TY - JOUR
AU1 - MD, Heike Bernhard,
AU2 - MD, Andrea Deutschmann,
AU3 - Leschnik, Bettina
AU4 - MD, Sabrina Schweintzger,
AU5 - MD, Michael Novak,
AU6 - MD, Almuthe Hauer,
AU7 - MD, Wolfgang Muntean,
AB - Background Patients with inflammatory bowel disease (IBD) have an increased risk of thromboembolic complications. The pathogenesis of IBD is not really clear and a high thrombin activity might contribute to the pathogenesis. We measured thrombin generation by means of calibrated automated thrombography (CAT), a new tool better reflecting overall hemostasis, in children with Crohn's disease (CD) during active and inactive disease and compared it to conventional markers of activity. We wanted to see whether children with CD have a higher potential for thrombin generation and if there is a correlation between hypercoagulability and disease activity. Methods Plasma samples were collected from 22 patients with CD and from 61 healthy children. Thrombin generation was measured by means of CAT. The disease activity was estimated using the Pediatric Crohn's Disease Activity Index (PCDAI). In addition, F1+2, TAT, tissue factor pathway inhibitor (TFPI), fibrinogen, prothrombin (FII), antithrombin (AT), erythrocyte sedimentation rate (ESR), platelet count, α2-globulin, and orosomucoide were measured. Results In all patients we found a significantly higher endogenous thrombin potential (ETP) and higher peak values during active disease. In accordance with this we also found significantly higher mean ETP values during active disease compared with the control group. We observed a significantly positive correlation between PCDAI and thrombin generation parameters. Conclusions Our study clearly shows that the active state of CD in children is associated with the potential for high thrombin generation, but this seems to be caused mainly by the inflammatory process and not by a preexisting propensity for high thrombin generation. (Inflamm Bowel Dis 2011;) Crohn's disease, thrombin generation, pediatric Crohn's disease (CD) is one of the major forms of inflammatory bowel disease (IBD). Thromboembolism is a disease-specific extraintestinal manifestation of IBD.1,2 In adults, IBD is associated with an increased risk of vascular complications. In clinical studies, thromboembolic events are demonstrated to occur in 1%–8% and disease activity may be a risk factor for thromboembolic events. Bench research and clinical experience have demonstrated that in IBD a hypercoagulable state and a prothrombotic condition exist.3,4 This might be caused by increased levels of hemostatic parameters (factors V, VII, VIII, fibrinogen), by reduced levels of anticoagulation factors (antithrombin, protein C, protein S, tissue factor pathway inhibitor), and by impaired fibrinolytic activity caused by the chronic inflammation.5 The pathogenesis of IBD is not really clear and a high thrombin activity might contribute to disease progression. The role of endothelial lesions and microthrombic episodes in the development of intestinal lesions has been discussed.6,7 Conventional global tests, such as prothrombin time (PT) and activated partial thromboplastin time (aPTT), do not reflect this hypercoagulable condition. Prothrombin fragments 1+2 (F1+2) are released from prothrombin during activation by factor Xa. Therefore, F1+2 concentrations are specific indicators for the amount of thrombin generated and consequently are sensitive markers of coagulation activation. Another good marker of coagulation is the thrombin-antithrombin complex (TAT), which is formed during inactivation of thrombin by its inhibitor antithrombin. Indeed, F1+2, TAT, and other sensitive markers of activation of coagulation such as fibrinopeptide A and B are high in IBD.5 Thrombin generation is the only test that is sensitive to hypercoagulable changes in the plasma. A new method to investigate thrombin generation is the calibrated automated thrombography (CAT) developed by Hemker et al.8,12 The major advantage of this method is that thrombin generation can be monitored automatically in clotting platelet-poor plasma (PPP). The resulting thrombogram visualizes the entire process of the overlapping steps of initiation, amplification, propagation, and termination of coagulation. The area under the curve of generated thrombin represents the endogenous thrombin potential (ETP) and has been shown to correlate with plasma-based hypercoagulable states and the individual's risk of possibly being affected by thrombosis. In contrast to F1+2 and TAT, which are indicators of actually ongoing thrombin generation in plasma, the ETP quantifies the enzyme thrombin activity that can be triggered in the plasma, and allows the detection of the influence of procoagulants and anticoagulants on the formation of thrombin.8,–10 We measured thrombin generation by means of CAT in children with CD during active and inactive disease and compared it to conventional markers of activity. We wanted to see whether children with CD have a higher potential of thrombin generation and if there is a relation between hypercoagulability and disease activity. Patients and Methods Patients In this study we compared 22 pediatric patients with CD followed at our gastroenterology outpatient clinics with 61 healthy children (29 girls, mean age ±SD: 13.67 ± 3.01 years). Our controls were recruited from the general pediatric outpatient clinics. They were healthy children who had routine coagulation screening before minor elective surgery such as tonsillectomy, adenoidectomy, or circumcision. The patients with CD were recruited at the Department of Pediatrics, Medical University of Graz, during episodes of active and inactive disease states. The age of our patients ranged from 7 to 19 years (mean age ±SD 13.40 ± 3.18 years), 13 patients were female. The disease activity was estimated for CD using the Pediatric Crohn's Disease Activity Index (PCDAI).11 Inactive state was defined by a score <150. The clinical scoring was done by an experienced physician. CD was confirmed by clinical, endoscopic, and histopathological findings. None of the patients had any clinical signs of thrombosis. During the study period, three patients received no treatment (two patients because of nonadherence and one patient because of disease onset), four patients were treated by enteral nutrition only, and the others received oral corticosteroids and/or 5-aminosalicylate (5-ASA), azathioprine/methotrexate. No other medication that might have interfered with test results (e.g., nonsteroidal antiinflammatory drugs, oral contraceptives) were administered. The clinical features of patients with CD are summarized in Table 1. Table 1 Characteristics of Study Group and Controls     View Large Table 1 Characteristics of Study Group and Controls     View Large This study was approved by the local ethics committee. After informed consent, blood samples for coagulation and inflammation markers were collected. Blood was collected into plastic tubes containing sodium citrate (0.1 M end concentration) using S-Monovette tubes from Sarstedt (Nümbrecht, Germany). Our control group consisted of 61 apparently healthy subjects. Immediately after collection, platelet-poor plasma (PPP) was prepared by centrifugation of whole blood at 2800g for 10 minutes at room temperature and stored at −70°C until further examination. Reagents and Devices Continuous Automated Thrombin Generation (CAT) For the fluorogenic assay via CAT, imidazole buffer solution was obtained from Dade Behring (Marburg, Germany). Fluobuffer contained 20 mM per L HEPES and 60 mg per mL bovine serum albumin (BSA), both from Sigma (St. Louis, MO). Working buffer consisted of 140 mM per L NaCl, purchased from Merck (Darmstadt, Germany); 20 mM per L HEPES; and 5 mg per mL human serum albumin, purchased from Sigma. The flurogenic substrate Z-Gly-Gly-Arg-amino-methyl-coumarin (AMC) was purchased from Bachem (Bubendorf, Switzerland) and was solubilized in pure dimethyl sulfoxide (DSMO), purchased from Sigma. Calcium chloride was obtained from Merck. Thrombin calibrator and the PPP reagent with a content of 5 pM tissue factor (TF) and 4μM phospholipids were purchased from Thrombinoscope (Maastricht, The Netherlands). For automated fluorogenic measurement of thrombin generation we used the method developed and described by Hemker et al.8,12 A mixture of 2625 μL of fluobuffer and 300 μL of 1 M per L CaCl2 solution was prepared and incubated for 5 minutes at 37°C for each experiment. After 5 minutes, 75 μL of the Fluo-DSMO solution was added, mixed, and incubated again for 5 minutes. The resulting clear solution was referred to as FluCa. PPP reagent was solubilized with 2 mL of deionized water; 20 μL of this TF containing trigger solution was put into each sample well of a 96-well round-bottom microtiter plate made of polypropylene, purchased from Nunc (Roskilde, Denmark). After reconstitution with 1 mL of sterile water, the thrombin calibrator was used in each experiment to compare the simultaneously measured thrombin activity in the sample to that from a known and stable concentration in the calibrator well. Finally, 80 μL of PPP was put into each well. Before starting the experiment all reagents were warmed to 37°C. The plate was placed in the fluorometer (Fluoroskan Ascent, Thermo Labsystems, Helsinki, Finland) with an excitation filter at 390 nm and an emission filter at 460 nm. Automatically dispensing 20 μL of FluCa started the measurement process. This continuous measurement is based on the conversion of a thrombin-specific substrate Z-Gly-Gly-Arg-AMC. For 60 minutes, each well was measured three times and the analysis software from Thrombinoscope was used to assess our results. Thrombin activity was calculated as a function of time by comparing the fluorescent signal with that from a known and stable sample. During the measurement the program calculates and displays the thrombin concentration in time. Prothrombin Fragment 1+2 (F1+2), Thrombin-Antithrombin Complex (TAT) Commercially available enzyme-linked immunosorbent assay (ELISA) systems (Enzygnost F1+F2 and Enzygnost TAT, Dade Behring, Marburg, Germany) were used for measurement of prothrombin F1+2 and TAT complex. Tissue Factor Pathway Inhibitor (TFPI) Actichrome TFPI activity assay was obtained from American Diagnostica (Greenwich, CT). TFPI antigen levels were determined by means of the Imubind Total TFPI ELISA Kit. Prothrombin Prothrombin was determined using coagulation factor deficient plasma. Fifty μL of a complete thromboplastin (Thromborel S) were added to 50 μL of the citrated plasma samples. For determination of the prothrombin values we used the Behring Coagulation Timer from Behring Diagnostics. Antithrombin Activity Antithrombin was measured using the chemistry analyzer “Hitachi 917” and Reagent Antithrombin III, Roche/Hitachi (Holliston, MA. Fibrinogen, erythrocyte sedimentation rate (ESR), platelet count, α2-globulin, and orosomucoid were measured by standard laboratory methods. Statistical Analyses All statistical analyses were performed with SPSS software (SPSS, Chicago, IL) and P-values less than 0.05 were considered significant. According to the normality Kolmogorov–Smirnov test, continuous variables were performed by means of parametric tests. Paired Student's t-test was used to assess mean values. Correlations between disease activity and thrombin generation values and different variables were calculated using Pearson's correlation. Results Thrombin generation was measured in PPP by means of CAT. The thrombogram describes the concentration of thrombin in clotting plasma. It starts with the lag time in which no thrombin is formed. After a steep increase, the thrombin generation curve arrives at its peak, the maximum concentration of thrombin. The ETP or the area under the curve represents the amount of thrombin built over the time during the whole process of thrombin generation and stands for a real-time monitoring of the coagulability of blood. The time for reaching the peak is the time to peak (TTP). Figure 1 shows the typical curves in a 10-year-old girl with CD in the active and inactive state. Figure 1 View largeDownload slide Thrombin generation curves in an active compared to inactive state of a 10-year-old girl with CD. Figure 1 View largeDownload slide Thrombin generation curves in an active compared to inactive state of a 10-year-old girl with CD. For our study we analyzed the thrombin generation in patients with CD in the active state and in inactive state. Thrombin generation was measured in healthy subjects as a control. The results of thrombin generation measurements, markers of coagulation activation, and several laboratory parameters of inflammation in the active state compared to the inactive state are shown in Table 2. We found significantly higher mean ETP values in the active state than in the inactive state of CD patients (P < 0.001) (Table 2). Mean ETP values were also significantly higher in active disease compared to the control group (P < 0.001). No significant difference in ETP was found between inactive state of CD patients compared to our control group (Fig. 2). The peak (P = 0.001) was significantly higher in patients with active CD compared to inactive CD. In contrast to this, there were no significant differences when comparing the lag time and TTP in our different groups. Table 2 Results of Thrombin Generation Measurements and Laboratory Parameters     View Large Table 2 Results of Thrombin Generation Measurements and Laboratory Parameters     View Large Figure 2 View largeDownload slide Boxplots of ETP levels in patients in the active and inactive state and healthy controls. The bottom of the boxes mark the 25th percentile, the median lines mark the 50th percentile, the top of the vertical lines mark the 5th and 95th percentile, respectively. Figure 2 View largeDownload slide Boxplots of ETP levels in patients in the active and inactive state and healthy controls. The bottom of the boxes mark the 25th percentile, the median lines mark the 50th percentile, the top of the vertical lines mark the 5th and 95th percentile, respectively. We observed a significant positive correlation between PCDAI and thrombin generation parameters ETP (P < 0.001, r = 0.662), peak height (P = 0.001, r = 0.465), and TTP (P = 0.008, r = 0.368) in our pediatric patients with CD (Fig. 3). No significant correlation was seen between PCDAI and lag time (P = 0.054, r = 0.271). Figure 3 View largeDownload slide Correlations between (A) ETP (P < 0.001), (B) lag time (P = 0.054) NS, (C) peak (P = 0.001), (D) time to peak (TTP) (P = 0.008) and PCDAI. NS, not significant. Figure 3 View largeDownload slide Correlations between (A) ETP (P < 0.001), (B) lag time (P = 0.054) NS, (C) peak (P = 0.001), (D) time to peak (TTP) (P = 0.008) and PCDAI. NS, not significant. In the whole cohort a significant positive correlation was seen between ETP and markers of inflammation such as FII (P = 0.001), ESR (P < 0.001), platelets (P = 0.011), fibrinogen (P < 0.001), orosomucoid (P = 0.01), and α2-globulin (P < 0.001) as well as between peak height and FII (P = 0.002), ESR (P < 0.001), fibrinogen (P = 0.002), orosomucoid (P = 0.005), and α2-globulin (P < 0.001) (Table 3). No statistically significant correlation was found between ETP and parameters of actual activation of thrombin generation TAT, F1+2, AT, and TFPI. Table 3 Correlations Between ETP and Coagulation, Fibrinolytic, and Inflammatory Parameters Using Pearson's Correlation     View Large Table 3 Correlations Between ETP and Coagulation, Fibrinolytic, and Inflammatory Parameters Using Pearson's Correlation     View Large A significant correlation between the TFPI values and FII (P = 0.016, r = 0.347) and also between the TFPI values and AT (P < 0.001, r = 0.565) was seen. Higher levels of F1+2 (P = 0.001) and TAT (P = 0.001) were observed in our patients compared to the control group. A significantly lower level of mean TFPI values were observed in patients in the active and inactive state (P < 0.001) compared to the control group. In patients with active disease ETP was not significantly different between subgroups with or without steroid administration. Discussion In this study we investigated thrombin generation for the first time in patients with IBD. We measured continuous thrombin generation by means of CAT in pediatric patients with active and inactive CD. Because of the key enzyme role of thrombin in hemostasis, CAT measurement is a new tool better reflecting overall hemostasis. CAT reflects the influence of all plasma pro- and anticoagulation factors and is appropriate to detect hypercoagulability. Results of CAT show a high interindividual variability, but measures rather constant over time in one person.8 There is some evidence that high ETP might reflect risk of thrombosis. Besser et al13 demonstrated an association between high ETP and recurrent venous thrombosis. Hemker et al12 demonstrated that an increased ETP is associated with certain congenital deficiencies as well as with acquired disorders with high risk of thrombosis. In recent studies we have shown an age dependency of thrombin generation measured by CAT14 and an increased ETP in obese compared with normal weight children, confirming a hypercoagulable state existing in obese children.15 So-called markers of clotting activation such as F1+2, TAT, or D-Dimer demonstrate that thrombin has been formed in vitro to a higher extent than normal (elevated F1+2) or has led to increased fibrin formation (elevated D-Dimer). In vitro measurements of thrombin generation can demonstrate that a high potential exists to form thrombin, therefore the term “endogenous thrombin potential.” This does not necessarily mean that increased thrombin formation actually exists in this patient, but that there is a high risk if an additional trigger exists. Endogenous thrombin generation describes the composite plasma phenotype of a person. The coagulation system is known to be activated in IBD.3 Our study supports these findings. We found a positive association between both ETP and PCDAI, indicating that a high thrombin generation potential could be the cause for the coagulation activation observed in active disease. Significant higher levels of ETP and peak height were observed, while no significant difference was found for lag time and TTP in the phases of active disease. These data show that children with CD in the active state have the potential to generate significantly higher amounts of thrombin represented by the high ETP. That the lag time and TTP was not significantly shortened suggests that actual activation of the hemostatic system has not yet taken place in most of the investigated children, also shown in the low correlation between parameters of actual activation of thrombin generation and ETP. F1+2 concentrations are a specific indicator of the amount of thrombin generated and consequently a sensitive marker for actual coagulation activation at a specific timepoint. However, F1+2 rising with disease activity has been previously reported.16 Tissue factor pathway inhibitor is a specific inflammatory inhibitor of the TF-FVIIa complex regulating both its procoagulant and proinflammatory properties. TFPI was significantly lower in our patients with CD in active as well in inactive compared to control group. We observed a significant positive correlation between FII and ETP. This is in good agreement with Butenas et al,10 who demonstrated in a “synthetic plasma model” that the most limiting factors for the ETP are the levels of prothrombin and antithrombin. None of our patients had AT levels below the age-specific normal ranges. A significant correlation between the TFPI values and FII and also between the TFPI values and AT was seen in our study. This is in agreement with in vitro data suggesting that TFPI is an important factor for the thrombin generation and once TFPI becomes low, the thrombin generation is increased. There is in vitro and epidemiologic evidence that corticosteroids increase the risk of hypercoagulability. Corticosteroids increased the activity of clotting factors in healthy men but an increasing clotting activity in vivo was not shown.17 There are no data available about F1+2, TAT, or ETP during corticosteroid treatment in healthy controls. We observed in our patients with CD that, by lowering the disease activity index, under therapy with corticosteroids, ETP decreased, but corticosteroids should have, if any, the opposite effect. There are no data about procoagulant effects of azathioprine. Therefore, our findings that ETP is high during states of active disease in patients with CD, whereas in inactive disease it is not higher than in controls, has two major implications: It explains very well the fact that signs of hypercoagulability and clinical manifest thromboembolic complications are frequently found in patients and argue for a prophylactic anticoagulation during active disease,18 but not during phases of well-controlled disease. But our results argue against the intriguing idea that individuals with IBD belong to the group of persons with a high thrombin generation potential and, therefore, have an increased inflammatory response because of their high amount of thrombin. Thrombin increases production of tumor necrosis factor, IL-6, IL-10 by PAR-1 signaling and therefore is able to amplify and modify inflammation.19 Our findings of a high ETP in active disease support the notion that anticoagulation might not only be a measure against thromboembolic complications but also might add to the antiinflammatory regimen; however, there is no support for this from clinical studies.20 Conclusion Our study shows that the active state of CD in children is associated with the potential for high thrombin generation, but this seems to be caused mainly by the inflammatory process and not by an individual disposition. References 1 Koutroubakis IE. Therapy insight: vascular complications in patients with inflammatory bowel disease. Nat Clin Pract Gastroenterol Hepatol . 2005; 2: 266– 272. CrossRef Search ADS PubMed  2 van Bodegraven AA. Haemostasis in inflammatory bowel diseases: clinical relevance. 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Thromb Res . 2006; 118: 247– 252. CrossRef Search ADS PubMed  18 Grainge MJ, West J, Card TR. Venous thromboembolism during active disease and remission in inflammatory bowel disease: a cohort study. Lancet . 2010; 375: 657– 653. CrossRef Search ADS PubMed  19 Chen D, Dorling A. Critical roles for thrombin in acute and chronic inflammation. J Thromb Haemost . 2009; 7( suppl 1): 122– 126. CrossRef Search ADS PubMed  20 Chande N, McDonald JW, Macdonald JK. Unfractionated or low molecular weight heparin for induction of remission in ulcerative colitis. Cochrane Database Syst Rev . 2008; CD0066774. Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.
TI - Thrombin generation in pediatric patients with Crohn's disease
JF - Inflammatory Bowel Diseases
DO - 10.1002/ibd.21631
DA - 2011-11-01
UR - https://www.deepdyve.com/lp/oxford-university-press/thrombin-generation-in-pediatric-patients-with-crohn-s-disease-1XbUuE1LfE
SP - 2333
EP - 2339
VL - 17
IS - 11
DP - DeepDyve
ER -