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J Thromb ThrombolysisDOI 10.1007/s11239-006-9046-z Thrombin generation in mesalazine refractory ulcerative colitisand the influence of low molecular weight heparin Anton A. Vrij Æ Ardi Oberndorff-Klein-Woolthuis Æ Gerard Dijkstra ÆAndrea E. de Jong Æ Rob Wagenvoord Æ Hendrik C. Hemker ÆReinhold W. Stockbru¨gger Ó Springer Science+Business Media, LLC 2007 differences were observed on the clinical, endoscop- state of hypercoagulation has frequently been observed.
ical and histological outcome, as compared to pla- Low molecular weight heparin (LMWH) has shown cebo. A high intrinsic and extrinsic thrombin potential beneficial effects as an adjuvant treatment of steroid was found before LMWH therapy. However, the refractory UC in open trials. We assessed potential significant reduction in the thrombin generation by therapeutic effects of the LMWH reviparin in hospita- LMWH was not related to the reduction in disease lised patients with mesalazine refractory UC, as well as its influence on haemostasis factors. Methods nine patients with mild-to-moderately active UC were bin generation in patients with mild-to-moderately included in a double-blind placebo controlled trial. All active, mesalazine refractory UC, but is not associated patients had a flare-up of disease under mesalazine with a reduction in disease activity.
treatment. Reviparin (ClivarinÒ) 3,436 IU anti-Xa/0.6ml or placebo s.c. was added, and self-administered twice daily for 8 weeks. Patients were monitored for possible Reviparin Á Thrombin Á Ulcerative colitis adverse events and changes in clinical symptoms.
Endoscopical, histological, biochemical and haemosta-sis parameters were analysed.
Tolerability and compliance were excellent and no serious adverse events occurred. No significant In inflammatory bowel disease (IBD), histologic andsystemic signs of enhanced coagulation are well doc-umented [–], and an increased risk of thrombo- embolic complications is well recognised in patients Department of Internal Medicine and Gastroenterology,Twenteborg Hospital Almelo, Zilvermeeuw 1, PB 7600, with ulcerative colitis (UC) and Crohn’s disease (CD) –The use of anticoagulants like low molecular weight heparin (LMWH) seems paradoxical as atherapy for a disorder characterised by bleeding.
A. Oberndorff-Klein-Woolthuis Á R. W. Stockbru¨ggerDepartment of Gastroenterology, University Hospital However, ‘‘fibrinoid’’ mucosal capillary thrombi have Maastricht, P. Debyelaan 25, PB 5800, 6202 AZ Maastricht, been detected in rectal biopsies of patients with UC ]. As the micro-vascular thrombi were found with-out a clear relationship to the degree of surrounding G. Dijkstra Á A. E. de JongUniversity Medical Center Groningen, Hanzeplein 1, inflammation, they might represent an association with PB 30001, 9700 RB Groningen, The Netherlands underlying pathophysiological mechanisms , The finding of raised levels of plasma coagulation and fibrinolysis end-products indirectly reflects a state of Department of Biochemistry, Maastricht University, 6229ER Maastricht, The Netherlands hypercoagulation in patients with IBD ].
More direct thrombin activity is difficult to measure, known active ulcer disease, serious hepatic disease as it is a transient phenomenon during the clotting (ASAT > 3· upper limit) or renal failure (serum process. Currently, thrombin activity can be quantified creatinin > 300 lmol/l), as well as pregnancy or breast- by measuring the amount of product that is produced feeding in female patients, were other exclusion from an artificial thrombin substrate during coagula- criteria. Written informed consent was obtained from tion. The time course of the thrombin generation curve all patients. The ethical committees of both participat- reflects the function of the ensemble of plasmatic pro- ing hospitals approved the protocol.
and anticoagulant factors in platelet-poor plasma(PPP), as well as the procoagulant role of platelets in platelet-rich plasma (PRP) [Both the extrinsic andintrinsic thrombin potential (ETP and ITP) seems to After randomisation (random allocation), the patients be increased in other thrombosis-prone states investi- received either reviparin (ClivarinÒ) 3,436 IU Pharm gated , ], but in IBD patients, the level of Eur/0.6 ml (corresponding to 10,000 U of unfraction- thrombin generation has not been reported before.
ated calcium heparin) or placebo subcutaneously twice As LMWH therapy may produce a benefit by affecting micro-vascular thrombi in the gut [its admin- The drug and placebo were made available in istration could be rational in patients with UC or CD- individually packed disposable syringes. Both were resistant to conventional forms of treatment.
administered through self-injection. All patients were The aim of this study was to clarify whether an on stable treatment with either salazopyrine (n = 4) or improvement of clinical disease activity after LMWH mesalazine (n = 20) 1 g 2–3 times daily or olsalazine therapy was associated with a reduced thrombin generation in patients with mild-to-moderately active Clinical disease activity was determined by the UC, unresponsive to mesalazine. We additionally Colitis Activity Index (CAI) ] (from 0 to 21; severe investigated the relationship of thrombin generation disease defined as > 12, inactive disease < 4) on week 0 with other haemostasis factors in these patients.
or just before treatment, and after 1, 2, 4, 6 and 8 weeksof treatment. Quality of life was assessed at week 1, 4and 8 by means of the Inflammatory Bowel Disease The endoscopic severity of disease was scored by means of the Endoscopic Grading System ] or EGS(from 0 to 26; severe disease >18) within 2 weeks prior In a prospective randomised double-blind placebo to LMWH treatment. Also, a grading of histologic controlled trial, 29 patients with mild-to-moderately disease activity (HDA, from 0 to 12; severe inflamma- active mesalazine refractory UC were seen at the tion >10) was performed ]. Duplicate biopsies University Hospitals of Maastricht and Groningen, (3 mm) were taken from the most severely affected The Netherlands, and enrolled in this study from area, at 10 cm from the anus in the rectum and at 25 cm August 1996 to February 2000. The diagnosis UC was in the sigmoid. The recording of endoscopic and based on the Lennard-Jones criteria [and patients histological scores was done with reference to the with a severity score of 4–14 according to the modified most inflamed site in the colon or rectum. The EGS Truelove classification ], were eligible. The active and HDA score were repeated after 8 weeks of colitis could either be the first manifestation or an treatment, and one experienced pathologist assessed exacerbation of the disease. Sigmoidoscopy had to be performed less than 2 weeks before start of treatment.
At regular intervals (week 0 or just before treat- Excluded from the trial were patients with proven CD, ment, and after 1, 2, 4, 6 and 8 weeks of treatment), infectious colitis, ischaemic colitis or irradiation colitis.
laboratory tests were performed: the erythrocyte sed- Use of oral or rectal corticosteroids or other immuno- imentation rate, red and white blood cell counts, suppressive drugs was prohibited within 4 weeks before platelet counts (normal values: 130–350 · 109/l), mean study entry. Also excluded were patients with known platelet volume (MPV; 8.6–9.7 fl), platelet distribution thrombo-embolic disposition or current use of antico- width (PDW; 15–18%), serum levels of C-reactive agulants, patients with known or suspected general protein (CRP; 0–9 mg/l), alkaline phosphatase (AP; bleeding tendency, and patients with regular use of 30–125 IU/l), gamma-glutamyl-transpeptidase (GGT; non-steroidal anti-inflammatory drugs or aminosalicy- 10–50 IU/l), lactate dehydrogenase (100–250 IU/l), and lates. Previous adverse events to heparin therapy, serum levels of aspartate aminotransferase (AST; 5–40 IU/l) and alanine aminotransferase (ALT; 5–40 IU/l) buffer containing phospholipid vesicles (20 mol% and creatinin (creat; 40–90 lmol/l).
phosphatidylserine and 80 mol% phosphatidylcholine).
Different markers of coagulation like the APTT (24– For the extrinsic system, 4 lM phospholipid with 15 35 s, thrombin–antithrombin complex (TAT; 0.00–1.90 pM recombinant human tissue factor was added, and lg/ml), prothrombin fragment 1 and 2 (F1.2; 0.40–1.45 for the intrinsic system 4 lM phospholipid and 1/6 nmol/l), as well as anti-IIa activity (0.0–0.07 U/ml), anti- volume of Actin-FSL (Dade BehringÒ). The ETP and Xa activity (0.0–0.02 U/ml) and the ITP and ETP were ITP level is expressed as a percentage of the ETP and determined. Treatment was intended to last 8 weeks.
ITP of normal pool plasma, measured simultaneously.
Control visits were planned at 1, 2, 4, 6 and 8 weeks.
The mean value of ETP in PPP in healthy controls was Treatment was discontinued if there was no 737 nmol/l min (range 720–790), the mean ITP level in improvement after 4 weeks according to the CAI PPP in healthy controls was 798 nmol/l min (range [or in any patient with progression of disease 780–850) []. The normal values of ETP, ITP, as activity at any control visit. Other predefined reasons well as TAT and F1.2, were obtained from ten healthy for discontinuation were heparin-induced thrombocy- controls (five male, five female), and were compared topenia (HIT) type 2 [and/or severe bleeding (defined as Hb < 5.0 mmol/l, Hb > 2.0 mmol/l belowbaseline value, blood loss with blood pressure <80/50 mmHg and/or need for blood transfusion). In patientsin whom the study treatment was discontinued, a The sample size of the clinical trial was based on treatment with corticosteroids was initiated.
categorical data (‘‘did the patient improve?’’). For theexpected proportion with specified outcome was cho- sen: p1 = improved on LMWH 0.80, p2 = improved onplacebo 0.20. The calculated SD was 1.2. Taking the The collection of blood was strictly timed, i.e. 30 min power to be 0.85 and a one-sided significance level of before the next dose of LMWH in the morning, and 30 0.05, the sample size was calculated to be 24 for each min before laboratory analysis started.
group. Analysis was performed on an intention-to-treat Fresh citrated blood (nine parts of blood to one part basis with last value carried forward in case of of 0.13 mol/1 trisodiumcitrate) was centrifuged at 250g, premature discontinuation. To test differences for 15°C for 10 min. The platelet count was adjusted to 3 · significance, the Mann–Whitney–Wilcoxon rank–sum 108/ml using autologous PPP, made by double centri- test was performed. A Bonferroni adjustment was used fugation of PRP at 1,000g, 15°C for 10 min. Before for multiple comparisons, and an alpha level of 0.017 storage at –80°C, PPP was centrifuged twice at l,000g was considered significant with tests performed at 0, 4 for 10 min. Plasma was defibrinated by adding 1/50 and 8 weeks [Also, the Spearman rank correlation volume of Ancrod and clot dissolution. After the coefficient and the two-tailed significance of the thrombin generation test had been performed, the correlation were calculated between the coagulation remaining serum was put on ice and centrifuged at factors and other biochemical and disease parameters.
15,000g for 2 min. The supernatant was stored at –80°C.
SPSS 13.0 software (SPSS Inc., USA) was used for Normal pool plasma was pooled PPP from at least ten apparently healthy male and female donors and storedat –80°C for less than 4 months.
Thrombin generation was measured with a subsam-pling technique, as previously described in detail – Fifteen patients were randomised to receive the either triggered by tissue factor (extrinsically) or LMWH reviparin and 14 to receive placebo (19 by contact activation (intrinsically). In short, thrombin patients were studied in the University Hospital was determined by monitoring optical density of the Maastricht and 10 in the University Hospital Gronin- pNA at 405 nm, released from a slow-reacting chro- gen). Demographic data and clinical characteristics of mogenic thrombin substrate (DEMZ-Gly-Arg-pNA) patients randomised to treatment are shown in which was added to the defibrinated plasma upon Table . There was no difference between the two recalcification. The reacting mixture for the measure- groups with regard to age, gender, smoking habits or ment of thrombin generation consisted of one part of disease severity as measured by CAI. Mean duration Table 1 Baseline characteristics of placebo and reviparin-trea- (p = 0.02). Also, patients in the placebo group had higher levels of ETP (784; SD 87) and ITP (833; SD 100), than the normal control values of ETP (737; 19with p < 0.001), and ITP (798; 19 with p < 0.01).
At baseline, TAT and F1.2 were significantly raised in the LMWH and placebo group, compared to the healthy controls (p < 0.0001 and p < 0.0001 respec- tively). The APTT, the anti-IIa and the anti-Xa activity was within normal limits in the LMWH and placebo At 4 weeks, the ETP and ITP values were signifi- cantly lower in the LMWH-treated group, as comparedto the placebo group (p = 0.014 and 0.015), as well as and extent of disease, previous steroid treatment and the healthy controls (p = 0.001 and 0.001). However, individual or family history of thrombosis or bleeding TAT and F1.2 levels were higher, both in the LMWH tendency were similar in both groups.
group (p < 0.0001 and p = 0.004) and placebo group(p < 0.0001 and p = 0.004), as compared to the healthy controls. Also, anti-IIa (p < 0.0001) and anti-Xa activity (p < 0.0001), as well as APTT (p = 0.007)and ALT levels (p = 0.008), were significantly higher in In the reviparin group 11/15 (73.3%) patients com- the LMWH-treated patients, as compared to placebo.
pleted the 8 weeks of treatment, in the placebo group At 8 weeks, the ETP and ITP values were not 9/14 (64.3%; p = 0.70). One patient in the placebo significantly lower in the LMWH-treated group, as group was lost to follow-up after 2 weeks. In all other compared to the placebo group (p = 0.536 and 0.887), or patients, reason for discontinuation was either lack of the healthy controls (p = 0.238 and p = 0.892). However, efficacy or exacerbation. At 8 weeks the improvement TAT (p < 0.0001) and F1.2 (p = 0.039) were higher in in CAI was 3.80 points in the reviparin group and 2.08 the placebo group, as well as was TAT in the LMWH points in the patients treated with placebo (p = 0.20).
group (p < 0.0001), as compared to the healthy controls.
In the reviparin group 12/15 (80%) patients had Also, the anti-Xa activity, but not the anti-IIa, improved, compared to 11/13 (85%) in the placebo APTT or ALAT levels, was significantly higher at 8 weeks in the LMWH-treated patients, as compared to There were no significant differences in IBDQ, placebo (p = 0.002). Table summarises the differ- ESG, or HDA between both groups (Table ).
ences of laboratory data between the reviparin andplacebo-treated groups. The time course of ETP and Haemostasis characteristics and the influence ITP is shown in Figs. (reviparin) and (placebo). The of LMWH treatment on thrombin generation, time course of TAT and F1.2 is shown in Figs. haemostasis and fibrinolysis factors at baseline, (reviparin) and (placebo). The box-whisker plots represent the median (line between the 25th and 75thpercentile box) and min–max values (whisker lines At baseline, patients in the LMWH group had above extended from the box as smallest and largest values), normal levels of ETP (777; 171) and ITP (824; 188) as well as outliers (0) and extremes (*).
Table 2 Clinical (CAI, IBDQ), endoscopical (EGS) and histological (HDA) activity indices, before and after reviparin therapy orplacebo, are shown Means and standard deviations ([ ]) between the groups with reviparin and those with placebo are presented at week 0, 4 and 8. Nosignificant differences were found between these groups Table 3 Laboratory data with means, standard deviations ([ ]) and significant differences (*p < 0.017) between the groups treated withreviparin or placebo, at weeks 0, 4 and 8 Fig. 1 Box-whisker plots of ETP and ITP (nmol/l min; normalmean values of ETP and ITP are 737 and 798), during 8 weeks of Fig. 2 Box-whisker plots of ETP and ITP (nmol/l min; normal follow-up in patients with UC, treated with mesalazine and mean values of ETP and ITP are 737 and 798), during 8 weeks of follow-up in patients with UC, treated with mesalazine andplacebo Relationship between haemostasis parametersand CAI, CRP, endoscopic and histologic p = 0.018). The endoscopy and histology score EGS and HDA did not correlate with the haemostasis parametersin the placebo group.
In the placebo-treated patients, the clinical activity score In the LMWH-treated group, CAI correlated CAI was positively correlated with the ITP (r = 0.267; inversely with MPV (r = –0.293; p=0.008), and inver- p = 0.038), as well as platelet count (r = 0.506; p < sely with the APTT (r = –0.271; p = 0.016). CRP 0.0001) and APTT (r = 0.336; p = 0.007), and inversely correlated with platelet count (r = 0.344; p = 0.001), correlated to PDW (r = –0.552; Spearman’s rho, two- ETP (r = 0.472; p < 0.0001), ITP (r = 0.491; p < 0.0001), tailed significance p < 0.0001). CRP correlated positively TAT (r = 0.283; p = 0.014) and APTT (r = 0.315; with ETP (r = 0.531; p < 0.0001), ITP (r = 0.664; p < p = 0.004). The EGS did not correlate with ETP, and 0.0001) and anti-Xa (0.336; p = 0.007), and inversely the HDA correlated inversely with ETP (r = –0.456; with MPV (r = –0.361; p = 0.002) and PDW (r = –0.382; on patients with active thrombo-embolic diseases In the reviparin-treated group, the anti-IIa activity and APTT were raised after 4 weeks of treatment.
However, both normalised after 8 weeks, while the anti-Xa activity was still raised. Although we did not find a clear explanation for the gradual loss of anti-IIaactivity in our patients, a more selective LMWH anti- Xa activity was expected from literature ]. Surpris- ingly, the administration of a therapeutic dose of reviparin was accompanied by a rise in the thrombin potential at week 8, not different from baseline levels.
On the one hand, this questions the effectiveness of selective anti-Xa blocking agents on reducing the thrombin potential over time in patients with UC.
The size of the effect on thrombin generation might Fig. 3 Box-whisker plots of TAT (lg/ml; normal upper limit 1.90 depend on the type of activation used to initiate the lg/ml) and F1.2 (nmol/l; normal upper limit 1.45 nmol/l) during 8 clotting, which could be dependent of higher amounts weeks of follow-up in patients with UC, treated with mesalazine On the other hand, the scheduled dose of reviparin (3,436 IU/12 h) corresponds to 10,000 IU/12 h ofunfractionated heparin, and this might be considered We do not know if a higher dose (e.d. 15,000 IU/ 12h) would be effective in improving the clinical outcomes of UC, as the reached anti-Xa plasma levelat 8 weeks was below the generally accepted thera- peutic range of 0.4–1.0 aXa IU/ml.
No correlation was found between the (level of) reduction of the thrombin potential and both clinical (CAI; IBDQ), endoscopy and histology scores. How- ever, a positive correlation was found between ETPand ITP with CRP, suggesting a pattern of acute phase reactivity in thrombin generation. The more stable end-products F1.2 and TAT did not correlate with CAIand CRP in the placebo-treated patients and this might favour the use of ITP or ETP as indices of UC diseaseactivity over that of F1.2 and TAT.
Fig. 4 Box-whisker plots of TAT (lg/ml; normal upper limit 1.90 No additional benefit of reviparin was found com- lg/ml) and F1.2 (nmol/l; normal upper limit 1.45 nmol/l) during 8 pared to the placebo-treated patients, on CAI, IBDQ, weeks of follow-up in patients with UC, treated with mesalazineand placebo EGS and HDA. This is in contrast with the positiveresults found in a previous but open study done withnadroparin, in corticosteroid-resistant UC patients ]. It suggests that LMWH, in combination withaminosalicylates but in the absence of corticosteroids, To our knowledge, this is the first study to present data does not have any therapeutic advantage above a on direct thrombin generation in patients with UC. The placebo, as was recently also shown by other investi- study demonstrates that the thrombin potential, both intrinsic and extrinsic, is above the norm in patients A masking effect due to the use of aminosalicylate with active UC, confirming more indirect data on therapy remains possible, although 8 weeks of therapy hypercoagulation in these patients [The adminis- should have led to induction of remission. Also, the tration of LMWH diminished the ITP and ETP in 8-week observation period following LMWH and patients with UC, comparative to the effect of LMWH placebo might have been too short to evaluate if a reduced thrombin generation has any influence with respect to the HDA score. However, the combinationof LMWH and corticosteroids did reduce the HDA 1. Lam A, Borda IT, Inwood MJ, Thomson S (1975) Coagu- lation studies in ulcerative colitis and Crohn’s disease.
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