Int J Clin Exp Pathol 2018;11(3):1629-1636 www.ijcep.com /ISSN:1936-2625/IJCEP0069407

Original Article Fibrinogen-like protein 2 prothrombinase may contribute to the progression of inflammatory bowel disease by mediating immune

Xiu-Li Dong1*, Hai-Hua Lin2*, Ren-Pin Chen1, Huan-Dong Zhou3, Wan-Dong Hong1, Xiang-Rong Chen1, Qing-Ke Huang1, Xue-Cheng Sun1, Zhi-Ming Huang1

1Department of Gastroenterology, The First Affiliated Hospital of Medical University, Ouhai , Wen- zhou, , ; 2Department of Pediatrics, The Children’s Hospital, Zhejiang University School of Medicine, , , China; 3Department of Vascular Surgery, Hangzhou Third Hospital, , Hangzhou, China. *Equal contributors and co-first authors. Received November 19, 2017; Accepted December 2, 2017; Epub March 1, 2018; Published March 15, 2018

Abstract: Inflammation and coagulation are interdependent processes that enable each process to activate and propagate the other in inflammatory bowel disease (IBD). Thus, we investigated the role of a novel immune co- agulant, fibrinogen-like protein 2 prothrombinase (FGL2), in patients and mice with IBD. 83 IBD patients and 40 normal controls were enrolled, and trinitro-benzene-sulfonic acid (TNBS)-induced colitis mice were used. Expression of FGL2 in the intestine was detected by immunohistochemistry. Using serial sections, staining was performed to detect tumor necrosis factor α (TNF-α) expression, and to demonstrate co-localization of FGL2 with and fibrin. Correlations between FGL2 expression with some common laboratory parameters were examined. FGL2 was seen primarily in inflammatory infiltrating cells, mainly macrophages, and microvascular vessels and had a strong co-localization with fibrin deposition. IBD patients and mice had increased expression of FGL2 compared with controls. Furthermore, FGL2 expression was correlated with intestinal and plasmatic TNF-α expression, mean volume (MPV), platelet count (PLT), platelet-crit (PCT), and fibrinogen. Our data indicate that FGL2 may me- diate immune coagulation in IBD patients. It may be considered as a novel molecule that contributes to the onset and development of IBD.

Keywords: Inflammatory bowel disease, fibrinogen-like protein 2 prothrombinase, coagulation, inflammation

Introduction lation are two interdependent processes, which is crucial for the progression of IBD [8, 9]. Inflammatory bowel disease (IBD) comprises a group of chronic and relapsing-remitting im- Fibrinogen-likeprotein 2 prothrombinase (FG- mune-mediated disorders of the gastrointesti- L2), also called fibroleukin, has been identified nal tract, which mainly includes Crohn’s dis- as a new member of fibrinogen-related protein ease (CD) and ulcerative colitis (UC). It has superfamily. FGL2 has been shown previously been shown that the coagulation cascade and to have the attributes to a serine protease fibrinolysis are activated in patients with IBD capable of directly cleaving prothrombin into and then initiate a cascade coagulat- and increased thromboembolic events are ing reaction [10]. Several studies have indicat- extra-intestinal and potentially life-threatening ed that FGL2 is involved in fulminant hepatitis, complications [1-3]. The prothrombotic state in spontaneous abortion and xenograft rejection IBD may be promoted by the inflammatory by mediating “immune coagulation”, such as response and cytokines [4]. As subsequent fibrin deposition and microthrombus formation anticoagulant mechanisms fail to control the [11-15]. clotting process, the pro-inflammatory state is maintained or even increased [5-7]. There is While the IBD-associated prothrombotic state growing evidence that inflammation and coagu- has been linked to the inflammatory response, Fibrinogen-like protein 2 may contribute to progression of IBD by immune coagulation

Table 1. Characteristics of the patients and Laboratory parameters of patients and con- control groups trols Characteristics NC IBD Venous blood samples were collected from all Number 40 83 the participants with tubes containing 3.8% tri- Male 22 42 sodium, then centrifuged (2000 rpm) for 10 Female 18 41 min at 4°C, and finally plasma was separated. Mean age, years 42 (20-79) 41.7 (15-79) Plasma levels of ESR and C-reaction protein Smoking 10 29 (CRP), PLT, MPV, PCT, prothrombin time (PT), Oral contraceptive use 4 13 activated partial thromboplastin time (APTT), NC: normal control; IBD: Inflammatory bowel disease. TNF-α, and fibrinogen were routinely me- asured. the mediators that link inflammation and coag- TNBS-induced colitis in mice ulation remain poorly defined. Therefore, we propose that FGL2 may contribute to the inflam- 6- to 8-week-old BALB/c male mice were pur- mation-induced coagulation or thrombosis in chased from Shanghai SLAC Laboratory Animal IBD through a clotting-dependent pathway. Co., Ltd (License Number: SCXK (Shanghai) 2012-0002). Mice were kept under specific Materials and methods pathogen-free conditions (Laboratory Animal Research Center of Wenzhou Medical Uni- Patients and observation versity), and all procedures were conducted ethically according to the Guide for the Care The study was performed at the Department of and Use of the Administration Committee of Gastroenterology and Hepatology of the First Experimental Animals of Wenzhou Medical Affiliated Hospital of Wenzhou Medical Un- University. After a week of adaptive feeding, iversity from December 2006 to December mice were weighed and anesthetized by intra- 2015. All the patients were followed up for 6-12 peritoneal injection with 80 ml of ketamine/ months. Informed consent was obtained for xylazine solution per 10 g body weight. TNBS each patient included in the study. The research was dissolved in alcohol (50:50 vol/vol). 100 μl protocol was reviewed and approved by the clin- of TNBS solution (vol (TNBS)/vol (alcohol) was ical research Ethics Committee of the First 50:50) was administered intra-rectally (via 3.5 Affiliated Hospital of Wenzhou Medical Un- F-catheter) to mice maintained for 60 s in a ver- iversity. tical position. The dose was 100 mg/kg body IBD patients were diagnosed according to the weight. The insert depth was 4 cm proximal to article written by Lennard-Jones JE [16]. 83 the anus. Control mice received 100 μl of 0.9% Patients with active disease enrolled in our saline intra-rectally [20]. Sample size in each study had not received any steroid medications group was 6. for their disease. For patients with CD, severity of disease was classified in accordance with Clinical and macroscopic analysis of colitis in the CDAI (activity indices of CD) score [17]. mice Active disease was defined as a CDAI score ≥150, in inactive disease <150. Disease activi- The clinical analysis of colitis in mice was moni- ty in UC patients (activity indices of UC, UCAI) tored daily for body weight, diarrhea, and was evaluated by using of the Truelove-Witts hemafecia [25]. Loss of body weight was calcu- criteria [18]. For statistical purposes, the clas- lated as percent difference relative to initial sification was quantitatively modified [19]. body weight. Diarrhea was scored as follows: 0, Active disease was defined as score >3 and normal; 2, loose stools; and 4, diarrhea that inactive disease as a score ≤3. Disease activity remained adhesive to the anus. Bleeding was evaluated at the time of sample collection. scores were assessed as follows: 0, negative 40 normal controls from the normal mucosal hemoccult; 2, positive hemoccult; and 4, obvi- areas of healthy subjects or the patients with ous bleeding [23]. Mice were euthanized 3 colonic polyp were included. days after TNBS administration. The colon was removed and opened longitudinally. The mac- The characteristics of patients and controls are roscopic damage was measured by a blinded shown in Table 1. observer with the following score system [5,

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controls and mice were fixed in 4% paraform, dehydrated, embedded in paraffin, sec- tioned (4 μm thickness) and stained with hematoxylin and eosin. The pathological slices were observed under a light microscope by two blinded pathologists.

Immunohistochemistry stain- ing FGL2 expression in local intestine

Mucosal specimens of pa- tients and controls and the colonic fragments of mice were fixed with 4% parafor- maldehyde, processed into paraffin, sectioned, and depa- raffinated in xylene and rehy- drated through ethanol to water. Nonspecific binding was blocked by sequential incubation of the sections in citrate buffer for 4 min at 100°C and 16 min at 20°C, Figure 1. Mice were treated with TNBS (100 mg/kg), then observed every and then 3% hydrogen peroxi- day until euthanasia. (A) Body weight changes (percentage of original body dase solution for 15 min fol- weight); (B) Bleeding score; (C) Diarrhea score; (D) Macroscopic score. Rep- resentative macroscopic images of normal control mice (I) and IBD mice lowed by 5% normal goat (J). **P<0.001, n=6 per each group. Representative H&E-stained colon serum in PBS at 37°C for 60 sections were shown: (E) Colon sections from the control patients (original min. Thereafter incubation in magnification, ×200). (F) Colon sections from IBD patients (original magnifi- a monoclonal antibody from cation, ×200). (G) Colon sections from the control mice (original magnifica- tion, ×100). (H) Colon sections from IBD mice (original magnification,× 100). mouse to human FGL2 (Ab- nova, Taiwan) was done at a dilution of 1:500 in PBS at 26]: 0, normal; 1, hyperemia, edema, no ulcer; 4°C for 16 h. After washing with PBS, sections 2, hyperemia, edema, small linear ulcers or were incubated with immunoperoxidase-conju- petechiae; 3, hyperemia, edema, wide ulcers, gated rabbit IgG fraction to mouse IgG Fc necrosis, or adhesions; 4, hyperemia, edema, (Zhongshan Company, Beijing, China) at 37°C megacolon, stenosis, or perforation. for 30 min, followed by three washes in PBS. Finally, the sections were incubated with Histology in patients and mice 3,3’-diaminobenzidine chromagen and counter- stained with hematoxylin. Negative control was For histological analysis, mucosal biopsy speci- set in the experiment. For the evaluation of mens, from macroscopically inflamed areas of FGL2 expression, a total of 10 random fields patients, and normal controls from the normal across each section were selected to conduct areas of healthy subjects were obtained. the semi-quantitative analysis of the mean Patients with a colonic polyp were seen at the absorbance at a magnification of ×200. Department of Gastroenterology and Hepa- tology of First Affiliated Hospital of Wenzhou Fibrin deposition in local intestine Medical University, and samples were collected in this study from December 2006 to December For fibrin detection, serial sections were stained 2015. Furthermore, the colons of mice were with a rabbit-anti-fibrinogen Ab (Abcam, Ca- removed and divided into fragments after sacri- mbridge, USA, at a dilution of 1:100). This fice. The colonic fragments (0.5 cm) of patients, reagent is known to react with fibrinogen and

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Figure 2. Immunohistochemical staining of various indicators. TNF-α in IBD mice and patients (B and F, ×400), and normal controls (A and E ×400). FGL2 in IBD mice and patients (C and J, ×400), and normal controls (D ×200 and I ×400). Fibrin in normal control and IBD patients (H ×200 and G ×400). CD68 in normal control and IBD patients (L and K, ×400). fibrin in mouse and human tissues. The tech- SEM and P<0.05 were considered significant. nique used for detection of fibrin was the meth- The associations between FGL2 expression od as previously described. and disease activity indices or other Laboratory parameters, including CRP, ESR, PLT, MPV, PCT, TNF-α expression in local intestine fibrinogen, D-dimer, PT, APTT, TNF-α were exam- ined by non-parametric correlation (Spearman Serial sections were stained with a rabbit-anti- TNF-α Ab (Abcam, Cambridge, USA, at a dilution r). of 1:150). The technique used for detection of Results TNF-α was the method as previously described.

Immunoperoxidase staining of macrophages TNBS-induced mice develop serious clinical manifestation with general and histological A CD68 Ab (ProteinTech, at a dilution of 1:100) changes similar to IBD patients was used to detect macrophages using the similar methodology described above. The TNBS-induced colitis mice acquired a severe sickness characterized by body weight Statistical analysis loss, diarrhea, defecate occult blood (OB) or hemafecia after TNBS administration (Figure Statistical Analysis was carried out by SPSS16.0 1A-C). Subsequently, the colonic samples were software. All results were presented as mean ± generally observed and scored by a blinded

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Table 2. Correlation of FGL2 expression with inflammation parameters TNF-α (intestine) TNF-α (plasma) ESR (mm/h) CRP (mg/l) Pearson Correlation 0.00199 ± 0.00146 84.8 ± 92.2 pg/ml 46.4 ± 34.3 23.8 ± 32.0 FGL2 0.00146 ± 0.00115 0.628*** 0.425*** 0.161 n.s. -0.175 n.s. IBD: Inflammatory bowel disease; ESR: sedimentation rate; CRP: C-reaction protein; TNF-α: tumor necrosis factor α. All results were expressed as mean ± SD. A level of P<0.050 was considered to be statistically significant. ***P<0.001; n.s. = not signifi- cant.

Table 3. Correlation of FGL2 expression with coagulation parameters Plt (10^9/l) MPV (fl) PCT (µg/l) PT (s) aPTT (s) Fibrinogen (g/l) Pearson Correlation 301.8 ± 119.6 7.77 ± 1.25 0.233 ± 0.097 14.2 ± 1.93 39.2 ± 4.69 4.23 ± 1.22 FGL2 0.00146 ± 0.00115 -0.330** -0.304** -0.404*** 0.005 n.s 0.002 n.s. 0.224* PLT: platelet count; MPV: mean platelet volume; PCT: platelet-crit; PT: prothrombin time; APTT: activated partial thromboplastin time. All results were expressed as mean ± SD. A level of P<0.050 was considered to be statistically significant. *P<0.05; **P<0.01; ***P<0.001; n.s. = no significance. observer (Figure 1D, 1I, 1J). We observed that Fibrin deposition was detected in the matrix the colon of IBD model was accompanied by and around the FGL2-positive infiltrating cells hyperemia, edema, ulcers, necrosis, and adhe- and microvascular vessels (Figure 2G, 2H) and sion. The clinical manifestation and general was strongly co-localized with FGL2 expre- changes were similar to patients with IBD. ssion. Simultaneously, histological examination sh- owed that the changes of TNBS-induced mice Correlations between FGL2 expression and covered decrease of goblet cells, loss of crypts, inflammation damage of crypts, infiltration by inflammatory cells, and extensive destruction of the mucosal There was association between FGL2 expres- layer (Figure 1H) compared with normal con- sion and inflammatory parameters. There was trols (Figure 1G). Similarly, IBD patients had the a strong correlation between FGL2 expression same histopathology (Figure 1E, 1F). and TNF-α expression in the intestine and plas- ma of IBD patients (r=0.628, P<0.001; r=0.425, FGL2 expression was increased in patients P<0.001). But the enhanced FGL2 expression and mice with IBD was not correlated with ESR levels or CRP lev- els. Moreover, an amount of TNF-α expression Compared with normal controls, there was sig- was observed in areas of inflammatory infiltra- nificantly up-regulation of FGL2 expression in tion and in the vessels region of IBD patients inflamed mucosal specimens from patients (Figure 2E, 2F) and colitis mice (Figure 2A, 2B). and mice with IBD (Figure 2C, 2D, 2J, 2K). The Spearman-rank correlation coefficient values expression of FGL2 was not only in the areas of and the two-tailed significance of the correla- inflammatory infiltration and the vessels, but tion between FGL2 expression and inflamma- also in the mucosal lamina propria. There was tion parameters in IBD patients are shown in no significant difference between the mean Table 2. ages of patients and controls. Gender distribu- tion was similar in patients and controls. Correlations between FGL2 expression and coagulation parameters Cellular source of FGL2 and fibrin deposition in IBD patients Intestinal FGL2 expression in patients with IBD negatively correlated with platelet count (r=- By serial section staining, the majority of 0.330, P<0.01), MPV (r=-0.304, P<0.01) and CD68+ cells showed high expression of FGL2 PCT (r=-0.404, P<0.001), but was not correlat- protein in patients with IBD (Figure 2K, 2L). ed with the levels of PT or aPTT. There was cor-

1633 Int J Clin Exp Pathol 2018;11(3):1629-1636 Fibrinogen-like protein 2 may contribute to progression of IBD by immune coagulation relation between FGL2 expression and fibrino- polysaccharide or cytokines like TNF-α, IL-2. gen level (r=0.224, P<0.05). Spearman-rank The direct prothrombinase activity of FGL2 has correlation coefficient values and the two-tailed implicated in the pathogenesis of several significance of the correlation between FGL2 inflammatory disorders including fulminant expression and coagulation parameters in IBD hepatitis and severe hepatitis, allo- and xeno- are shown in Table 3. graft rejection, as well as murine and human cytokine induced fetal loss [10-15]. FGL2 con- Discussion tributes to these diseases by mediating “immune coagulation” such as fibrin deposition Despite numerous studies, to date, the exact and microthrombus formation. Furthermore, pathogenesis of IBD has not been clearly eluci- neutralizing antibodies to FGL2 prevent both dated. The most prevailing factors include host fibrin deposition and death from MHV-3 infec- genetic susceptibility and immune abnormali- tion. Gene-targeted FGL2-deficient (FGL2-/-) ties [21-23]. However, recently, a large number mice or targeted FGL2 gene with antisense of studies that focus on the role of endothelial FGL2 result in the prevention of MHV-3-induced damage and coagulation disturbances as IBD- fibrin deposition, liver injury, and death [11]. triggering factors [24-27]. Inflammation and The observation that xenografts from FGL2-/- coagulation are interdependent processes that mice transplanted into rats are devoid of throm- enable each process to activate and propagate bosis also supports FGL2 as a coagulant [13]. the other [6-8]. The persistent pro-coagulant state likely involves endothelial cells, leuko- Our study first demonstrates the role of FGL2 in cytes, and , which are activated in IBD patients. We found that there was signifi- response to the inflammatory stimulus. The cantly higher FGL2 expression in patients and activated endothelial cells, leukocytes, and mice with IBD than normal controls. The up- platelets during inflammation phase exhibit an regulated expression of FGL2 in IBD patients increased expression, downregu- may be promoted by a inflammatory response lation of the anticoagulant pathway, or pro-inflammatory cytokines like TNF-α [32]. and generation/activation of coagulation fac- This is consistent with our result that there was tors (e.g., factor Xa), as well as enhanced throm- parallel high expression of intestinal TNF-α. bin production [28-30]. In addition, several find- FGL2 was primarily seen in macrophages and ings have demonstrated a role for cytokines in endothelial cells in IBD patients and was co- mediating inflammation-induced coagulation or localization with fibrin deposition. This con- thrombosis, such as interleukin (IL)-1β, TNF-α forms to the cellular source and coagulable and IL-6. Blocking antibodies of IL-1β or TNF-α, characteristic of FGL2 as previous studies [11- or lacking the gene for the TNF-α receptor (TNF 15]. Disease activity indices, ESR, and CRP are r-/-) largely abolish the extra-intestinal thrombo- important for disease assessment in IBD. sis associated with experimental IBD [31, 32]. Platelet count, MPV, PCT, PT, and APTT are also While all these studies have clearly shown that biomarkers of coagulation and fibrinolysis as distinct pro-inflammatory stimuli activates the measures of disease activity in active IBD [9]. clotting cascade, parallels have also indicated In our results, correlation of FGL2 expression that coagulation in turn propagates the inflam- with CDAI or UCAI, ESR, and MPV, PCT, as well matory state, by activating of signaling path- as fibrinogen was found. All these findings sup- ways, or recruiting more inflammatory cells to port our assumption that FGL2 may particip- the inflamed tissue [7-9]. ate in inflammation-induced coagulation and thrombosis in IBD through a clotting-dependent FGL2 prothrombinase (FGL2) is a 64-70kDa, pathway. type 2 transmembrane protein containing a C-terminal FRED (fibrinogen related extracellu- In conclusion, our study of FGL2 expression in lar domain). It was classified as a member of IBD has important theoretical implications for fibrinogen superfamily due to its homology the understanding of the “inflammation-coagu- (36%) with fibrinogen β and γ chains. It has lation” crosstalk that may result in novel diag- been demonstrated that FGL2 is significantly nostic and therapeutic strategies that targ- up-regulated in activated macrophages, T cells, et the inflammation-induced hypercoagulable and endothelial cells under the stimuli of lipo- state. Measurement of FGL2 may be used as a

1634 Int J Clin Exp Pathol 2018;11(3):1629-1636 Fibrinogen-like protein 2 may contribute to progression of IBD by immune coagulation helpful biomarker in the pathogenesis and dis- in inflammatory bowel disease: the clot thick- ease assessment of IBD. To confirm this, we ens. Am J Gastroenterol 2007; 102: 174-186. have now initiated further investigation of the [8] Yoshida H and Granger DN. Inflammatory bow- role of FGL2 in patients with IBD and in experi- el disease: a paradigm for the link between mental IBD models. coagulation and inflammation. Inflamm Bowel Dis 2009; 15: 1245-1255. Acknowledgements [9] Lipinski S, Bremer L, Lammers T, Thieme F, Schreiber S and Rosenstiel P. Coagulation and inflammation. Molecular insights and diagnos- The authors are grateful to the Department tic implications. Hamostaseologie 2011; 31: of Surgical Laboratory, the First Affiliated 94-102, 104. Hospital of Wenzhou Medical University, for [10] Levy GA, Liu MF, Ding JW, Yuwaraj S, Leibowitz assistance with technical support and cons- J, Marsden PA, Ning Q, Kovalinka A and Phillips ulting. This work was supported by the Scien- MJ. Molecular and functional analysis of the ce and Technology project of Wenzhou, China human prothrombinase gene (HFGL2) and its (Y20150163) and the Natural Science Foun- role in viral hepatitis. Am J Pathol 2000; 156: dation of China (81570495). 1217-1225. [11] Zhu CL, Yan WM, Zhu F, Zhu YF, Xi D, Tian DY, Disclosure of conflict of interest Levy G, Luo XP and Ning Q. Fibrinogen-like pro- tein 2 fibroleukin expression and its correla- None. tion with disease progression in murine hepati- tis virus type 3-induced fulminant hepatitis Address correspondence to: Zhi-Ming Huang, De- and in patients with severe viral hepatitis B. partment of Gastroenterology, The First Affiliated World J Gastroenterol 2005; 11: 6936-6940. Hospital of Wenzhou Medical University, South Bai- [12] Clark DA, Foerster K, Fung LS, He W, Lee L, Mendicino M, Markert UR, Gorczynski RM, xiang Street, Shangcai Village, Ouhai District, Wen- Marsden PA and Levy GA. The FGL2 prothrom- zhou, Zhejiang, China. Tel: +86-13706658620; Fax: binase/fibroleukin gene is required for lipo- +86-577-55578033; E-mail: wzhospitalhzm@126. polysaccharide-triggered abortions and for com normal mouse reproduction. Mol Hum Reprod 2004; 10: 99-108. References [13] Ning Q, Sun Y, Han M, Zhang L, Zhu C, Zhang W, Guo H, Li J, Yan W, Gong F, Chen Z, He W, [1] Miehsler W, Reinisch W, Valic E, Osterode W, Koscik C, Smith R, Gorczynski R, Levy G and Tillinger W, Feichtenschlager T, Grisar J, Ma- Luo X. Role of fibrinogen-like protein 2 pro- chold K, Scholz S, Vogelsang H and Novacek G. thrombinase/fibroleukin in experimental and Is inflammatory bowel disease an independent human allograft rejection. J Immunol 2005; and disease specific risk factor for thrombo- 174: 7403-7411. embolism? Gut 2004; 53: 542-548. [14] Su G, Liu K, Wang Y, Wang J, Li X, Li W, Liao Y [2] Vrij AA, Rijken J, van Wersch JW and Stockbrug- and Wang Z. Fibrinogen-like protein 2 expres- ger RW. Coagulation and fibrinolysis in inflam- sion correlates with microthrombosis in rats matory bowel disease and in arteri- with type 2 diabetic nephropathy. J Biomed tis. Pathophysiol Haemost Thromb 2003; 33: Res 2011; 25: 120-127. 75-83. [15] Su K, Chen F, Yan WM, Zeng QL, Xu L, Xi D, Pi [3] Folwaczny C. Coagulation system and inflam- B, Luo XP and Ning Q. Fibrinogen-like protein matory bowel disease: therapeutic and pa- 2/fibroleukin prothrombinase contributes to thophysiologic implications. Z Gastroenterol tumor hypercoagulability via IL-2 and IFN-gam- 2002; 40: 991-998. ma. World J Gastroenterol 2008; 14: 5980- [4] Grignani G and Maiolo A. Cytokines and hemo- 5989. stasis. Haematologica 2000; 85: 967-972. [16] Lennard-Jones JE. Classification of inflamma- [5] Esmon CT. The impact of the inflammatory re- tory bowel disease. Scand J Gastroenterol Sup- sponse on coagulation. Thrombosis Res 2004; pl 1989; 170: 2-6; discussion 16-19. 114: 321-327. [17] Best WR, Becktel JM, Singleton JW and Kern F [6] Esmon CT. Crosstalk between inflammation Jr. Development of a Crohn’s disease activity and thrombosis (reprinted from Maturitas, vol index. National Cooperative Crohn’s Disease 47, pg 305-314, 2004). Maturitas 2008; 61: Study. Gastroenterology 1976; 70: 439-444. 122-131. [18] Truelove SC and Witts LJ. Cortisone in ulcer- [7] Danese S, Papa A, Saibeni S, Repici A, Malesci ative colitis; final report on a therapeutic trial. A and Vecchi M. Inflammation and coagulation Br Med J 1955; 2: 1041-1048.

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