Plasma thrombomodulin as a candidate biomarker for the diagnosis and prognosis of HBV-related acute-on-chronic liver failure

Figures & data

Figure 1 PBMC TM expression analysis. (A) PBMC TM expression levels in patients at different disease stages. (B) ROC curve of PBMC TM expression levels for distinguishing HBV-ACLF from ACHD. (C) PBMC TM expression levels in HBV-ACLF patients with different outcomes at 28 days. (D) ROC curve of PBMC TM expression levels for predicting the 28-day mortality rates of HBV-ACLF patients. (E) PBMC TM expression levels in HBV-ACLF patients with different outcomes at 90 days. (F) ROC curve of PBMC TM expression levels for predicting the 90-day mortality rates of HBV-ACLF patients.

Abbreviations: PBMC, peripheral blood mononuclear cell; TM, thrombomodulin; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; ACHD, acute-on-chronic hepatic dysfunction; LC, liver cirrhosis; CHB, chronic hepatitis B; NC, normal controls; AUROC, area under the receiver operating characteristic; ACLF-D, ACLF-deceased; ACLF-S, ACLF-surviving.

Figure 2 Functional analysis of TM. Histogram showing the Pearson correlation coefficients for the relationships between TM mRNA expression levels and BTMs (left) and GO biological processes (right). The terms that are positively or negatively correlated are shown in red or blue, respectively. The top 20 terms with high correlation coefficients are displayed.

Table 1 Clinical Characteristics of the Patients in the ELISA Group

Characteristics	HBV-ACLF (n=213)	ACHD (n=50)	LC (n=50)	CHB (n=50)	NC (n=30)
Age (years)	50 [38, 58]	48 [44, 55]	47 [42, 53]	46 [41, 51]*	37 [32, 45]**
Male (no.)	83.1% (177)	78.0% (39)	64.0% (32)**	58.0% (29)***	80.0% (24)
MAP (mmHg)	83.7 [75.5, 93.8]	85.3 [77.4, 96.9]	/	/	/
Cirrhosis (no.)	59.2% (126)	74.0% (47)*	100.0% (50)***	0***	0***
Complication
Hepatic encephalopathy	13.1% (28)	2.0% (1)*	/	/	/
Gastrointestinal bleeding	4.2% (9)	30.0% (15)***	/	/	/
Ascites	61.0% (130)	44.0% (22)***	/	/	/
Bacterial infection	26.3% (56)	20.0% (10)	/	/	/
HBV DNA level (IU/mL)		***	***	***	***
≤ 2×10^2	16% (34)	52.0% (26)	94.0% (47)	66.0% (33)	100% (30)
2×10^2–2×10^6	60.1% (128)	34.0% (17)	6.0% (3)	26.0% (13)	0
> 2×10^6	23.9% (51)	14.0% (7)	0	8.0% (4)	0
Laboratory data
ALT (U/L)	191.0 [83.5, 371.5]	36.5 [23.0, 187.0]***	23.5 [16.0, 38.0]***	23.0 [16.0, 32.3]***	20.0 [14.8, 36.3]***
AST (U/L)	122.0 [80.0, 224.5]	51.5 [30.0, 161.0]***	22.0 [19.0, 28.0]***	21.5 [18.0, 28.0]***	22.0 [18.0, 22.5]***
ALB (g/L)	30.6±3.8	30.3±4.5	46.9±2.4***	46.0±3.2***	44.0±1.3***
TB (μmol/l)	354.9 [284.3, 428.0]	39.5 [23.8, 143.0]***	11.4 [8.7, 15.5]***	11.6 [8.9, 15.3]***	14.3 [9.2, 17.1]***
GGT (U/L)	70.0 [49.0, 109.0]	52.0 [21.5, 103.0]*	24.0 [14.8, 37.0]***	15.0 [12.0, 19.3]***	23.0 [16.0, 35.5]***
Cr (μmol/L)	61.0 [52.0, 74.5]	70.5 [62.0, 80.0]***	69.5 [61.8, 79.0]**	73.5 [63.8, 88.0]***	74.0 [62.3, 79.0]**
Serum urea (mmol/L)	4.0 [2.9, 5.6]	4.4 [3.5, 5.8]	4.5 [4.1, 5.6]*	4.6 [4.1, 5.5]**	4.9 [4.4, 5.4]**
Sodium (mmol/L)	138.0 [136.0, 140.0]	140.0 [138.0, 142.0]***	140.0 [141.0, 143.0]***	141.0 [140.0, 142.0]***	/
WBC (10^9/L)	6.9 [5.4, 9.1]	4.0 [2.4, 5.1]***	4.4 [3.6, 6.4]***	5.1 [4.1, 5.9]***	5.6 [5.1, 5.9]**
Neutrophil count (10^9/L)	4.6 [3.3, 6.7]	2.1 [1.1, 3.2]***	2.5 [2.2, 3.8]***	3.0 [2.4, 3.4]***	3.1 [2.6, 3.5]***
PLT (10^9/L)	94.0 [65.5, 130.0]	61.0 [45.0, 138.3]	165.0 [123.8, 213.8]***	181.5 [152.0, 219.0]***	212.0 [199.3, 259.3]***
HGB (g/L)	119.3±21.8	106.3±27.9**	145.8±17.6***	149.8±13.6***	149.8±3.6***
FIB (g/L)	1.3 [1.0, 1.6]	1.5 [1.4, 1.9]**	1.3 [1.0, 1.6]***	2.4 [2.2, 2.6]***	/
PT (s)	21.7 [19.5, 26.7]	15.8 [14.6, 17.5]***	21.7 [19.5, 26.7]***	11.5 [11.1, 12.1]***	/
D dimer (μg/L)	2157 [1088, 3882]	818.5 [438.8, 3290.8]**	/	/	/
INR	1.9 [1.7, 2.4]	1.4 [1.3, 1.5]***	1.9 [1.7, 2.4]***	1.0 [1.0, 1.1]***	/
TM (ng/mL)	8.9 [6.0, 12.9]	3.2 [1.9, 7.0]***	1.5 [0.9, 2.1]***	1.5 [1.0, 2.2]***	1.3 [0.9, 2.4]***
Organ failure (No.)
Liver	97.7% (208)	/	/	/	/
Coagulation	23.5% (50)	/	/	/	/
Kidney	2.8% (6)	/	/	/	/
Cerebral	6.1% (13)	/	/	/	/
Lung	0.9% (2)	/	/	/	/
Circulation	0.9% (2)	/	/	/	/
Severity score
MELDs	22.0 [20.0, 24.9]	/	/	/	/
MELD-Nas	22.9 [21.0, 26.1]	/	/	/	/
COSSH-ACLF IIs	7.0 [6.5, 7.6]	/	/	/	/

Notes: Categorical variables are expressed as % (n); continuous variables are expressed as either the mean ± SD or median (Q1-Q3). (Student’s t test, Mann–Whitney U-test, χ² test or Fisher’s exact test). *P value (<0.05), **P value (<0.01) and ***P value (<0.001) for comparisons between groups.

Abbreviations: HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; ACHD, acute-on-chronic hepatic dysfunction; LC, liver cirrhosis; CHB, chronic hepatitis B; NC, normal controls; MAP, mean arterial pressure; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALB, albumin; TB, total bilirubin; GGT, gamma glutamyl transferase; Cr, creatinine; WBC, white blood cell count; PLT, platelet count; HGB, haemoglobin; FIB, fibrinogen; PT, prothrombin time; INR, international normalized ratio; TM, thrombomodulin; MELDs, model for end-stage liver disease score; MELD-Nas, MELD-sodium score; COSSH-ACLF IIs, Chinese Group on the Study of Severe Hepatitis B-ACLF II score.

Figure 3 The use of plasma TM concentrations for diagnosing HBV-ACLF at admission. (A) Plasma TM concentrations in patients at different disease stages. (B) ROC curve of plasma TM concentrations for distinguishing HBV-ACLF from ACHD. ****P < 0.0001.

Abbreviations: TM, thrombomodulin; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; ACHD, acute-on-chronic hepatic dysfunction; LC, liver cirrhosis; CHB, chronic hepatitis B; NC, normal controls; AUROC, area under the receiver operating characteristic.

Table 2 Associations of Clinical Parameters and Prognostic Scoring Systems with Plasma TM Concentration in Patients with HBV-ACLF

Variable	Regression Coefficient	P
Laboratory data
ALT (U/L)	−0.067	0.334
AST (U/L)	0.001	0.991
ALB (g/L)	−0.237	<0.001
TB (μmol/L)	0.117	0.088
GGT (U/L)	−0.239	<0.001
Cr (μmol/L)	0.261	<0.001
Serum urea (mmol/L)	0.477	<0.001
Sodium (mmol/L)	−0.126	0.066
WBC (10^9/L)	0.102	0.140
Neutrophil count (10^9/L)	0.040	0.558
HGB (g/L)	−0.164	0.017
PLT (10^9/L)	−0.165	0.016
FIB (g/L)	−0.180	0.009
PT (s)	0.225	<0.001
D dimer (μg/L)	0.339	<0.001
INR	0.227	<0.001
Severity score
MELDs	0.411	<0.001
MELD-Nas	0.406	<0.001
COSSH-ACLF IIs	0.411	<0.001

Note: Data are P-value (r-value).

Abbreviations: TM, thrombomodulin; HBV-ACLF, hepatitis B virus-related acute-on-chronic liver failure; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALB, albumin; TB, total bilirubin; GGT, glutamyl transferase; Cr, creatinine; WBC, white blood cell count; PLT, platelet count; HGB, hemoglobin; FIB, fibrinogen; PT, prothrombin time; INR, international normalized ratio; MELDs, model for end-stage liver disease score; MELD-Nas, MELD-sodium score; COSSH-ACLF IIs, Chinese Group on the Study of Severe Hepatitis B-ACLF II score.

Figure 4 Plasma TM concentrations are related to the severity of HBV‐ACLF. (A) Plasma TM concentrations in ACLF‐1 and ACLF‐2/3 patients. (B) Plasma TM concentrations in patients stratified by COSSH-ACLF IIs. (C) Plasma TM concentrations and coagulation failure. (D) Plasma TM concentrations and kidney failure. (E) Plasma TM concentrations and cerebral failure. *P < 0.5; ***P < 0.001; ****P < 0.0001.

Abbreviations: TM, thrombomodulin; ACLF, acute-on-chronic liver failure; COSSH-ACLF IIs, Chinese Group on the Study of Severe Hepatitis B-ACLF II score.

Table 3 Characteristics of Patients in ACLF-D and ACLF-S Group at 28 Days

Characteristics	ACLF-D (n=64)	ACLF-S (n=149)	P value
Age (years)	55 [48, 60]	47 [37, 57]	<0.001
Male (no.)	73.4% (47)	87.2% (130)	0.014
MAP (mmHg)	84.1 [76.8, 96.6]	83.0 [75.0, 92.2]	0.300
Cirrhosis (no.)	67.2% (43)	55.7% (83)	0.118
Complication
Hepatic encephalopathy	32.8% (21)	4.7% (7)	<0.001
Gastrointestinal bleeding	7.8% (5)	2.7% (4)	0.088
Ascites	67.2% (43)	58.4% (87)	0.227
Bacterial infection	29.7% (19)	24.8% (37)	0.461
HBV DNA level (IU/mL)			0.070
≤ 2×10^2	10.9% (7)	18.9% (27)
2×10^2–2×10^6	59.4% (38)	61.7% (92)
> 2×10^6	32.8% (21)	20.1% (30)
Laboratory data
ALT (U/L)	214.0 [91.8, 551.0]	188.0 [81.0, 335.5]	0.159
AST (U/L)	145.0 [76.0, 312.8]	118.0 [80.5, 196.5]	0.104
ALB (g/L)	29.9 [28.0, 32.5]	30.9 [28.2, 33.6]	0.160
TB (μmol/L)	390.6±119.2	344.9±116.7	0.004
GGT (U/L)	61.5 [38.8, 98.0]	77.0 [51.0, 116.5]	0.136
Cr (μmol/L)	58.5 [52.0, 75.5]	62.0 [53.0, 74.5]	0.394
Serum urea (mmol/L)	4.6 [3.1, 6.7]	4.3 [2.8, 5.0]	0.032
Sodium (mmol/L)	138.0 [136.0, 140.8]	138.0 [136.0, 140.0]	0.626
WBC (10^9/L)	7.7 [6.1, 9.5]	6.6 [4.9, 8.8]	0.016
Neutrophil count (10^9/L)	5.7 [4, 7.4]	4.4 [2.9, 6.4]	0.001
PLT (10^9/L)	91.0 [65.5, 118.0]	97.0 [65.5, 134.0]	0.194
HGB (g/L)	116.7±22.6	120.4±21.4	0.266
FIB (g/L)	1.1 [0.9, 1.4]	1.4 [1.1, 1.7]	<0.001
PT (s)	27.4 [22.7, 3.4]	21.0 [18.6, 23.4]	<0.001
D dimer (μg/L)	3184 [2122, 5266]	1742 [751, 3469]	<0.001
INR	2.5 [2.1, 3.2]	1.8 [1.6, 2.1]	<0.001
TM (ng/mL)	11.7 [8.5, 18.1]	7.5 [5.4, 11.9]	<0.001
Organ failure (No.)
Liver	96.9% (62)	98.0% (146)	0.623
Coagulation	51.6% (33)	11.4% (17)	<0.001
Kidney	6.3% (4)	1.3% (2)	0.047
Cerebral	17.2% (11)	1.3% (2)	<0.001
Lung	3.1% (2)	0	0.030
Circulation	1.6% (1)	0.7% (1)	0.536
Severity score
MELDs	24.7 [21.5, 28.7]	21.3 [19.5, 23.3]	<0.001
MELD-Nas	25.9 [22.4, 29.7]	22.2 [20.6, 24.5]	<0.001
COSSH-ACLF IIs	8.0 [7.3, 8.4]	6.8 [6.3, 7.2]	<0.001

Notes: Categorical variables are expressed as % (n); continuous variables are expressed as either the mean ± SD or median (Q1-Q3). (Student’s t-test, Mann–Whitney U-test, χ2 test or Fisher’s exact test). P-values for comparisons between patients in ACLF-D and ACLF-S group.

Abbreviations: ACLF-D, ACLF-deceased; ACLF-S, ACLF-survived; MAP, mean arterial pressure; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALB, albumin; TB, total bilirubin; GGT, glutamyl transferase; Cr, creatinine; WBC, white blood cell count; PLT, platelet count; HGB, hemoglobin; FIB, fibrinogen; PT, prothrombin time; INR, international normalized ratio; TM, thrombomodulin; MELDs, model for end-stage liver disease score; MELD-Nas, MELD-sodium score; COSSH-ACLF IIs, Chinese Group on the Study of Severe Hepatitis B-ACLF II score.

Figure 5 The use of plasma TM concentrations for predicting the 28-day and 90-day mortality rates of HBV-ACLF patients. (A) Plasma TM concentrations in HBV-ACLF patients with different outcomes at 28 days. (B) Plasma TM concentrations in HBV-ACLF patients with different outcomes at 90 days. (C) ROC curve of plasma TM concentrations for predicting the 28-day mortality rates of HBV-ACLF patients compared to other clinical scores. (D) ROC curve of plasma TM concentrations for predicting the 90-day mortality rates of HBV-ACLF patients compared to other clinical scores. (E) Cumulative incidence of death (high‐/low‐risk: plasma TM concentrations ≥8.4/<8.4 ng/mL) in patients with HBV-ACLF in 28 days. (F) Cumulative incidence of death in patients with HBV-ACLF in 90 days. ****P < 0.0001.

Abbreviations: TM, thrombomodulin; ACLF-D, ACLF-deceased; ACLF-S, ACLF-surviving; COSSH-ACLF IIs, Chinese Group on the Study of Severe Hepatitis B-ACLF II score; MELD, Model for End-stage Liver Disease; MELD-Na, MELD-sodium; AUROC, area under the receiver operating characteristic.