BASIC LIVER, PANCREAS, AND BILIARY TRACT. An Imbalance of Pro- vs Anti-Coagulation Factors in Plasma From Patients With Cirrhosis

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1 GASTROENTEROLOGY 2009;137: BASIC LIVER, BILIARY TRACT An Imbalance of Pro- vs Anti-Coagulation Factors in Plasma From Patients With Cirrhosis ARMANDO TRIPODI,* MASSIMO PRIMIGNANI, VEENA CHANTARANGKUL,* ALESSANDRA DELL ERA, MARIGRAZIA CLERICI,* ROBERTO DE FRANCHIS, MASSIMO COLOMBO, and PIER MANNUCCIO MANNUCCI* *Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Department of Internal Medicine and Medical Specialties, and Third Division and First Division of Gastroenterology, University and IRCCS Ospedale Maggiore, Mangiagalli and Regina Elena Foundation, Milano, Italy BACKGROUND & AIMS: Patients with cirrhosis have an increased tendency to develop thromboses despite the longer coagulation times of their plasma, compared with that of healthy individuals. We investigated whether plasma from cirrhotic patients has an imbalance of provs anti-coagulation factors. METHODS: We analyzed blood samples from 134 cirrhotic patients and 131 healthy subjects (controls) for levels of pro- and anticoagulants and for thrombin generation in the presence or absence of thrombomodulin (the main physiologic activator of the protein C anticoagulant pathway). RESULTS: The median ratio of thrombin generation (with/without thrombomodulin) was higher in patients (0.80; range, ) than controls (0.66; range, ), indicating that cirrhotic patients are resistant to the action of thrombomodulin. This resistance resulted in greater hypercoagulability of plasma from patients of Child Pugh class C than of class A or B. The hypercoagulability of plasma from patients of Child Pugh class C (0.86; range, ) was slightly greater than that observed under the same conditions in patients with congenital protein C deficiency (0.76; range, ). Levels of factor VIII, a potent pro-coagulant involved in thrombin generation, increased progressively with Child Pugh score (from Child Pugh class A to C). Levels of protein C, one of the most potent naturally occurring anti-coagulants, showed the opposite trend. CONCLUSIONS: The hypercoagulability of plasma from patients with cirrhosis appears to result from increased levels of factor VIII and decreased levels of protein C typical features of patients with cirrhosis. These findings might explain the risk for venous thromboembolism in patients with chronic liver disease. Coagulation is a highly integrated cellular/humoral mechanism that acts through 2 opposing drivers. The first, represented by the pro-coagulant factors, is triggered by tissue factor when this cellular receptor forms a complex with plasma factor VII at the site of injury. 1 This initiates a series of reactions mediated by negatively charged phospholipids on the membranes of activated platelets that ultimately lead to thrombin generation and fibrinogen-to-fibrin conversion 2 (Figure 1). The anti-coagulant driver originates from thrombin itself that once complexed with its endothelial receptor thrombomodulin activates plasma protein C. 3 Activated protein C is a potent anti-coagulant that, in combination with its plasma cofactor protein S, down-regulates thrombin generation through the inhibition of the activated forms of factors VIII and V 3 (Figure 1). The anti-coagulant driver is also potentiated by the presence of plasma antithrombin, 4 which, in complex with endothelial heparin-like substances, inhibits thrombin directly and indirectly through the inactivation of the activated forms of the coagulation factors XI, IX, and X 5 (Figure 1). Thrombin formation is also down-regulated by the tissue factor pathway inhibitor (TFPI) that inhibits the complex tissue factor-factor VII and activated factor X 6 (Figure 1). The balance between the pro- and anti-coagulant drivers is essential to ensure unwanted thrombin formation in physiologic conditions. Chronic liver disease is characterized by a defective synthesis of coagulation factors leading to prolonged conventional global coagulation tests such as the prothrombin time (PT). 7 The causal relationship between the abnormality of this test and the risk of bleeding has been for long time taken for granted, as shown by the common practice of measuring the PT and attempting its correction by means of fresh frozen plasma prior to liver biopsy or other potentially hemorrhagic procedures. 8,9 Recently, we challenged this paradigm by showing differ- Abbreviations used in this paper: ETP, endogenous thrombin potential; PT, prothrombin time by the AGA Institute /09/$36.00 doi: /j.gastro

2 2106 TRIPODI ET AL GASTROENTEROLOGY Vol. 137, No. 6 Figure 1. Simplified scheme of the reactions leading to thrombin generation and inhibition. Roman numbers represent coagulation factors. Solid and broken arrows represent pro- and anti-coagulant drivers, respectively. TF, tissue factor; TFPI, tissue factor pathway inhibitor; PC, protein C; PS, protein S; APC, activated protein C; PL, negatively charged phospholipids on platelet membranes; AT, antithrombin. ent patterns of thrombin generation in patients with chronic liver disease according to whether or not thrombomodulin (the main physiologic activator of the naturally occurring anticoagulant protein C) was incorporated into the assay system. Accordingly, thrombin generation was reduced in the absence, but much less in the presence of thrombomodulin. 10,11 We surmised that the test in the presence of thrombomodulin, by ensuring optimal activation of the reduced levels of protein C, restores the balance between pro- and anti-coagulants whose concomitant reduction is the common feature of patients with chronic liver disease. On the contrary, the test performed in the absence of thrombomodulin resembles the PT in which protein C activation is suboptimal because reagents and plasma employed for its performance do not contain sufficient amounts of thrombomodulin. Accordingly, the PT test is more responsive to the pro-coagulant driver and less to its anti-coagulant counterpart. 12 Remarkably, the percentage reduction of thrombin generation after addition of thrombomodulin into the test system was smaller for patients than for healthy subjects (ie, 38% vs 58%, respectively). 11 We surmise that this difference has some practical relevance because it may indicate that patients with chronic liver disease have higher than normal thrombin generation when assessed as the ratio between the tests performed in the presence or absence of thrombomodulin. The heightened thrombin generation might explain the relatively high risk of venous thromboembolism observed in patients with chronic liver disease. 13,14 We present results of a study evaluating a large cohort of patients with chronic liver disease to assess whether and to what extent the aforementioned ratio of thrombin generation is higher than normal and to look for possible explanations. Patients and Methods Patients One hundred thirty-four adult patients with cirrhosis (108 males and 26 females; median age, 63 years, range, 32 82) were enrolled in this study after approval of our Institutional Review Board and informed consent of the patients. Cirrhosis was diagnosed on the basis of clinical, laboratory, and ultrasound evidence. Criteria for exclusion were the use of drugs known to interfere with blood coagulation, bacterial infections, hepatocellular carcinoma, extrahepatic malignancy, and known hemostatic disorders other than cirrhosis. The severity of the disease was estimated according to Child Turcotte Pugh score. 15 Six patients with an established diagnosis of congenital protein C deficiency and 131 healthy individuals (100 males and 31 females; median age, 51 years, range 30 80) were enrolled in this study as controls. Blood for laboratory analyses was drawn by clean venepuncture and collected in vacuum tubes (Becton and Dickinson, Meylan, France) containing 109 mmol/l trisodium citrate as anticoagulant in the proportion of 1:9 parts of anticoagulant/blood. Blood was centrifuged within 30 minutes from collection at 2880g for 15 minutes at room temperature. Platelet-free plasma was then harvested, quick frozen in liquid nitrogen, and stored at 70 C until tested. Measurements Thrombin generation was assessed as endogenous thrombin potential (ETP) according to Hemker et al 16 as described in details by Chantarangkul et al. 17 The test is based on the activation of coagulation in platelet-free plasma after addition of human relipidated recombinant tissue factor (Recombiplastin; Instrumentation Laboratory, Orangeburg, NY), which acts as a coagulation trigger in the presence of synthetic phospholipids 1,2-dioleoyl-snglycero-3-phosphoserine, 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine and 1,2-dioleoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids Inc, Alabaster, AL) in the proportion of 20/20/60 (mol/l/mol/l). The concentrations of tissue factor and phospholipids in the test system were 1 pmol/l and 1.0 mol/l, respectively. Tests were repeated in a second aliquot of plasma by adding to the test system soluble thrombomodulin (ICN Biomedicals, Aurora, OH) at a final concentration of 4 nmol/l. Continuous registration of the generated thrombin was achieved with a fluorogenic synthetic substrate (Z-Gly- Gly-Arg-AMC HCl, Bachem, Bubendorf, Switzerland) added to the test system at a final concentration of 617 mol/l. The procedure was carried out with an automated fluorometer (Fluoroskan Ascent; ThermoLabsystem, Helsinki, Finland). Readings from the fluorometer were automatically recorded and calculated by a dedicated software (Thrombinoscope, Thrombinoscope BV, Maastricht, The Netherlands), which displays throm-

3 December 2009 HYPERCOAGULABILITY IN CIRRHOSIS 2107 Table 1. Demographic Characteristics of the Study Population Separated According to Child Pugh Classification Characteristics All (n 134) Child Pugh A (n 54) Child Pugh B (n 57) Child Pugh C (n 23) Age (y) 63 (32 82) 64 (39 82) 64 (32 79) 51 (39 78) Etiology HCV (n) HBV (n) Alcoholic (n) Other (n) Bilirubin (mg/dl) 1.6 ( ) 1.2 ( ) 1.8 ( ) 4.6 ( ) Albumin (g/l) 3.4 ( ) 3.9 ( ) 3.3 ( ) 2.7 ( ) Creatinine (mg/dl) 0.8 ( ) 0.8 ( ) 0.8 ( ) 0.9 ( ) Hemoglobin (g/dl) 12.8 ( ) 13.4 ( ) 12.8 ( ) 10.8 ( ) Leukocytes (10 9 /L) 3.8 ( ) 3.8 ( ) 3.5 ( ) 4.8 ( ) Platelets (10 9 /L) 66 (19 203) 81 (26 203) 55 (24 192) 74 (19 171) NOTE. Values are expressed as median (range). HCV, hepatitis C virus; HBV, hepatitis B virus. bin generation curves (time vs generated thrombin) and calculates the area under the curve, defined as ETP and expressed as nanomolars thrombin times minutes (nmol/l min). Thrombin generation is measured as function of an internal calibrator for thrombin (Thrombin Calibrator; Thrombinoscope BV). ETP represents the balance between the actions of pro- and anti-coagulants in plasma. To minimize analytical variability, equal numbers of samples from patients and healthy subjects were included on each test occasion. Factor II was measured as pro-coagulant activity upon activation with Taipan snake venom. Other parameters to assess pro- (factors VIII and V) and anti-coagulant factors (antithrombin and protein C) were measured as previously reported with results expressed as percentage of a normal pooled plasma arbitrarily set at 100% of normal. 10 Statistical Analysis ETP levels for patients and healthy subjects were used to calculate the ratios between the values obtained with and without thrombomodulin. These ratios denote the efficiency of thrombomodulin in the activation of protein C and were taken as indexes of hypercoagulability (the greater the ratios the higher the hypercoagulability). Likewise, the ratios between the levels of the individual pro-coagulants (factors II, V, and VIII) and anti-coagulants (protein C and antithrombin) were taken as indexes of the coagulation imbalance (the greater the ratios, the higher the procoagulant imbalance). Continuous variables were expressed as medians and ranges and tested for statistical significance with the nonparametric Mann Whitney U and Wilcoxon tests, and the P value of.05 or less was considered as statistically significant. Statistical analyses were performed with the SPSS software package, version 17.0 (SPSS, Inc, Chicago, IL). Results The main characteristics of the patient population are reported in Table 1. The distribution of severity of liver disease according to the Child Pugh classification identified 3 groups of 54 (Child Pugh A), 57 (Child Pugh B), and 23 (Child Pugh C) patients. Information on the patient population pertaining to the plasma levels of proand anti-coagulant factors is given in Table 2 and Figure 2. When compared with healthy subjects, patients had significantly prolonged PT, reduced levels of antithrombin, protein C, factor V and factor II, and increased levels of factor VIII (P.001) (Table 2). Factors II and V, antithrombin, and protein C decreased progressively from Table 2. Hemostatic Parameters for Patients With Cirrhosis and Healthy Subjects Patients Controls Parameters N Median (range) N Median (range) P value PT ratio a ( ) ( ).001 Factor VIII (%) b (87 378) (84 160).001 Factor V (%) b (19 177) (60 209).001 Protein C (%) b (11 95) (76 134).001 Antithrombin (%) b (13 96) (80 124).001 Factor II (%) b (10 89) (55 139).001 N, number of observations. a Ratio of patient-to-reference normal plasma clotting time. The conventional PT-INR is not reported because of the known inter-laboratory variability. 25 The INR liver 26 for these patients was not available. b Percent activity relative to the reference normal plasma set at 100%.

4 2108 TRIPODI ET AL GASTROENTEROLOGY Vol. 137, No. 6 Figure 2. Box plots of the distribution of values (median, lower, and upper quartile, and outliers identified as dots) for individual pro- and anti-coagulant factors for healthy subjects and patients with cirrhosis stratified according to classes of Child Pugh. Child Pugh A to C (P.001) (Figure 2), with Child Pugh C patients displaying median protein C levels smaller than those recorded for the population of patients with congenital protein C deficiency (34% vs 46%, respectively, P.03) (Figure 2). Factor VIII increased from Child Pugh A to C (P.02), reaching a median value close to 200% (Figure 2). Thrombin Generation in Platelet-Free Plasma The median (range) ETP values for the healthy subjects were 1856 ( ) nmol/l * min and 1202 ( ) nmol/l * min when the test was performed in the absence or presence of thrombomodulin (average ETP reduction, 35%). The corresponding values for the patients population were 1380 ( ) nmol/l * min and 1095 ( ) nmol/l * min (average ETP reduction, 21%). Ratios of ETP Performed in the Presence/ Absence of Thrombomodulin Figure 3 shows ETP ratios for patients and healthy subjects. The median [range] ETP ratio value for the patient population (0.80 [ ]) was significantly higher than that for the healthy subjects (0.66 [ ]) (P.001) and similar to that for a population of patients with congenital protein C deficiency (ie, 0.76 [ ] (P.47). Figure 4 shows ETP ratios for the patient population subdivided according to the Child Pugh score. ETP ratios increased progressively from Child Pugh A (0.72 [ ]) to C (P.001) with Child Pugh C displaying slightly higher median value than that for patients with congenital protein C deficiency (0.86 [ ] vs 0.76 [ ], P.03). Ratios of Pro- vs Anti-Coagulant Factors Figure 5 shows ratios of pro- vs anti-coagulant factors for patients and healthy subjects. The ratios of Figure 3. Box plots of the distribution of values for ratios between thrombin generation with/without thrombomodulin for healthy subjects, patients with cirrhosis, or with protein C congenital deficiency. Thrombin generation has been assessed as endogenous thrombin potential (ETP) (see text for more details).

5 December 2009 HYPERCOAGULABILITY IN CIRRHOSIS 2109 Figure 4. Box plots of the distribution of values for ratios between thrombin generation assessed with/without thrombomodulin for healthy subjects, patients with cirrhosis (stratified according to classes of Child Pugh), or with protein C congenital deficiency. Thrombin generation has been assessed as endogenous thrombin potential (ETP) (see text for more details). factor II-to-protein C were close to the unity, and there were neither differences between patients and healthy subjects (P.94) nor between Child Pugh categories (P.90). The ratios of factor V-to-protein C were significantly higher for patients than for healthy subjects (P.001) and increased slightly, but not significantly, from Child Pugh A to C (P.19). The ratios of factor VIII-to-protein C were significantly higher for patients than healthy subjects (P.001) and increased progressively from Child Pugh A to C (P.001) reaching a value of 5.6. Likewise, the ratios of factor VIII-to-antithrombin were higher for patients than healthy subjects (P.001) and increased progressively from Child Pugh AtoC(P.001) reaching a value of 5.3. Discussion Chronic liver disease has been considered for many years the epitome of acquired coagulation disorder, and the abnormal PT test, which is frequently observed in this condition, has been taken until recently as an index of the bleeding tendency. Accordingly, its numerical value was incorporated (in spite of the lack of evidence) into clinical guidelines recommending specific values as the target of correction before performing liver biopsy or other potentially hemorrhagic procedures. 8 More recently, this issue has been reevaluated and specific target values are no longer recommended. 9 On the other hand, less attention has been paid over the years to the risk of thrombosis in this category of patients because it was widely accepted that they were protected because of the prolonged coagulation tests. The observation of a normal thrombin generation in these patients opened new venues in the interpretation of the mechanisms regulating the balance between pro- and anti-coagulants, suggesting that patients with stable chronic liver disease behave essentially as normal individuals. 18 Accordingly, one may expect that patients with cirrhosis may develop thrombosis as healthy individuals. 13 A recent, Danish, nationwide, population-based, case-control study confirmed this impression, showing that patients with liver disease had a relative risk of venous thromboembolism ranging from 1.74 (95% confidence interval [CI]: ) for cirrhotic to 1.87 (95% CI: ) for non-cirrhotic liver disease. 14 The risk was even greater when the analysis was restricted to patients with unprovoked venous thromboembolism (2.06 [95% CI: ] for cirrhotic and 2.10 [95% CI: ] for non-cirrhotic liver disease). 14 In this study we now provide in vitro evidence supporting the above epidemiologic findings. Plasmas from patients with cirrhosis are resistant to the action of thrombomodulin, which is less efficient in the activation of protein C in vitro as compared with plasma from healthy individuals. In general, the in vitro resistance to thrombomodulin may be explained by the presence of the gain-of-function factor V Leiden mutation or to an imbalance favoring pro-coagulant factors. The latter may result from a net excess of such pro-coagulant factors as VIII or to a deficiency of the naturally occurring anticoagulants, especially protein C. 19 Although, we did not assess the genotype for factor V in our cohort, it is unlikely that the frequency of factor V Leiden mutation in patients with cirrhosis is different from that of the general population. We surmise that the resistance to thrombomodulin is due to an imbalance of coagulation favoring some of the pro-coagulant factors that occurs in patients with cirrhosis. Remarkably, factor VIII, ie, the most powerful rate limiting factor in the amplification of thrombin generation in plasma, 20 is indeed considerably increased in cirrhosis 21 (see Figure 2). Factor VIII is also 1 of the 2 factors inhibited by activated protein C. 3 As shown in this study, factor VIII levels are higher in Child Pugh C than Child Pugh A (see Figure 2). Furthermore, the ratio of the 2 most powerful pro- and anti-coagulants operating in plasma (ie, factor VIII and protein C) is considerably in favor of the former (see Figure 5), thus leading to a state of hypercoagulability. Also, the ratio between factor VIII and antithrombin is in favor of the former, thus substantiating the existence of hypercoagulability (see Figure 5). These findings might explain why the incidence of thrombotic events increases with the severity of cirrhosis, 22 being less than 1% in patients with compensated cirrhosis, 23 but from 8% to 25% in candidates for liver transplantation. 24 The ETP ratio (with/ without thrombomodulin) recorded in this study for patients with cirrhosis classified as Child Pugh C is slightly higher than that recorded for patients with congenital protein C deficiency (see Figure 4). Whether this entails a thrombotic risk similar to that of protein C congenital deficiency is unknown. However, our findings

6 2110 TRIPODI ET AL GASTROENTEROLOGY Vol. 137, No. 6 Figure 5. Box plots of the distribution of values for ratios between pro- and anti-coagulants for healthy subjects and patients with cirrhosis stratified according to classes of Child Pugh. may pave the way to clinical studies employing the ratio of ETP with/without thrombomodulin or other similar assays to see whether or not the patients with the highest ratios are those who are more likely to develop thrombosis. Among the limitations of this study one should recognize that the patients investigated were on the stable phase of the disease and free from recent thrombotic and/or hemorrhagic events. Whether the purported hypercoagulability also applies to other categories of patients (ie, cirrhotic patients with the occurrence of portal vein thrombosis, those with non-cirrhotic portal hypertension such as non-hepatic portal fibrosis or Budd Chiari) is presently unknown. Prospective studies with clinical end points on the above categories of patients at high risk would be of special interest to validate the hypothesis that hypercoagulability as detected in this study is of value to identify those at increased risk. In conclusion, we found that plasma from patients with cirrhosis is resistant to the action of thrombomodulin, which is the main activator of the protein C anticoagulant pathway in vivo. This resistance translates into a state of hypercoagulability that is more pronounced in those patients classified as Child Pugh C than Child Pugh A or B. The extent of hypercoagulability in Child Pugh C is similar to that observed under the same experimental conditions in plasma from patients with congenital protein C deficiency. We surmise that hypercoagulability is due to the increase of factor VIII combined with the marked decrease of protein C, which are typical features of patients with cirrhosis. These findings might explain the relatively high risk of venous thromboembolism in this setting earlier reported in a retrospective case-control study 13 and recently documented in a larger nationwide, population-based, case-control study. 14 References 1. Morrissey JH. Tissue factor: a key molecule in hemostatic and nonhemostatic systems. Int J Hematol 2004;79: Mann KG. Thrombin formation. Chest 2003;124(3 Suppl):S4 S Dahlback B. Progress in the understanding of the protein C anticoagulant pathway. Int J Hematol 2004;79: Lane DA, Caso R. Antithrombin: structure, genomic organization, function and inherited deficiency. Baillieres Clin Haematol 1989; 2:

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