients with liver disease

In summary, we report for the first time coagulation assessment by thromboelastometric testing after in-vitro activation of protein C. In the present study, Protac®, as a protein C activator, was added to the blood samples from healthy individuals and patients with liver disease. We demonstrated that specific combination of standard, velocity, and fibrinolysis thromboelastometric parameters accurately defines the haemostasis profile of patients with liver disease consisting in slow clot formation and reduced clot firmness. We also demonstrated that the addition of Protac® to the thromboelastometry test did not result in a better definition of this profile. Future studies with bigger cohorts should be conducted to confirm these data. A multicenter observational trial based on the data of this pilot study regarding complications is planned.

Finally, the findings suggest that haemostasis re-balance could changes across the severity of the liver disease, showing once again, that one size does not fit all, and that specific subgroups needs to be defined in order to provide a more precise and personalized treatment.

Ethics approval and consent to participate: This study was approved by the Ethical Committee of the Hospital Clinic of Barcelona (approval number HCB/2017/0948) on April 4^th, 2018. Healthy individuals and patients were enrolled after giving their written informed consent.

Consent for publication: Not applicable

Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests: Klaus Görlinger works as the medical director of TEM innovations gmbh, Munich, Germany. The others authors declare that they have no competing interests

Funding_This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authors’ contributions: All authors contributed to the study conception and design. The study was designed by Dr A. Blasi, Dr JC.Reverter and K. Görlinger. Preparation,patient recruitment and data collection were performed by Dr A.Calvo, Dr M. Torrente and Dr J. Vidal. The first draft of the manuscript was written by Dr A. Blasi, Dr K.Görlinger and Dr A. Calvo, all the authors commented on previous versions of the manuscript. Dr A. Blasi performed the data analysis. Dr D. Tassies, Dr E. Reverter,Dr J.colmenero and Dr J. Fernandez , reviewed the different contributions of all the authors and contributed to the structure of this manuscript. All the authors read and approved the final manuscript. Dr A. Blasi and Dr JC. Reverter share the senior authorship of this manuscript.

Acknowledge: Not applicable

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Competing interest reported. Klaus Görlinger works as the medical director of TEM innovations gmbh, Munich, Germany. The others authors declare that they have no competing interests

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Haemostasis Patterns in Patients With Acute-on-chronic Liver Failure and Acute Decompensation of Cirrhosis Including Thromboelastometric Tests With and Without the Addition of Protac: a Pilot Study

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Abstract

Introduction

Material And Methods

Study Design

Study Population and Data Collection

Outcome Measurements

Pre-test Study

Sample Preparation

Main Study

Thromboelastometry (ROTEM) Assays and Parameter

Definition of a Hypocoagulable Profile

Definition of a Hypercoagulable profile

Statistical Analysis

Results

Discussion

Declarations

References

Additional Declarations

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