Increased thrombin generation in inflammatory bowel diseases

Increased thrombin generation in inflammatory bowel diseases

Thrombosis Research 125 (2010) 278–282 Contents lists available at ScienceDirect Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e ...

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Thrombosis Research 125 (2010) 278–282

Contents lists available at ScienceDirect

Thrombosis Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / t h r o m r e s

Regular Article

Increased thrombin generation in inflammatory bowel diseases Simone Saibeni a, Valeria Saladino b, Veena Chantarangkul c, Federica Villa b, Savino Bruno a, Maurizio Vecchi d, Roberto de Franchis b, Cinzia Sei c, Armando Tripodi c,⁎ a

Department of Internal Medicine and Hepatology, Azienda Ospedaliera Fatebenefratelli e Oftalmico, Milan Gastroenterology and Gastrointestinal Endoscopy Service, Department of Medical Sciences, IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena and University of Milan c Angelo Bianchi Bonomi Haemophilia and Thrombosis Center, Department of Internal Medicine, IRCCS Fondazione Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena and University of Milan d Gastroenterology and Gastrointestinal Endoscopy Unit, IRCCS Policlinico San Donato and University of Milan, San Donato Milanese, Italy b

a r t i c l e

i n f o

Article history: Received 26 June 2009 Received in revised form 19 October 2009 Accepted 21 October 2009 Available online 14 November 2009 Keywords: Inflammatory bowel diseases Thrombin generation Inflammation

a b s t r a c t Background: Inflammatory bowel diseases (IBD) are characterized by an increased thrombotic risk of uncertain etiology. Endogenous thrombin potential (ETP), a parameter of the thrombin generation curve, represents a new tool in the evaluation of thrombotic and bleeding disorders. Aims: To study ETP in IBD patients and to correlate the results with clinical and biochemical features. Methods: Seventy-four IBD patients (37 ulcerative colitis and 37 Crohn's disease) and 74 sex- and agematched healthy individuals. ETP was measured upon activation of coagulation with small amounts of tissue factor and phospholipids in the presence or absence of thrombomodulin; results were expressed as nM thrombin·minutes. Results: Mean±SD ETP values were significantly higher in patients (1,499 ± 454) than controls (1,261 ± 385) (p b 0.001) only when the test was performed in the presence of thrombomodulin. ETP evaluated as ratio (with/without thrombomodulin), taken as an index of hypercoagulability, was significantly higher in patients (0.69 ± 0.14) than controls (0.62 ± 0.18) (p b 0.006). Patients with increased C-reactive protein (CRP) had significantly higher mean ETP (1,721 ± 458) than those with normal CRP (1,357 ± 394) or controls (1,261 ± 385) (p b 0.001). Patients who at the time of blood sampling were classified as having a clinically active disease had ETP higher than those who were quiescent (1,655 ± 451 versus 1,388 ± 427, p b 0.001) or controls (1,261 ± 385, p b 0.001). Conclusions: ETP measured in the presence of thrombomodulin or as ratio (with/without thrombomodulin) is increased in IBD patients, mainly in those with increased CRP or active disease. It may be considered as a candidate test for prospective studies aimed at assessing the risk of thrombosis in IBD patients. © 2009 Elsevier Ltd. All rights reserved.

Introduction Crohn's disease and ulcerative colitis, the two major forms of Inflammatory Bowel Diseases (IBD), are chronic illnesses characterized by local and systemic inflammation as well as by an increased risk of thrombotic events [1]. Thrombosis complicating IBD course may affect both the venous [2,3] and the arterial [4] district; they usually occur during active phases of the disease [1] and represent a relevant cause of morbidity and mortality due to the relatively young age of the affected patients [5,6]. Despite awareness is rising and the medical therapy of IBD is largely improved, the thrombotic risk does not appear to decline [7].

Abbreviations: ETP, Endogenous thrombin potential; PT, prothrombin time; APTT, activated partial thromboplastin time; CRP, C-reactive protein. ⁎ Corresponding author. Via Pace 9, 20122-Milano, Italy. Tel.: +39 02 503 20725; fax: +39 02 503 20723. E-mail address: [email protected] (A. Tripodi). 0049-3848/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.thromres.2009.10.012

Several prothrombotic alterations have been described in IBD patients, such as quantitative and qualitative impairments of platelets [8], procoagulant [9] or fibrinolytic proteins [10] as well as hyperhomocysteinemia [11] or decreased naturally-occurring anticoagulant factors [12]. However, the exact pathogenic mechanisms of the thrombotic events are so far not fully elucidated. Thrombin generation represents the final step of the coagulation cascade. It is triggered by the tissue factor in complex with activated factor VII and down-regulated by the naturally occurring anticoagulants [13]. In physiological conditions the balance between the procoagulant and anticoagulant drivers is essential to prevent excessive thrombin formation. Protein C and antithrombin are the two main anticoagulant proteins; the first is activated by thrombin in complex with the endothelial receptor thrombomodulin [14], the second mainly upon interaction with the glycosoaminoglycans located on endothelial cells [15]. The conventional coagulation tests such as the prothrombin and activated partial thromboplastin times (PT and APTT) do not contain

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sufficient amounts of thrombomodulin or glycosaminoglycans and are, therefore, unable to represent the balance of coagulation as it occurs in vivo [16]. In contrast, the last generation of assays that monitor the tissue factor induced thrombin generation in the presence of thrombomodulin are credited as better laboratory tools to represent the balance of pro- and anti-coagulant forces operating in plasma [16–18]. To the best of our knowledge, only one study assessed so far thrombin generation in a small group of patients with active ulcerative colitis [19] and the test was performed in the absence of thrombomodulin. This study aims to evaluate thrombin generation in the absence or presence of thrombomodulin in ulcerative colitis and Crohn's disease patients and to correlate the results with their biochemical and clinical features, with particular attention to the levels of the acute phase reactant C-reactive protein (CRP) and clinical disease activity. A more precise elucidation of the pathogenetic mechanisms underlying the thrombotic complications would be of value to identify patients at higher risk. Materials and Methods The Ethical Committee of our Institutions approved the study. All enrolled subjects were of Italian descent and gave their informed consent to participate in the study. Criteria of exclusion from the study were the previous history of thrombosis, anticoagulant treatment and the presence of cirrhosis or chronic pathologic conditions such as diabetes mellitus or renal failure. Patients We enrolled 74 consecutive IBD patients (42 men and 32 women, mean age ± SD: 44.7 ± 14.0 years) followed at our outpatient gastrointestinal clinics; 37 had ulcerative colitis and 37 Crohn's disease. The diagnosis of IBD was made by means of clinical, endoscopic, radiological, and histological findings. The clinical history of IBD patients was obtained from their clinical records. Disease activity was assessed by means of Crohn's disease activity index (CDAI) [20] and Truelove and Witts criteria [21] for ulcerative colitis; the clinical type of Crohn's disease was classified according to the Vienna classification [22]. The demographic and clinical features of IBD patients are summarized in Table 1.

Table 1 Demographic and clinical features of IBD patients. Crohn's disease

Ulcerative colitis

n = 37

n = 37

Men/women Mean age at sampling ± SD (years) Mean disease duration ± SD (months) Active disease activity at sampling, n (%) CRP ≥ 0.5 mg/dl, n (%)

19/18 40.4 ± 12.3 70.0 ± 55.9 11 (29.7) 13 (35.1)

23/14 48.9 ± 14.3 69.3 ± 55.8 20 (54.1) 16 (43.2)

Location/extension, n (%) ileum ileum+colon colon pancolitis left colitis procto-sigmoiditis

8 (21.6) 17 (45.9) 12 (32.4) – – –

– – – 9 (24.3) 16 (43.2) 12 (32.4)

Behavior, n (%) non-stricturing, non-penetrating penetrating stricturing

11 (29.7) 10 (27.0) 16 (43.2)

– – –

Treatment, n (%) no therapy only 5-ASA steroids ± other drugs

5 (13.5) 17 (46.0) 15 (40.5)

2 (5.4) 25 (67.6) 10 (27.0)

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Healthy subjects As a control population we enrolled 74 sex- and age-matched healthy subjects (mean age ± SD: 43.4 ± 13.7 years) randomly picked up from a larger cohort of healthy individuals enrolled as controls for the studies of thrombophilia and thrombin generation in other clinical conditions carried out in the same period as the study in IBD patients. Their characteristics have been previously described [23]. Briefly, they were free from present and past thrombotic events, their prevalences of prothrombotic mutations were those observed in the general Italian population and carriers were not excluded from the study. None of the control subjects was on oral anticoagulants or other medications (including oral contraceptives) known to interfere with blood coagulation. Blood collection and plasma preparation Blood was obtained by clean venipuncture and collected in vacuum tubes containing 109 mmol/L trisodium citrate as an anticoagulant (Vacutainer; Becton Dickinson, Meylan, France) at a blood to anticoagulant ratio of 9:1. Blood was centrifuged within 30 minutes at (controlled) room temperature for 15 minutes at 2,880 ×g. The platelet-free plasma was subsequently aliquoted in plastic-capped tubes, quickly frozen in liquid nitrogen, and stored at -70 °C. Methods Coagulation parameters Protein C was measured with a commercially available anticoagulant assays (PC Clot, Instrumentation Laboratory, Orangeburg, NY, USA). Factors VIII and II were measured as clotting activities by modifications of the APTT or the PT, respectively, and factor VIII or factor II deficient plasmas. Results for protein C, factors VIII and II were expressed as percentage activity relatively to a pooled normal plasma set arbitrarily at 100% activity. C-reactive protein (CRP) CRP was measured by a standard laboratory method and results were considered as increased when ≥0.5 mg/dL. Thrombin Generation Thrombin generation was evaluated according to Hemker et al. [24] as described in detail by Chantarangkul et al. [25]. The test is based on the activation of coagulation in test plasmas after addition of human relipidated recombinant tissue factor (Recombiplastin; Instrumentation Laboratory, Orangeburg, NY, USA) which acts as a coagulation trigger in the presence of the synthetic phospholipids 1,2-dioleoyl-sn-glycero3-phosphoserine (DOPS), 1,2-dioleoyl-sn-glycero-3-phosphoetanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) (Avanti Polar Lipids Inc., Alabaster, AL) in the proportion of 20/20/60 (M/M). The concentrations of tissue factor and phospholipids in the test system were 1 pM and 1.0 μΜ, respectively. The experiments were also carried out by adding soluble rabbit thrombomodulin (ICN Biomedicals, Aurora, OH, USA) in the reaction mixture at a final concentration of 4 nM. This concentration was arbitrarily chosen upon experiments that showed the best discrimination of thrombin generated upon triggering coagulation for healthy subjects and patients with congenital protein C deficiency. Continuous registration of the generated thrombin was obtained by means of a fluorogenic synthetic substrate (Z-Gly-Gly-Arg-AMC HCl, Bachem, Bubendorf, Switzerland) added to the test system at a final concentration of 417 μΜ. The testing procedure was performed with an automated fluorometer (Fluoroskan Ascent; ThermoLabsystem, Helsinki, Finland) that is able to handle simultaneously several samples in an automated fashion. Readings from the fluorometer are recorded and calculated by a dedicated software (Thrombinoscope™, Thrombinoscope BV, Maastricht,

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The Netherlands), which displays thrombin generation curves [nM thrombin versus time (minute)] and calculates the area under the curve, defined as endogenous thrombin potential (ETP) and expressed as nM thrombin times minutes (nM·min). Thrombin is measured as function of a calibrator (Thrombin Calibrator, Thrombinoscope BV). ETP represents the plasmatic balance between the action of pro- and anticoagulants. To minimize analytical variability equal numbers of plasmas from patients and healthy subjects were included on each test occasion. Statistical analyses Results are reported as means and standard deviations (SD). The Statistical Package for Social Sciences (SPSS Inc., v. 11.0 for Windows, Chicago, IL, USA) and GraphPad Instat package software (GraphPad Software Inc., San Diego, CA, USA) were used for statistical analyses. According to the normality Kolmogorov-Smirnov test, continuous variables were normally-distributed, therefore statistical analyses were performed by means of parametric tests. In particular, the twosided Student's t test and the one-way ANOVA (with StudentNewman-Keuls multiple comparison) were used to assess for differences between mean values and the Pearson coefficient of correlation was used to test for correlation between different variables (clinical activity was considered as a categorical variable). Comparison of prevalences was assessed by means of Fisher's exact test and Chi-square for independence, as appropriate. The level of significance was set at p = 0.05; the p value was two-tailed. Results Conventional coagulation parameters Mean ± SD factor VIII levels were significantly higher in IBD (118.1 ± 40.8%) than in healthy controls (103.5 ± 12.7%) (p ≤ 0.008). Mean ± SD factor II levels in IBD patients (108.7 ± 11.5%) were not significantly different from those of healthy controls (111.2 ± 33.8%) (p = 0.571). In IBD patients mean ± SD protein C activity (123.7 ± 29.5%) were slightly higher than in healthy subjects (109.5 ± 24.4%) (p ≤ 0.003). ETP assessment in the absence of thrombomodulin No significant differences were observed between mean±SD ETP values in IBD (2,099.8 ± 447.2 nM·min) and healthy subjects (2,057.7 ± 385.8 nM·min) populations (p= 0.543). However, when stratified according to CRP levels, IBD patients with increased CRP levels showed mean ETP values (2,272.6 ± 437.2 nM·min) significantly higher than IBD patients with normal CRP levels (1,988.4± 421.6 nM·min) or healthy subjects (2,057.7 ± 385.8 nM·min) (p= 0.013). No difference was observed between Crohn's disease and ulcerative colitis patients (data not shown).

Fig. 1. ETP values (nM thrombin x min) measured in the presence of thrombomodulin in IBD patients and healthy subjects. Solid horizontal bars and broken line represent mean values and the 95th percentile of the distribution of healthy subjects.

In IBD patients, there was a slight but significant correlation between ETP and CRP values (r = 0.28, p = 0.015) and disease activity (r = 0.29, p = 0.011). Overall, the prevalence of high ETP levels (defined as values higher than the 95th percentile of values of controls i.e., 1,821.5 nM·min) was significantly higher in IBD patients (19 of 74, 25.7%) than in healthy controls (4 of 74, 5.4%) (RR 6.04, 95% C.I. 1.94-18.80; p b 0.002) (Fig. 1). The prevalence of IBD patients with high ETP levels (stratified according to CRP and clinical disease activity) and healthy subjects showed significant differences (Fig. 2). IBD type, disease location, disease duration, drug treatment and Crohn's disease behaviour did not significantly influence the ETP values nor the prevalence of high ETP values, both when the experiments were performed in the presence or absence of thrombomodulin (data not shown). ETP assessment as ratio (with/without thrombomodulin) ETP was also evaluated as ratio of values obtained with and without thrombomodulin in patients with IBD and healthy individuals. Mean±SD values were significantly higher in IBD patients (0.69 ± 0.14) than those recorded in healthy individuals (0.62 ± 0.18), (pb 0.006). Discussion Despite the increased risk for thrombosis is known for a long time [26] and several studies demonstrated qualitative and quantitative Table 2 ETP values (mean ± SD) in the presence of thrombomodulin in healthy subjects and in IBD patients according to C-reactive protein (CRP) levels and clinical disease activity. ETP (nM·min)

ETP assessment in the presence of thrombomodulin When tested in the presence of thrombomodulin, mean±SD ETP values were significantly higher in IBD patients (1,499.7±454.3 nM·min) than in healthy subjects (1,261.2±384.8 nM·min) (p b 0.001) (Fig. 1). When stratified according to CRP levels, IBD patients with increased CRP levels showed mean ETP values significantly higher than IBD patients with normal CRP levels or healthy subjects (p b 0.001) (Table 2a). When stratified according to clinical disease activity IBD patients who had a clinically-active disease state had mean ETP values significantly higher compared to those who were quiescent (p b 0.01) or healthy subjects (p b 0.001) (Table 2b). No difference was observed between Crohn's disease and ulcerative colitis patients (data not shown).

a) CRP levels (increased when ≥ 0.5 mg/dl) IBD with increased CRP 1,721.3 ± 458.0 IBD with normal CRP 1,356.6 ± 394.5 Healthy subjects 1,261.2 ± 384.8 IBD with increased CRP vs. IBD with normal CRP p b 0.001 IBD with increased CRP vs. healthy subjects p b 0.001 IBD with normal CRP vs. healthy subjects p = n.s. b) Clinical disease activity IBD clinically active IBD clinically quiescent Healthy subjects Active IBD vs. quiescent IBD p b 0.01 Active IBD vs. healthy subjects p b 0.001 Quiescent IBD vs. healthy subjects p = n.s.

1,654.9 ± 451.3 1,387.6 ± 427.3 1,261.2 ± 384.8

p b 0.001

b 0.001

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Fig. 2. Prevalence of high (N 95th percentile of the distribution of healthy subjects) ETP values measured in the presence of thrombomodulin in IBD patients (stratified for CRP levels and clinical disease activity) and healthy subjects.

abnormalities of the hemostatic system [8–12], the exact pathogenetic mechanisms underlying thromboembolic events in IBD are not yet fully elucidated. Literature data about haemostatic parameters and risk factors for thrombosis in IBD are not always unequivocal and results are contrasting. This may be due to the different hemostatic parameters and to differences in IBD populations studied (i.e., clinical and demographic features as well as disease activity definition and evaluation) [27]. Furthermore, single-factor determination or conventional global coagulation tests are not suitable to represent the coagulation balance as it occurs in vivo [16]. The assessment of the ETP, a parameter of the thrombin generation curve, represents a new promising laboratory tool in the study of clinical conditions characterised by a state of hyper- or hypo-coagulability because it can be considered as an overall function test of the haemostatic-thrombotic system [24]. In particular, previous studies showed that the thrombin generation tests when performed upon addition of thrombomodulin [18,28,29] proved more suitable than other conventional global coagulation tests to represent the balance of coagulation which occurs in vivo. The concept that conditions which are associated with an increased thrombin generation may cause a thrombotic tendency [17] prompted us to evaluate ETP in IBD patients. The most interesting findings were observed when thrombomodulin was added in the experiments. Our results showed that thrombin generation observed in IBD patients was significantly higher than that observed in healthy subjects (Fig. 1). When IBD patients were stratified according to clinical, demographical and biochemical features, there was an association between the ETP values and CRP as well as clinical activity, suggesting a relationship between increased thrombin generation and systemic inflammation. Subsequent analyses of our data have shown that the ratio of ETP performed with/without thrombomodulin for patients was significantly greater than that for the control population. This indicates that patients with IBD generate relatively more thrombin than controls after thrombomodulin addition and full protein C activation and are, therefore, resistant to the action of thrombomodulin. We surmise that the resistance to thrombomodulin

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translates into hypercoagulability, which may contribute to the increased risk of thromboembolism described in these patients. Thrombomodulin resistance, in the face of normal protein C activity, may be a consequence of factor V Leiden mutations or increased levels of such procoagulant factors as VIII [30] and others. The prevalence of factor V Leiden or prothrombin mutations in patients with IBD does not appear to be different from that of the general population [31,32] and it has been described that the occurrence of the above mutations is significantly less frequent in thrombotic IBD patients than in thrombotic non-IBD patients [33]; this suggests that other acquired risk factors play the most relevant role in determining the thromboembolic events observed in IBD patients especially during the active phases of the diseases. Factor VIII is one of the pro-coagulant drivers of thrombin generation and although contrasting results on this factor have been described in IBD patients [34–37] we observed slightly, but significantly increased levels that might play a role in the observed resistance to thrombomodulin and increased thrombin generation. Activation of factor VIII and/or other pro-coagulants may be triggered by the interaction of the inflammatory response and the haemostatic process through the mediation of cytokines [38,39], thus resulting in increased thrombin generation. The association between the hypercoagulability assessed as thrombin generation and the inflammatory state as shown in this study indirectly confirm the observations that thromboembolic events occur more frequently when IBD patients are in an active state [1,9,33] and that the majority of the hemostatic abnormalities are observed during the active phases of the disease [40]. Very few data exist about thrombin generation in IBD. ETP has been evaluated only in a small-size placebo-controlled trial of active ulcerative colitis patients treated with low molecular weight heparin [19]. Also in that study the authors reported a significant correlation between ETP and CRP. Our study, performed in a larger IBD population including also quiescent patients, extends the findings to Crohn's disease patients and strengthens the relationship between ETP levels and systemic inflammation. In conclusion, thrombin generation evaluated both as ETP measured in the presence of thrombomodulin or as ratio (with/without thrombomodulin) is increased in IBD patients, mainly in those with increased CRP or active disease. These findings are consistent with the concept of hypercoagulability due to a procoagulant imbalance. Although thrombin generation assays are not yet routinely performed in clinical practice they have the potential to become useful global tests for the estimation of the risk of thrombosis. Thrombin generation could help to identify IBD patients with hypercoagulability at increased risk of thrombosis. Prospective studies are, however, needed to assess its clinical value in stratifying the risk of thrombosis in this setting. Conflict of interest statement None to declare. References [1] Miehsler W, Reinisch W, Valic E, Osterode W, Tillinger W, Feichtenschlager T, et al. Is inflammatory bowel disease an independent and disease specific risk factor for thromboembolism? Gut 2004;53:542–8. [2] Bernstein CN, Blanchard JF, Houston DS, Waida A. The incidence of deep venous thrombosis and pulmonary embolism among patients with inflammatory bowel disease: A population-based cohort study. Thromb Haemost 2001;85:430–4. [3] Huerta C, Johansson S, Wallander Ma, Garcia Rodriquez LA. Risk factors and shortterm mortality of venous thromboembolism diagnosed in the primary care setting in the United Kingdom. Arch Intern Med 2007;167:935–43. [4] Ha C, Magowan S, Accortt NA, Chen J, Stone CD. Risk of arterial thrombotic events in inflammatory bowel disease. Am J Gastroenterol 2009;104:1445–51. [5] Quera R, Shanahan F. Thromboembolism – An important manifestation of inflammatory bowel disease. Am J Gastroenterol 2004;99:1971–3. [6] Grip O, Svensson PJ, Lindgren S. Inflammatory bowel disease promotes venous thrombosis earlier in life. Scand J Gastroenterol 2000;35:619–23. [7] Nguyen GC, Sam J. Rising prevalence of venous thromboembolism and its impact on mortality among hospitalized inflammatory bowel disease patients. Am J Gastroenterol 2008;103:2272–80.

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