IN FOCUS
Increased factor VIII plays a significant role in plasma
hypercoagulability phenotype of patients with cirrhosis
T . S I N E G R E , *† C . D U R O N ,‡ § T . L E C O M P T E , ¶ * * B . P E R E I R A , † † S . M A S S O U L I E R , ‡ G . L A M B L I N , ‡ A . A B E R G E L‡ § and A . L E B R E T O N * †
*Service d’Hematologie Biologique, CHU Clermont-Ferrand; †Universite Clermont Auvergne, INRA, UMR 1019; ‡Service d’Hepato-Gastro-Enterologie, CHU Clermont-Ferrand; §Universite Clermont Auvergne, CNRS, UMR 6284, Clermont-Ferrand, France; ¶H^opitaux Universitaires de Geneve, Unite d’hemostase, Departement des specialites de medecine; **Universite de Geneve, GpG, Geneva, Switzerland; and ††CHU Clermont-Ferrand, Unite de Biostatistiques (Direction de la recherche clinique et de l’innovation), Clermont-Ferrand, France
To cite this article: Sinegre T, Duron C, Lecompte T, Pereira B, Massoulier S, Lamblin G, Abergel A, Lebreton A. Increased factor VIII plays a significant role in plasma hypercoagulability phenotype of patients with cirrhosis. J Thromb Haemost 2018; 16: 1132–40.
See also Lisman T, Bos S, Intagliata NM. Mechanisms of enhanced thrombin-generating capacity in patients with cirrhosis. This issue, pp 1128–31.
Essentials
• The role of increased factor VIII in cirrhosis-induced hypercoagulability has never been demonstrated. • Factor VIII and protein C effects were characterized by
thrombin generation with thrombomodulin.
• Factor VIII elevation plays a significant role in cirrho-sis-induced plasma hypercoagulability.
• Only protein C and factor VIII normalization led to thrombin generation similar to controls.
Summary. Background: In cirrhosis, thrombin generation (TG) studied in the presence of thrombomodulin (TM) indicates plasma hypercoagulability. Although the role of protein C (PC) deficiency has been investigated, the influ-ence of an increase in the factor VIII level has never been addressed. Objectives: We investigated the roles of high FVIII and low PC levels in increased TG in the presence of TM. Methods: Blood samples were prospectively col-lected from 35 healthy controls and 93 patients with cir-rhosis (Child–Turcotte–Pugh [CTP]-A, n = 61; CTP-B, n= 19; and CTP-C, n = 13) and FVIII levels > 150% (n= 48) and/or PC levels < 70% (n = 88). TG was per-formed with tissue factor (5 pM), phospholipids, and TM
(4 nM). FVIII and PC levels were normalized by adding
an inhibitory anti-FVIII antibody and exogenous PC, respectively. Results: The endogenous thrombin potential (ETP) in the presence of TM was higher in patients than in controls. After FVIII normalization, the ETP (median) decreased from 929 nMmin to 621 nM min (CTP-A),
1122 nMmin to 1082 nMmin (CTP-B), and 1221 nMmin
to 1143 nM min (CTP-C); after PC normalization, it
decreased from 776 nMmin to 566 nM min (CTP-A),
1120 nMmin to 790 nMmin (CTP-B), and 995 nMmin to
790 nMmin (CTP-C). The ETP was reduced by 17% and
30%, respectively, but normal TG was not restored. When both FVIII and PC levels were normalized, the ETP decreased from 929 nMmin to 340 nM min (CTP-A),
1122 nMmin to 506 nMmin (CTP-B), and 1226 nMmin
to 586 nMmin (CTP-C), becoming similar to control
levels. Conclusion: Cirrhosis-induced plasma hypercoagu-lability, as demonstrated in these experimental conditions, can be partly explained by opposite changes in two fac-tors: PC level (decrease) and FVIII level (increase).
Keywords: cirrhosis; factor VIII; hypercoagulability; protein C; thrombin generation.
Introduction
Patients with cirrhosis are at risk of bleeding but also of thromboembolic events, such as deep vein thrombosis, pulmonary embolism, and portal vein thrombosis [1–3]. Besides local factors such as reduced portal venous flow [4], cirrhosis-induced coagulopathy also could be an important determinant of the thrombotic process. Cirrho-sis is associated with decreases in the levels of both proco-agulant and anticoproco-agulant factors synthesized by the liver, including antithrombin (AT) and protein C (PC),
Correspondence: Thomas Sinegre, Service d’Hematologie Biologique, CHU Estaing, 1 place Lucie et Raymond Aubrac, 63003 Clermont-Ferrand, France
Tel.: +33 47 375 0200
E-mail: tsinegre@chu-clermontferrand.fr Received: 11 October 2017
Manuscript handled by: T. Lisman
but not in that of factor VIII, the level of which is increased [5–7]. This results in a fragile hemostasis bal-ance that can tilt either towards bleeding or towards thrombotic events, depending on the challenge to the coagulation system and additional circumstantial factors [8,9]. Global coagulation assays, such as those investigat-ing thrombin generation (TG), have allowed us to improve our knowledge on the coagulation alterations in patients with cirrhosis (i.e. a hypercoagulable laboratory phenotype, at least in some experimental conditions), and also to study the underlying mechanisms [10]. Indeed, TG assays (TGAs), such as calibrated automated thrombog-raphy (CAT), have been designed to be closer to the in vivo conditions than traditional laboratory tests, by taking into account coagulation inhibitors [11]. The TGA results are displayed as a TG curve, and the area under the curve represents the endogenous thrombin potential (ETP) (i.e. the total amount of thrombin generated in the studied plasma) [12]. Tripodi et al. [13,14] were the first to investigate TG in plasma of patients with cirrhosis and to challenge the prevalent view at that time that coagula-tion was defective. Since then, TG has been found to be rebalanced in such patients, because both prothrombin conversion and thrombin inactivation are reduced, the latter in relation to the reduced AT level [15]. If studied in the presence of thrombomodulin (TM), which is a thrombin cofactor for PC activation, plasma of patients with cirrhosis produces even more thrombin activity over time than that of healthy subjects [15–19]. The determi-nants are not fully elucidated. Acquired PC deficiency and increased FVIII levels are likely to play a key role [15,19]. Moreover, it has been shown that the addition of a (fixed) amount of purified PC can attenuate such a hypercoagulable phenotype [20]. The role of increased FVIII levels has never been assessed in this setting. Indeed, an elevated FVIII level is a well-established risk factor for thrombosis, but its impact on TG is not well documented [21–23]. However, the underlying mecha-nisms deserve to be fully deciphered, because of the potential clinical impact of plasma hypercoagulability in patients with cirrhosis.
Thus, the aim of this study was to investigate the effect of in vitro normalization of FVIII or/and PC levels on TG in the presence of TM in plasma of patients with cir-rhosis, to determine their respective roles.
Patients and methods
Patients and controls
For this study, 35 healthy controls and 93 patients with cirrhosis, an FVIII level of> 150% and/or a PC level of < 70% were prospectively enrolled between July 2012 and July 2015. This study was reviewed and approved by the local ethics committee (AU949 and AU765, Sud-Est VI France). The diagnosis of uncomplicated cirrhosis was
confirmed by liver biopsy or by non-invasive tests: throm-bocytopenia (< 140 G L 1), an International Normalized
Ratio (INR) of > 1.2, dysmorphic liver, a hepatic elastic-ity of >19.5 kPa, and varicose veins. Cirrhosis severity was evaluated with the Child–Turcotte–Pugh (CTP) score. Exclusion criteria for patients with cirrhosis were: age < 18 years, anticoagulant therapy, the occurrence of infectious, inflammatory or bleeding events during the previous 2 months, a personal or familial (first-degree rel-atives) history of venous thromboembolism, portal vein thrombosis diagnosed by Doppler ultrasonography and confirmed by computed tomography or magnetic reso-nance imaging, and hepatocellular carcinoma. Exclusion criteria for healthy controls were: a history of liver dis-ease, pre-existing untreated medical conditions, a personal or familial (first-degree relatives) history of bleeding or thrombotic disorders, the presence of a portal vein throm-bosis, anticoagulant therapy, daily alcohol consumption higher than 20 g for females and 30 g for males in the week before blood sampling, and ongoing infections.
Blood sampling and plasma preparation
Blood was drawn from an antecubital vein with a light tourniquet and a 21G needle, and collected into a tube containing 0.109 Mcitrate (Beckton Dickinson, le Pont de
Claix, France), 9 : 1 v/v, after the first few milliliters had been discarded. No inhibitor of the contact phase, such as corn trypsin inhibitor, was added, because we used 5 pMtissue factor (TF) for TGAs to initiate coagulation,
an experimental condition in which the contact phase seems to play a minimal role [24]. Platelet-poor plasma was prepared by double centrifugation at 2500 9 g for 15 min within 2 h after blood collection. Samples were stored at 80 °C at the Centre de Ressources Biologi-ques (biobank) Auvergne (NF S96-900 certified) until being tested. Before use, plasma samples were thawed in a water bath at 37 °C for 5 min.
Coagulation assays
Coagulation assays were performed with a STA-R Evolu-tion coagulometer (Stago, Asnieres-sur-Seine, France) with the following reagents (all from Stago): Neoplastin CI+ for prothrombin time (expressed as the INR, which is widely used in the case of cirrhosis despite its well-known limitations) and related coagulation factors, PTT-A for the activated partial thromboplastin time (PTT-APTT) and factors of the intrinsic pathway, and STA-Fibrinogen for fibrinogen (Clauss method). Coagulation factors were measured with the following plasmas specifically deficient in the measured factor (all from Stago): STA-Deficient II, STA-Deficient V, STA-Deficient VII, STA-Deficient X, Immunodef VIII, Immunodef IX, and Factor XI defi-cient. FV Leiden screening was performed with the APC Resistance V Kit (Werfen, Le Pre Saint Gervais, France)
and an ACL-TOP 700 instrument. D-dimers were ana-lyzed with the Vidas D-Dimer Exclusion Kit (bioMerieux, Marcy l’Etoile, France). AT, PC and free protein S (PS) were measured with functional assays by use of the STA-ATIII, Staclot-PC and Staclot-PS reagents (all from Stago), respectively.
TGAs
TGAs were performed with the CAT method [25], by use of a fluorometer (Fluoroscan Ascent; ThermoLab Systems, Franklin, MA, USA) equipped with a dispenser. Coagula-tion was initiated with 5 pM TF in the presence of 4 lM
procoagulant phospholipids (PPP reagent; Stago). Calibra-tion for each patient was performed with the Thrombin Calibrator (Stago). Preliminary experiments allowed us to determine the final level (4 nM) of soluble rabbit lung TM
(American Diagnostica, Stamford, CT, USA), added together with the PPP reagent, needed to reduce by 50% the ETP of healthy controls’ plasmas. TG in the presence of TM will be referred to as ‘TG-TM’. The following exper-imental conditions were used to normalize in vitro FVIII and PC levels: before and after addition to plasma of an inhibitory murine monoclonal IgG2 antibody against the
C2 domain of FVIII (ESH8; Sekisui Diagnostics, Stam-ford, CT, USA), of purified PC (Hyphen, Neuville, France), or both. The appropriate amounts of ESH8 and PC were determined in preliminary experiments to bring the plasma levels of FVIII or PC to the normal range (80– 120%). FVIII levels were normalized by use of a 100 lg mL 1 stock solution of ESH8. The volume added to 400lL of plasma ranged from 4 lL to 12 lL. To nor-malize the PC level (4lg mL 1) in patients’ plasmas, 4– 15lL of purified PC 100 lg mL 1 stock solution was added to 400lL of plasma. Plasma was supplemented with vehicle to have similar plasma dilutions. These experiments were performed with the plasmas from patients with the relevant FVIII and PC alterations (flow chart in Fig. S1).
As FVIII inhibition by the anti-FVIII mAb ESH8 is time-dependent, the kinetics of FVIII inhibition by ESH8 were evaluated over time (Fig. S2). As a control, the TGA was performed with an isotypic control mAb, and the ESH8 effect in an FVIII-deficient plasma sample was also evaluated (Figs S3 and S4). For all experiments with ESH8, FVIII level normalization was confirmed with the coagulometer assay when TG was initiated to have always the same incubation time (1 h).
All plates (Immulon 2HB; Stago) were incubated at 37°C for 10 min before addition of the fluorogenic sub-strate and CaCl2(FluCa-Kit; Thrombinoscope BV,
Maas-tricht, the Netherlands). All tests were performed in duplicate with a< 10% difference for the ETP. Raw data were analyzed withTHROMBINOSCOPE V5 (Stago). For each
assay, the ETP (nMmin) was the primary endpoint,
because it represents the whole thrombin that the plasma under study can generate. The results of TG evaluation
with CAT were previously reported [19] for 26 healthy con-trols and 56 patients; however, all of these subjects were retested for this study, and the agreement between previous and current results was very good (data not shown).
Statistical analysis
Statistical analyses were performed with PRISM, version 6
(GraphPad Software, La Jolla, CA, USA). Tests were two-sided, with a type I error set ata = 0.05. Continuous data are presented as the median (Q1–Q3). Independent groups were compared by the use of ANOVA, or the
Kruskal–Wallis test when the ANOVA conditions were not
met (normality and homoscedasticity verified with the Bartlett test), followed by the appropriate multiple-com-parison post hoc tests (Tukey–Kramer or Dunn test, respectively). Owing to the multiple comparisons, a type I error correction was performed (a = 0.05).
Results
Characteristics of patients and controls
Table 1 shows the demographic, clinical and laboratory parameters of healthy controls (n= 35) and patients with cirrhosis (n= 93). Patients were divided in three CTP groups: A (n= 61), B (n = 19), and C (n = 13). PC levels were < 70% in 88 patients, FVIII levels were > 150% in 48 patients, and both alterations were present in 43 patients. The median age (59 years for patients and 41 years for controls) and sex ratio (83% men in the cir-rhosis group and 100% in the control group) were signifi-cantly different between patients and controls (P< 0.001), but we previously showed that age is not a strong determinant of TG variation when healthy con-trols and patients with cirrhosis are compared [19]. The causes of cirrhosis were alcohol (38%), hepatitis C virus infection (32%), or various combinations (30%).
Coagulation assays
The results for the main coagulation parameters are sum-marized in Table 2. The INR and APTT increased pro-gressively with cirrhosis severity (P< 0.0001). The levels of all coagulant factors except FVIII were decreased, with a gradient from healthy controls to CTP-C patients. Con-versely, FVIII levels were higher in patients than in controls (153% [131–179], 179% [163–212] and 204% [171–229] for CTP-A, CTB-B and CTP-C, respectively, as compared with 95% [79–116]) (P < 0.0001), indicating that the increase was related to cirrhosis severity. The levels of the natural coagulation inhibitors AT, PC and PS were significantly lower in patients than in controls (P< 0.0001), particularly PC levels, which gradually decreased from 109% (100–122) in controls to 16% (12–22) in patients with severe cirrhosis (CTP-C)
(P< 0.0001). It is of note that the decrease in PC levels was more pronounced than the decreases in AT and PS levels. D-dimer levels gradually increased from healthy controls to CTP-C patients. This could be explained by the reduced hepatic clearance in cirrhosis rather than by in vivo coagulation activation. FV Leiden polymorphism was not detected in controls or patients.
TGAs
TGAs performed without and with TM. When TGAs were performed without TM, the ETP values were comparable
to those in previous studies performed in the same condi-tions [16,17,26], and no difference was observed between healthy controls and patients (Table S1). This is consistent with the assumption that the marked decrease in AT level is counterbalanced by a decrease in the prothrombin level. When TGAs were performed in the presence of TM, the ETP tended to increase with cirrhosis severity (Table S2). TG-TM after FVIII normalization. TG-TM was studied with the plasma from the 48 patients with FVIII levels of > 150% (19 CTP-A, 16 CTP-B, and 13 CTP-C) before and after in vitro addition of the inhibitory anti-FVIII
Table 1 Demographic, clinical and laboratory parameters of patients with cirrhosis and healthy controls
Healthy controls (n= 35)
Cirrhotic patients (n= 93)
P-value CTP class A (n= 61) CTP class B (n= 19) CTP class C (n= 13)
Male sex, n (%) 35 (100) 49 (80) 15 (79) 13 (100) < 0.0001 Age (years) 41 (28–49) 59 (53–65) 59 (47–66) 57 (52–62) < 0.0001 Cause of cirrhosis, n (%) NA NA ALD 12 (20) 13 (68) 10 (77) HCV 28 (46) 2 (11) – ALD+ HCV 20 (33) 2 (11) 2 (15) ALD+ HBV – 1 (5) – ALD+ NASH 1 (2) 1 (5) 1 (8) ALP (IU L 1) 61 (55–64) 112 (85–128) 130 (102–137) 184 (118–227) < 0.0001 AST (IU L 1) 23 (20–27) 65 (35–79) 82 (40–91) 84 (43–115) < 0.0001 ALT (IU L 1) 30 (24–35) 74 (36–88) 60 (25–69) 42 (25–46) < 0.001 Bilirubin (lM) 12 (8–15) 17 (11–23) 30 (21–38) 117 (57–192) < 0.0001 Triglycerides (g L 1) 1.0 (0.7–1.3) 1.1 (0.8–1.4) 1.0 (0.7–1.1) 0.8 (0.7–1.0) 0.6 PLT (G L 1) 219 (198–249) 121 (63–159) 96 (69–111) 92 (60–123) < 0.0001
ALD, alcoholic liver disease; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CTP, Child –Tur-cotte–Pugh; HBV, hepatitis B virus; HCV, hepatitis C virus; NA, not applicable; NASH, non-alcoholic steatohepatitis; PLT, platelets. Continu-ous data are presented as median (Q1–Q3).
Table 2 Coagulation assay results in patients with cirrhosis and healthy controls
Healthy controls (n= 35)
Cirrhotic patients n= 93
P-value CTP class A (n= 61) CTP class B (n= 19) CTP class C (n= 13)
PT (s) 12.2 (11.1–13.2) 13.8 (13.0–15.3) 15.1 (14.5–16.4) 19.7 (17.9–26.3) < 0.001 INR 1.0 (0.9–1.0) 1.1 (1.0–1.2) 1.2 (1.2–1.4) 1.7 (1.5–2.4) < 0.001 APTT (ratio) 1.0 (0.9–1.1) 1.2 (1.1–1.3) 1.4 (1.2–1.5) 1.7 (1.5–2.1) < 0.001 FII (%) 106 (98–113) 78 (65–87) 62 (54–70) 39 (30–42) < 0.001 FV (%) 112 (90–117) 83 (74–106) 64 (56–79) 46 (36–50) < 0.001 FVII (%) 102 (88–119) 91 (57–114) 46 (31–56) 21 (14–28) < 0.001 FX (%) 103 (95–112) 87 (72–100) 65 (55–74) 48 (40–54) < 0.001 FVIII (%) 95 (79–116) 153 (131–179) 179 (163–212) 204 (171–229) < 0.001 FIX (%) 89 (79–97) 96 (72–109) 62 (54–73) 41 (33–58) < 0.001 FXI (%) 89 (80–99) 57 (44–71) 42 (33–58) 28 (21–41) < 0.001 Fibrinogen (g L 1) 2.8 (2.5–3.2) 2.8 (2.4–3.2) 2.6 (2.4–3.2) 2.0 (1.4–2.4) < 0.01 D-dimers (lg mL 1) 0.21 (0.17–0.30) 0.46 (0.28–0.87) 1.83 (0.85–3.14) 2.21 (1.45–4.65) < 0.001 AT (%) 107 (99–115) 82 (68–94) 58 (49–71) 35 (29–40) < 0.001 PC (%) 113 (100–123) 65 (51–79) 33 (26–44) 16 (12–22) < 0.001 PS (%) 111 (100–123) 70 (58–86) 62 (51–76) 58 (49–69) < 0.001
APTT, activated partial thromboplastin time; AT, antithrombin; CTP, Child–Turcotte–Pugh; FII, factor II; FV, factor V; FVII, factor VII; FVIII, factor VIII; FIX, factor IX; FX, factor X; FXI, factor XI; INR, International Normalized Ratio; PC, protein C; PS, protein S; PT, prothrombin time. Data are presented as median (Q1–Q3).
mAb. The mAb significantly reduced FVIII levels to 82% (64–92), 101% (76–119) and 99% (84–119) in the CTP-A, CTP-B and CTP-C groups, respectively (P< 0.0001); thus, FVIII levels were no longer different between patients and controls (P= 0.07) (Fig. 1A).
The ETP of TG-TM was reduced in all patient groups after FVIII normalization (Fig. 2). The ETP decreased from 929 nMmin (784–1086) to 621 nMmin (518–854) in
the CTP-A group (P< 0.0001), from 1122 nMmin (1035–
1360) to 1082 nMmin (957–1154) in the CTP-B group
(P< 0.01), and from 1221 nMmin (910–1407) to
1143 nMmin (1050–1211) in the CTP-C group (P < 0.05).
After FVIII normalization, no significant ETP difference was found between the control (508 nMmin [394–731]) and
CTP-A groups (P= 0.32). Conversely, the ETP remained significantly higher in the CTP-B and CTP-C groups than in controls (P< 0.0001). These data highlight the effect of elevated FVIII levels on TG-TM in the plasma of patients with cirrhosis. The mAb led to a mean ETP reduction of 17%; however, in the CTP-B and CTP-C groups, FVIII normalization was not sufficient to restore normal TG-TM. Moreover, when TG was studied in the absence of TM, FVIII normalization was accompanied by a slight but significant ETP decrease (Table S4).
TG-TM after PC normalization. TG-TM was studied in plasma from the 88 cirrhotic patients with PC levels of < 70% (61 CTP-A, 18 CTB-B, and nine CTP-C) before and after PC supplementation. PC levels increased from 50% (41–76) to 100% (94–110), which is a value comparable to that in controls (113%, 100–123) (P = 0.054) (Fig. 1B).
The ETP of TG-TM consistently decreased after PC normalization, regardless of the CTP class (Fig. 3). Specifically, the ETP decreased from 776 nMmin (626–
991) to 566 nMmin (384–769) in the CTP-A group, from 400 A B FVIII activity (%) PC activity (%) **** **** **** **** NS **** NS NS NS NS **** NS 300 200 200 150 100 50 0 100 0 Controls Controls CTP-A CTP-A n = 35 n = 35 n = 19 n = 61 n = 16 n = 18 n = 13 n = 9 CTP-A + anti-FVIII CTP-A + PC CTP-B CTP-B CTP-B + anti-FVIII CTP-B + PC CTP-C CTP-C CTP-C + anti-FVIII CTP-C + PC ¤¤¤¤ ¤¤¤¤ ¤¤¤¤ ¤¤¤¤ ¤¤¤¤ ¤¤¤¤
Fig. 1. Factor VIII and protein C (PC) levels of cirrhotic patients and healthy controls. (A) FVIII levels were increased in patients, whatever the stage of cirrhosis, as compared with controls (P< 0.0001). After addition of the inhibitory anti-FVIII antibody, FVIII levels were nor-malized (P> 0.05 as compared with controls). (B) PC levels were sig-nificantly lower in cirrhotic patients than in controls (P< 0.0001). After addition of PC, PC levels were normalized (P> 0.05 as com-pared with controls).****P < 0.0001 as compared with controls;¤¤¤ ¤P< 0.0001 for comparison with and without the addition of PC for each Child–Turcotte–Pugh (CTP) stage. Statistical analysis was per-formed with the Kruskal–Wallis test followed by the Dunn test. NS, not significant. 2000 1500 1000 500 ETP (n M .min) 0 Controls n = 35 n = 19 n = 16 n = 13 CTP-C + anti-FVIII CTP-B + anti-FVIII CTP-A CTP-A + anti-FVIII CTP-B CTP-C NS * **** ¤¤¤¤ ¤¤ ¤ **** **** ****
Fig. 2. Role of increased factor VIII levels in cirrhotic patients in thrombin generation in the presence of thrombomodulin (TM). Box plots show that normalization of FVIII levels by addition of the inhibitory anti-FVIII antibody decreased the endogenous thrombin potential (ETP) when thrombin generation assays were performed with TM. This reduction was less marked for Child– Turcotte–Pugh (CTP)-A than for CTP-B and CTP-C (P < 0.0001, P< 0.01, and P < 0.05, respectively). The ETP of CTP-A patients was comparable to that of controls, whereas the ETPs of CTB-B and CTP-C patients remained significantly higher than that of con-trols (P< 0.0001). ****P < 0.0001 and *P < 0.05 as compared with controls;¤¤¤¤P< 0.0001 and¤¤P< 0.01 for¤P< 0.05 comparison with and without the addition of anti-FVIII for each CTP stage. Statistical analysis was performed withANOVAfollowed by the Tukey test. NS, not significant.
1120 nMmin (1062–1184) to 790 nMmin (618–971) in the
CTP-B group, and from 995 nMmin (977–1442) to
790 nMmin (732–877) in the CTP-C group (P < 0.0001).
The average ETP reduction after PC normalization was 30%. No significant difference was found between healthy controls [508 nMmin (410–725)] and the CTP-A group
(P= 0.63). Conversely, ETP values remained higher in the CTP-B and CTP-C patients than in controls (P< 0.01 and P < 0.05, respectively). This indicates that, in these two groups, PC normalization alone is not suffi-cient to restore normal TG-TM.
TG-TM after concomitant FVIII and PC normaliza-tion. Finally, TG-TM was studied in plasma from the 43 patients with PC and FVIII plasma levels of < 70% and> 150%, respectively (19 CTP-A, 15 CTP-B, and nine CTP-C), before and after concomitant FVIII and PC nor-malization by addition of the anti-FVIII mAb and PC. After FVIII and PC normalization to 88% (75–101) and 109% (102–114), respectively, the ETP decreased from 929 nMmin (784–1086) to 340 nMmin (191–576) in the
CTP-A group (P< 0.0001), from 1122 nMmin (1035–
1360) to 506 nMmin (289–646) in the CTP-B group
(P< 0.0001), and from 1226 nMmin (1108–1488) to
586 nMmin (506–744) in the CTP-C group (Fig. 4). After
FVIII and PC normalization, the ETP was no longer sig-nificantly different between patients (all groups) and
healthy controls (P > 0.05). This demonstrates that, when an increase in FVIII level and PC deficiency are simulta-neously normalized in vitro, TG-TM in patients with cir-rhosis is restored to values comparable to those of controls.
Besides ETP, the values of other parameters used to refine the quantitative description of TG (e.g. lag time, peak, and time to peak) are shown in Table S3.
Discussion
Cirrhosis has been associated with the alteration of the dynamic inhibitory system involving activated PC (APC), in addition to decreased AT levels and dysregulation of the TF pathway inhibitor (TFPI)–PS anticoagulant sys-tem [15,27]. The APC pathway plays a central role in rebalancing the coagulation of patients with cirrhosis. Previous studies showed that, in experimental conditions that allow PC activation by generated thrombin, TG is normal or even increased, depending on the experimental conditions and the studied patients [13,14,16–19,28–30]. Here, we report that the ETP decreased after normaliza-tion of FVIII (by addinormaliza-tion of an inhibitory antibody) or PC (by addition of PC), and that a normal coagulation phenotype could be fully restored after normalization of both FVIII and PC levels. This is the first direct experi-mental demonstration of FVIII involvement.
2000 1500 1000 ETP (n M .min) 500 0 Controls CTP-A CTP-Bn = 18 CTP-B + PC CTP-Cn = 9 CTP-C + PC n = 35 n = 61 CTP-A + PC ** NS **** ** **** * ¤¤¤¤ ¤¤¤¤ ¤¤¤¤
Fig. 3. Role of protein C (PC) deficiency in cirrhotic patients in thrombin generation in the presence of thrombomodulin (TM). Box plots show that the endogenous thrombin potential (ETP) in the presence of TM was consistently higher in cirrhotic patients than in controls, regardless of the stage of cirrhosis. Addition of PC signifi-cantly reduced the ETP (P< 0.0001). The ETP of Child–Turcotte– Pugh (CTP)-A patients was therefore not significantly different from that of controls, but the ETPs of CTP-B and CTP-C patients were sig-nificantly different from that of controls.****P < 0.0001, **P < 0.01 and*P < 0.05 as compared with controls;¤¤¤¤P< 0.0001 for compar-ison with and without the addition of PC for each CTP stage. Statisti-cal analysis was performed withANOVAfollowed by the Tukey test. NS, not significant. 2000 1500 1000 500 ETP (n M .min) 0 Controls n = 35 CTP-A n = 19 CTP-A + anti-FVIII/PC CTP-B n = 15 CTP-B + anti-FVIII/PC CTP-C n = 9 CTP-C + anti-FVIII/PC * NS NS ¤¤¤¤ ¤¤¤¤ ¤¤¤¤ NS **** ****
Fig. 4. Effect of normalization of both factor VIII and protein C (PC) levels of patients with cirrhosis on thrombin generation in the presence of thrombomodulin. Box plots show that the normalization of both FVIII and PC levels significantly decreased the endogenous thrombin potentials (ETPs) of Child–Turcotte–Pugh (CTP)-A, CTB-B and CTCTB-B-C patients (P< 0.0001). The ETPs became non-signifi-cantly different from that of controls regardless of the CTP stage. ****P < 0.0001 and *P < 0.05 as compared with controls;¤¤¤ ¤P< 0.0001 for ncomparison with and without the addition of PC and anti-FVIII for each CTP stage. Statistical analysis was per-formed withANOVAfollowed by the Tukey test. NS, not significant.
We chose the ETP as the main parameter. Although ETP ratios, in the presence or absence of APC (added or endogenously formed), assess one particular aspect [31], it can be reasonably argued that the net result, relevant to the in vivo situation, is the amount of thrombin that can be generated in well-defined conditions.
Previous studies showed that addition of fixed amounts of purified PC significantly decreases the ETP when TGAs are performed with TM [16,20]. Furthermore, we previously found that, after addition of APC to bypass the PC activation step, TG is similar in the healthy and cirrhotic groups, irrespective of the liver disease severity [19]. Here, we report that the ETP decreased in the plasma of all patients with cirrhosis after PC normaliza-tion, i.e. when the experimental conditions were adjusted to the magnitude of the deficiency, at variance with previ-ous studies. However, plasma hypercoagulability persisted in the CTP-B and CTP-C groups, indicating that PC nor-malization alone is not sufficient. This is consistent with the observation that plasma hypercoagulability increases with disease severity [14,19].
An increased FVIII level is an independent risk factor for thrombosis and its recurrence [32], and high FVIII levels have been correlated with increased TG [24,33]. Extrahepatic FVIII synthesis together with decreased hep-atic FVIII clearance, secondary to liver disease, leads to increased FVIII plasma levels in patients with cirrhosis [34]. The precise role of this increase in this setting has not been accurately evaluated, partly because of technical limitations. Indeed, FVIII level reduction by anti-FVIII antibodies immobilized on Sepharose alters plasma [20], and the use of heat to inactivate FVIII [35] severely affects the TG capacity of plasma. To circumvent these problems, we used ESH8, a mAb against the FVIII C2 domain that prevents interaction with von Willebrand factor or negatively charged phospholipids [36,37], and can reduce FVIII-dependent TG [38]. Our results demon-strate that, after FVIII level reduction with adjusted amounts of anti-FVIII mAb, ETP values in the presence of TM were consistently decreased, but not always nor-malized. Moreover, ETP reduction was less important after FVIII normalization than after PC normalization (approximately 17% and 30%, respectively). This is con-sistent with our previous finding that, after APC addition, an elevated FVIII level does not seem to interfere with TG as indicated by the absence of differences between patients and controls [19]. One possible explanation is the artificial presence of relatively high amounts of APC at the beginning of the TG process, in contrast to the slower, progressive and more physiological formation of APC from endogenous PC during TGAs in the presence of TM. Indeed, the first experimental condition should be less sensitive to elevated FVIII levels than the second. We found that, when TGAs were performed without TM, FVIII normalization was also accompanied by ETP reduction, albeit very limited. As an elevated FVIII level
is associated with thrombotic risk, its influence on TG in various experimental conditions deserves to be better understood.
The main finding of our study is that an increased FVIII level plays a significant role in cirrhosis-induced plasma hypercoagulability. It has been shown that what is often referred to as acquired resistance to APC (i.e. not caused by FV Leiden polymorphism) is associated with thrombotic events [39,40]. High FVIII levels are also associated with a higher risk of thrombotic events [41]. Therefore, we suggest that FVIII levels should be taken into account in the assessment of the thrombotic risk of patients with cirrhosis, in combination with PC levels [42,43]. Importantly, despite the presence of complex changes in determinants of coagulation, plasma hyperco-agulability, as demonstrated in our experimental condi-tions, is explained by opposite changes in PC and FVIII. This does not rule out a role for AT deficiency or TFPI– PS dysregulation in the net coagulation phenotype, inves-tigated under different experimental conditions that might be as clinically relevant as the one that we chose for this study. In addition, they would make the rebalanced phe-notype more fragile.
The slight difference in the sex ratio between controls and patients is a limitation of our study. This could have skewed the results, because women might have lower sensi-tivity to PC, mainly because of hormone-dependent changes in PS levels. Literature data on the influence of gender on TG are rather inconsistent [44–46]. The limited sample size, particularly in the group with advanced cirrho-sis, is another limitation. Finally, TG is only one piece in the complex puzzle of hemostasis, and neither cellular aspects nor fibrinolysis were assessed in this study. TG does not give any information on fibrin formation and the struc-ture of fibrin clots, which are also altered in cirrhosis [47].
Clinical studies have found that patients with cirrhosis are not protected against thrombotic events [3,48]. More-over, de novo portal vein thrombosis has been associated with high TG in the presence of TM [49]. Thus, coagula-tion evaluacoagula-tion with TGAs might help us to better evalu-ate the risk of thrombosis in patients with cirrhosis.
Addendum
A. Lebreton, T. Sinegre, T. Lecompte, A. Abergel, and B. Pereira: designed the study. A. Abergel, G. Lamblin, and C. Duron recruited the patients. S. Massoulier carried out all of the data management. T. Sinegre performed experi-ments. B. Pereira performed the statistical analysis. T. Sinegre, A. Lebreton, A. Abergel, and T. Lecompte wrote the manuscript.
Acknowledgements
The authors sincerely thank the technical staff of the Hemostasis Department (University Hospital of
Clermont-Ferrand), the Centre de Ressources Biologiques Auvergne, and E. Andermarcher for help with English editing.
Disclosure of Conflict of Interests
The authors state that they have no conflict of interest.
Supporting Information
Additional supporting information may be found online in the Supporting Information section at the end of the article:
Fig. S1. Study flow diagram outlining the study design. Fig. S2. Kinetics of inhibition of FVIII level by inhibitory anti-FVIII mAb ESH8.
Fig. S3. Impact of an isotype IgG2acontrol on thrombin
generation.
Fig. S4. Impact of inhibitory anti-FVIII mAb ESH8 on thrombin generation in FVIII-deficient plasma.
Table S1. Results of thrombin generation assays per-formed in the absence of thrombomodulin for healthy controls and cirrhotic patients.
Table S2. Results of thrombin generation assays per-formed in the presence of thrombomodulin for healthy controls and cirrhotic patients.
Table S3. Results of thrombin generation assays per-formed in the presence of thrombomodulin for cirrhotic patients after supplementation with PC or/and addition of an anti-FVIII antibody.
Table S4. Results of thrombin generation assays per-formed in the absence of thrombomodulin for cirrhotic patients before and after FVIII normalization.
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