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ORIGINAL ARTICLE
Year : 2021 | Volume
: 46
| Issue : 4 | Page : 227-233
Assessment of Von Willebrand factor antigen and activity levels in inflammatory bowel diseases
Alaa Abozied1, Yousryeia A Ahmed1, Mostafa F. Mohammed Saleh MD 1, Hanan Galal2, Wael A Abbas3
1 Clinical Hematology Unit, Department of Internal Medicine, Assiut University, Assiut, Egypt
2 Clinical Pathology, Assiut University, Assiut, Egypt
3 Internal Medicine, Faculty of Medicine, Assiut University, Assiut, Egypt
Correspondence Address:
Mostafa F. Mohammed Saleh
Clinical Hematology Unit Internal Medicine Department Assiut University, 71515
Egypt
Source of Support: None, Conflict of Interest: None
CheckDOI: 10.4103/ejh.ejh_68_21
Background There is a close interaction between inflammation and coagulation. Hemostatic abnormalities are common in inflammatory bowel disease (IBD) with higher risk for a hypercoagulable state and prothrombotic conditions. In addition, a few cases of acquired coagulopathy with higher risk of bleeding have been reported. The involved pathophysiologic mechanisms are complex and incompletely understood.
Objective This is a case–control study that aimed to assess the levels of Von Willebrand factor (VWF) in IBD as a marker of disease activity and its relation to higher risk of bleeding or thrombotic events.
Patients and methods A total of 46 patients with IBD aged 18 years or older were enrolled in the study. After consenting, patients were divided into two groups: one group included 23 patients with active IBD and the other group included 23 patients with inactive IBD. Activity of Crohn’s disease was assessed through the Harvey-Bradshaw index and activity in patients with ulcerative colitis was evaluated with the simple clinical colitis activity index. The white blood cell count, hemoglobin level, platelet count, activated partial thromboplastin time, C-reactive protein, erythrocyte sedimentation rate, albumin, fecal calprotectin, VWF antigen level (VWF:Ag), and VWF ristocetin cofactor activity (VWF:RCo) were measured. The VWF:RCo/VWF:Ag ratio was calculated.
Results There were significant differences in the mean±SD of vWF antigen and vWF:RCo levels between active IBD group (189.30±62.83 and 101.73±23.42, respectively, P=0.001) and inactive IBD group 177.30±64.90 and 97.08±24.21, respectively, P=0.001). The IBD activity index was correlated with VWF antigen (r=0.78 P=0.001) and VWF:RCo levels (r=0.74 P=0.001). VWF antigen and VWF:RCo were correlated with fecal calprotectin (r=0.65, P=0.001, and r=0.67, P=0.001, respectively). The odds ratio of an elevated vWF antigen greater than 150% was 24 (95% confidence interval: 4.38–131.47) in the group with active IBD compared with the inactive IBD group. The VWF:RCo/VWF:Ag ratio of less than 0.7 which reflects the possibility of acquired Von Willebrand syndrome was detected in five patients (21.7%) with active IBD in comparison with only one patient (4.3%) in the inactive IBD group, with odds ratio of 6.1 (95% confidence interval: 0.65–57.1).
Conclusion VWF antigen and activity levels can be used as markers for evaluation of IBD activity. Assessment of VWF in IBD could be significant for better hemostatic control of such patients. Activated coagulation system in IBD is well known; however, precautions for coexisting acquired functional coagulopathy should be considered.
Keywords: activity, assessment, inflammatory bowel diseases, Von Willebrand factor
A.A., M.M.S., and W.A. designed the study, supervised the implementation, and drafted the manuscript. A.A. recruited patients and collected the data. H.G. supervised hemostatic analysis of specimens collected. Y.A., W.A., and H.G. supervised data analysis and result interpretation. All authors contributed to writing and editing of the final manuscript.
Introduction
Inflammatory bowel disease (IBD), which consists mainly of ulcerative colitis (UC) and Crohn’s disease (CD), is a chronic inflammatory condition characterized by local and systemic inflammation mainly affecting the gastrointestinal tract [1]. Patients with IBD are genetically predisposed to some pathological interactions between intestinal microflora and the immunological system [2].
During tissue damage, coagulation and inflammation are both triggered at the same time and respond in synergy as a conserved mechanism to repair the affected parts [3]. In IBD, there is a higher risk for venous thromboembolism (VTE), either primary or recurrent. The reasons are complex, and they are linked to prothrombotic changes during active disease, albeit the mechanisms are still unknown [4].
Von Willebrand factor (VWF) plays a key function in primary hemostasis, mediating platelet adherence to injured vascular subendothelium and, as a result, platelet aggregation. It could be a ‘connection bridge’ between the hemostatic and inflammatory pathways, mediating platelet and leukocyte adhesion and recruitment [5]. Growing evidence shows that inflammation can induce thrombosis by a VWF-mediated mechanism, which includes endothelial activation, secretion of VWF, assembly of hyperadhesive VWF strings and fibers, cleavage by ADAMTS13, and adhesion and deposition of VWF-platelet thrombi in the vasculature. This mechanism appears to have a role in thrombosis in both small and big blood arteries [6].
A few reports showed there is an association of IBD with acquired Von Willebrand Syndrome (AVWS). This could attributed to the etiology of gastrointestinal bleeding seen in IBD, with the autoimmune pathogenesis of IBD strongly supporting this claim [7],[8].
As inflammation and endothelial dysfunction are contributors in the etiopathology of IBD and as VWF can act as a link between inflammation and hemostasis, we have evaluated the plasma levels of VWF and assessed their associations with the inflammatory markers levels and disease activity index in patients with IBD.
Patients and methodsThe study design
The was a single-center observational study and was registered in clinicalTrials.gov with ID: NCT03715673. It was conducted in Assiut University Hospitals, Assiut, Egypt, from January 2019 to May 2021. A total of 46 patients with IBDs mainly CD and UC diagnosed by the clinical features combined with the characteristic endoscopic findings and histopathology who are followed up in the Internal Medicine Department of Assiut University Hospital were recruited. The sample size was calculated using Epi-info program (link; https://www.openepi.com/SampleSize/SSCohort.htm), assuming that power of the study set at 80% and confidence interval (CI) of 95%.
Ethics approval
The protocol of the study was approved by the Research Ethics Medical Review Board of Faculty of Medicine, Assiut University. The well-being of the subjects took priority over interest of science as stated by the declaration of Helsinki whose principles were followed in the study. The confidentiality of all patients admitted to the study was protected. Informed written consents were taken from all participants after thorough discussion and explanation.
Inclusion criteria
The study included adult patients with established diagnosis of UC or CD aged 18 years or older. Subjects were divided into two groups: one group included 23 patients with active IBD and the other group included 23 patients with inactive IBD.
Activity of UC was evaluated through simple clinical colitis activity index (SCCAI) and activity of CD was evaluated by the Harvey-Bradshaw index (HBI) for CD.
Exclusion criteria
Exclusion criteria included patients who have conditions that might affect the VWF antigen results such as pregnancy, concomitant inflammatory disorders, and severe diseases including myocardial infarction, stroke, thromboembolism, known hemorrhagic diathesis, cancer, renal insufficiency, liver injury, and diabetes. Patients who are on aspirin, heparin, oral anticoagulants, or oral contraceptives were excluded as well.
Clinical assessment
Patients were assessed for demographic data, comorbidities, cigarette-smoking history, and previous history of thromboembolism and/or medications.Height and weight were measured and BMI was calculated for all patients.We assessed disease duration, disease location, disease activity, complications, and past surgical procedures. Complications included abscesses, fistulae, perforations, and stenoses.Activity of CD was assessed through the HBI for symptom assessment given it is simple to use and correlates well the complex Crohn’s Disease Activity Index. HBI scores greater than 16 are consistent with severe disease activity, whereas scores between 5 and 7 suggest mild activity and 8 and 16 suggest moderate activity [9],[10].Activity in patients with UC was evaluated with the simple clinical colitis activity index. It is the best noninvasive index for validity, reliability, and feasibility with the added benefit of being able to measure responsiveness or change in disease activity. The calculated score ranges from 0 to 19, where active disease is a score of 5 or higher [10],[11].Laboratory assessment
Laboratory investigations were done as follows: 2 ml of blood was obtained in an EDTA tube for complete blood count, which was done using automated hematology analyzers, and 6 ml of blood was allowed to clot to separate the serum. Nonhemolyzed serum was separated by centrifugation, and a part was used for the determination of serum glucose level, blood urea, serum creatinine, liver function tests [albumin, aspartate transaminase, and alanine transaminase, erythrocyte sedimentation rate (ESR) using the Westergren method (mm/h), and C-reactive protein (CRP) by latex enhanced nephelometry (mg/l)]. Fecal calprotectin was done as well using enzyme-linked immunoassay (ELISA) (Epitope Diagnostics, Inc., San Diego, California, USA); a stool sample using a 10-μl inoculation loop was weighed and dispensed into an analysis pot. The exact weight was recorded, and 5 ml of extraction buffer was added. Samples were swirled for 30 min, to ensure dissolution, and then centrifuged. The supernatant was removed for analysis by ELISA. The assay utilizes the two-site ‘sandwich’ technique with two selected antibodies that bind to different epitopes of human calprotectin. The recommended normal cutoff for fecal calprotectin concentration using this ELISA and sample collection system is 120 ng/ml or 43.2 μg/g directly read from assay standard curve.
VWF:Ag was assessed turbidimetrically (Siemens Healthcare Diagnostics for the quantitative determination of VWF Ag in human plasma, Germany). The normal reference ranges for VWF Ag parameters were 50–150 IU/dl. VWF activity (vWF:Ricof) was assessed by automated coagulation analyzer (INNOVANCE, Siemens), and the results were reported as percentage of normal (100% of normal=1 IU/ml). The limit of Detection (LoD) of the INNOVANCE vWF Ac method is 2.2%. The normal reference range is between 50 and 150% VWF.
Statistical analysis
Statistical Package for the Social Sciences (SPSS) program windows (SPSS: An IBM Company, version 24.0, IBM Corporation, Armonk, NY, USA), version 24.0, was used for the analysis of the results. Results were reported as means±SD, medians, and interquartile ranges or percentages. A comparison of the patients’ properties was performed by the χ2 test for categorical variables and the independent t-test for normally distributed data or Mann–Whitney U-test when the variables were not normally distributed. Pearson’s correlation coefficients or Spearman’s rank correlation was evaluated to demonstrate associations among the variables followed by regression analysis for some variables.
ResultsThis study included 46 patients with IBD: 28 patients with UC (60.9%) and 18 patients with CD (30.1%). There were 19 male patients (41.3%) and 27 female patients (58.7%). Their ages ranged from 18 to 38 years, with mean±SD of 26.52±5.52 years. Disease duration ranged from 0.5 to 5 years, with mean±SD of 2.45±1.15. Nine patients (19.6%) had extraintestinal manifestations and 37 (80.4%) has no extraintestinal manifestations. Regarding the site distribution in the studied cases, 19 patients (41.3%) had colonic involvement, nine patients (19.6%) had left sided colonic involvement, eight patients (17.4%) had ileocolonic involvement, six patients (13%) had rectal involvement, and four patients (8.7%) had ileal involvement. The demographic data and clinical characteristics of the patients with IBD are presented in [Table 1].
Table 1 Demographic data and clinical characteristics of the studied patientsThe studied cases were classified into two groups: group 1 included 23 patients with inactive IBD and group 2 included 23 patients with active IBD. Comparison between the two groups regarding the age, BMI, disease duration, extraintestinal manifestations, and sex distribution were statistically insignificant as shown in [Table 2].
The comparison between the laboratory results in the two groups is presented in [Table 3]. There was a significant statistically difference (P=0.001) between mean±SD of white blood cells, hemoglobin level, platelet count, ESR, CRP, and fecal calprotectin in the inactive IBD group and active IBD group.
There were significant differences (P=0.001) between the mean±SD of the VWF Ag and VWF:RCo levels in the inactive IBD group (101.73±23.42 and 97.08±24.21, respectively) and active IBD group (189.30±62.83 and 177.30±64.90, respectively) ([Figure 1]). Approximately 69% of the patients with active IBD (n=16) had a VWF:Ag of greater than 150%, compared with about 8% (n=2) of the patients with inactive IBD ([Figure 2]). In the active IBD group, the odds ratio (OR) of a VWF:Ag greater than 150% was 24 (95% CI: 4.3–131.4).
Figure 1 Comparison between means of Von Willebrand factor Ag and Von Willebrand factor:RCo in the two groups.
Figure 2 Frequency of Von Willebrand factor Ag greater than 150 in the two groups.VWF Ag and VWF:RCo were positively correlated with disease activity (r=0.78, P=0.001, and r=0.74, P=0.001, respectively), fecal calprotectin (r=0.65, P=0.001, and r=0.67, P=0.001, respectively), ESR (r=0.64, P=0.001, and r=0.64, P=0.001, respectively), and CRP (r=0.62, P=0.001, and r=0.67, P=0.001, respectively) ([Table 4]). The VWF:RCo/VWF:Ag ratio of less than 0.7 was detected in 5 patients (21.7%) with active IBD in comparison with only one patient (4.3%) in inactive IBD group with OR of 6.1 (95% CI: 0.65–57.1; P=0.09). The patients with VWF:RCo/VWF:Ag ratio of less than 0.7 were UC cases and were observed to have more significant bleeding.
Table 4 Correlation between VWF Ag and VWF:RCo with activity index and other variables Discussion Hemostatic abnormalities are common in IBD with higher risk for a hypercoagulable state and prothrombotic conditions. IBD is associated with a roughly three-fold increase in the risk of VTE compared with the general population while ambulatory [12]. Our study evaluated the status of VWF antigen and its activity in patients with IBD and how it could be a contributing factor for assumed hypercoagulability or higher gastrointestinal bleeding risk and whether it might represent an indicator of IBD activity.
We have observed significant higher levels of VWF antigen and VWF:RCo in patients with active IBD disease than patients with inactive IBD disease, and both were positively correlated with the disease activity index and inflammatory markers such as CRP, ESR and fecal calprotectin. There was no significant difference between the two groups regarding the patients’ age, sex, BMI, or disease duration. Similar results were shown by Meucci et al. [13]. Furthermore, Zezos et al. [14] studied the plasma VWF concentrations in patients with UC, and they were significantly higher in patients with active UC than in healthy control and patients with inactive UC. Interestingly, patients with CD in clinical remission with endoscopic activity present endothelial lesion, inducing TF exposure and subsequent coagulation cascade activation with high level of VWF compared with healthy controls. The authors suggested thromboprophylaxis for endoscopic activity outpatient subgroups, but additional investigations are required for validation [15].
There is a wide-ranging cross-talk between hemostasis and inflammation, which is evidently seen in organ dysfunction in patients with sepsis. Inflammation not only leads to initiation and propagation of coagulation activity but also coagulation markedly influences inflammation [16].
The majority of individuals with IBD have active disease at the time of VTE, which is associated with high mortality [17],[18],[19]. A meta-analysis provided a correlation between IBD and VTE and demonstrated that IBD raised the incidence of VTE by around twofold. Additionally, patients with UC may be at higher risk; the illness was active in 60% to 80% of patients during the VTE event [20]. In an interesting review to assess the prothrombotic or antithrombotic manner by which pharmacological therapies and surgery modify the IBD clinical course, prolonged use of steroids, increased VTE risk, whereas the use of infliximab seemed to reduce such risk. The data related to tofacitinib were not enough to make definitive conclusions. Moreover, surgery has an increased prothrombotic risk [21].
We noted that in ∼16 patients (69%) of the active IBD cases, the VWF:Ag value was greater than 150%, compared with two patients (8%) in the group with inactive disease. The inflammatory nature of IBD is associated with increased levels of VWF:Ag, which our findings support. One of the patients with active CD was diagnosed with left lower limb deep venous thrombosis (4.4%). She was a female patient aged 26 years old with no other apparent risk factor for thrombosis.
The VWF:RCo/VWF:Ag ratio of less than 0.7 was detected in five patients (21.7%) with active IBD in comparison with only one patient (4.3%) in inactive IBD group with insignificant OR of 6.1. We observed that those patients who have VWF:RCo/VWF:Ag coefficients of less than 0.7 had UC and were associated with more frank bleeding. VWF:RCo/VWF:Ag coefficients of less than 0.7 are seen in patients with type 2A VWD, indicating substantial depletion of HMWM [22]. Thus, the findings of our study suggest that the risk for AVWS in patients with active IBD is higher than that in those with inactive disease. This is useful in patients receiving antithrombotic prophylaxis during the attacks of IBD exacerbation, as it allows for the identification of patients who could be at a higher risk of gastrointestinal bleeding. Cibor et al. [23] had published similar data regarding high VWF antigen above 150% and detection of patients with VWF:RCo/VWF:Ag less than 0.7 in patients with active IBD.
Few case series showed AVWS has been linked to IBD, including two cases of UC and two cases of CD [8],[24],[25],[26]. Di Sabatino et al. [7] also reported three cases of patients with IBD, one with CD, and two with UC, who turned out to be affected by AVWS. This is in contrast to prior research that found higher levels of circulating VWF in patients with active IBD as a result of vascular inflammation and damage [12].
Evaluation of the VWF:Ag concentration and VWF activity may help to detect patients with IBD at risk for bleeding consequences and the management of patients with active disease, specifically when there is a need to give anticoagulation prophylaxis. In this population, an AVWS-specific therapy could be used during invasive procedures, such as surgery. However, more research with larger patient populations and long-term follow-up is required to confirm our results regarding VWF’s role in IBD.
There are various shortcomings in this study. It is a single-center study with a small number of patients that did not allow to report higher rates of events. The existence of large VWF multimers in plasma also was not investigated. Because associations do not always imply causation, in vitro and animal model research are required to clarify the molecular mechanisms behind our findings.
ConclusionVWF antigen and activity levels can be used as markers for evaluation of IBD activity, and higher VWF antigen levels may predispose to thrombosis. Evaluation of the ratio of VWF activity to VWF antigen may help to diagnosis the rare cases of acquired von Willebrand disease in patients with IBD, and it can affect the further management plan.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
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