Anti-EPCR autoantibodies in ulcerative colitis show sex- and lipid-dependent patterns

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search Anti-EPCR autoantibodies in ulcerative colitis show sex- and lipid-dependent patterns Nerea Ugidos-Damboriena , Llùcia Jaime-Gómez , Cristina Rodríguez-Gutiérrez , Lucía Zabalza , Óscar Nantes-Castillejo , María Gilda Dichiara-Rodríguez , Jacinto López-Sagaseta doi: https://doi.org/10.1101/2025.10.18.25337568 Nerea Ugidos-Damboriena 1 Unit of Structural Immunology , Navarrabiomed, Navarra, Spain 2 Public University of Navarra (UPNA) , Pamplona, Navarra, Spain 3 Navarra University Hospital , Pamplona, Navarra, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Llùcia Jaime-Gómez 1 Unit of Structural Immunology , Navarrabiomed, Navarra, Spain 2 Public University of Navarra (UPNA) , Pamplona, Navarra, Spain 3 Navarra University Hospital , Pamplona, Navarra, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Cristina Rodríguez-Gutiérrez 3 Navarra University Hospital , Pamplona, Navarra, Spain 4 IdiSNA , Pamplona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lucía Zabalza 3 Navarra University Hospital , Pamplona, Navarra, Spain 4 IdiSNA , Pamplona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Óscar Nantes-Castillejo 3 Navarra University Hospital , Pamplona, Navarra, Spain 4 IdiSNA , Pamplona, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site María Gilda Dichiara-Rodríguez 1 Unit of Structural Immunology , Navarrabiomed, Navarra, Spain 2 Public University of Navarra (UPNA) , Pamplona, Navarra, Spain 3 Navarra University Hospital , Pamplona, Navarra, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jacinto López-Sagaseta 1 Unit of Structural Immunology , Navarrabiomed, Navarra, Spain 2 Public University of Navarra (UPNA) , Pamplona, Navarra, Spain 3 Navarra University Hospital , Pamplona, Navarra, Spain 5 Phase Solution Protein Discovery SL , 31621, Navarra, Spain Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: jacinto.lopez.sagaseta{at}navarra.es Abstract Full Text Info/History Metrics Preview PDF Abstract Autoantibodies targeting the endothelial protein C receptor (EPCR) have been associated with ulcerative colitis (UC). We aimed to assess a potential role of EPCR lipidation on the detection of anti-EPCR antibodies in inflammatory bowel disease (IBD). To this end, serum samples from patients with UC, Crohn’s disease, and healthy controls were analyzed using an in-house ELISA employing either native or delipidated EPCR. Overall, male UC patients showed significantly higher absorbance (mean=1.09) than CD patients (mean=0.50) and controls (mean=0.37). Remarkably, in males, the difference between UC patients and controls was highly significant (p=1.2e-07), as was the difference between UC and CD patients (p=5.7e-05). In women, the difference was less pronounced and only significant when comparing UC to controls (p=0.023). Replacement of native EPCR with delipidated EPCR in the ELISA procedure dropped detection and eliminated the UC-CD discrimination power (p=0.784). These findings indicate a role for the bound lipid as key determinant of male-biased, anti-EPCR reactivity, and support the diagnostic potential of this biomarker when assay conditions preserve the physiological lipid-bound state of EPCR. Results and discussion The presence of high anti-EPCR autoantibody titres has emerged as a potential biomarker in varied autoimmune diseases 1 , 2 . EPCR is pivotal for the integrity of the endothelium and plays critical roles in the anticoagulation system 3 . Moreover, abrogation of EPCR’s functions leads to a pro-inflammatory state, with a particular impact on chronic autoimmune disorders such as inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn’s disease (CD). Built on previous work by Mutoh et al 4 , which represents the first association of EPCR as a novel autoantigen in UC, a recent study by Kakuta and colleagues 5 claims the diagnostic potential of anti-EPCR autoantibodies in UC. The authors demonstrated that anti-EPCR titres are significantly increased in UC patients compared to both healthy individuals and those with other colorectal disorders. They also found a high anti-EPCR prevalence in patients with left-sided or extensive UC, and those presenting UC comorbidities, such as arthritis and erythema nodosum. Further, Sawahashi et al 6 performed a large prospective cohort, showing that anti-EPCR antibodies (alone or combined with anti-αvβ6) can predict UC years before clinical onset. Together, these results provide a solid ground for EPCR autoantibodies as promising biomarkers in IBD. Here, by using an in house-developed ELISA assay ( Figure 1A ) , we interrogated any sex bias associated with anti-EPCR levels, but also, a potential mechanistic basis underlying the pathogenic recognition of EPCR. Three cohorts were established by collecting blood samples from healthy subjects (controls) and patients with UC and CD ( Supplementary Table 1 ) . Analysis of anti-EPCR levels across the different groups indicate that the median absorbance values in the UC patients is notably higher than in CD and controls ( Figure 1B ) . This preliminary observation aligns with previous reports 4 – 6 . The interquartile range is wider in the UC cohort, suggesting stronger variability in the anti-EPCR levels. Both disease type (p=0.028) and sex (p=0.018) exhibited a notable impact on anti-EPCR autoantibody levels. A strong interaction was also noticed between disease type and sex (p=0.024), suggesting that disease status influences anti-EPCR titres differently in males and females. Overall, male UC patients showed significantly higher absorbance (mean=1.09) than CD patients (mean=0.50) and controls (mean=0.37) ( Figure 1C and Supplementary Table 2 ) . Remarkably, in males, the difference between UC patients and controls was highly significant (p=1.2×10 -7 ), as was the difference between UC and CD patients (p=5.7×10 -5 ). In women, while UC patients showed higher absorbance (mean=0.71) than controls (mean=0.45), the difference was less pronounced and only significant when comparing UC to controls (p=0.023). Comparison of CD and controls led to no significant differences. Thus, these observations indicate a strong and sex-specific immune response characterized by elevated anti-EPCR autoantibody levels in males with UC. Furthermore, the poor increase of anti-EPCR antibody levels in CD patients supports that the determination of anti-EPCR autoantibody titres is a promising biomarker to discriminate between UC and CD, particularly in males. Download figure Open in new tab Figure 1. Differences in EPCR reactivity by disease type, sex and antigen lipidation. (A) The specificity of the ELISA was assessed with a dose-dependent binding response to commercial, recombinant anti-EPCR (5 ng/ml and 100 ng/ml) compared with isotype control. (B) Box plots displaying anti-EPCR absorbance levels (450 nm) in samples from patients suffering of ulcerative colitis (UC), Crohn’s disease (CD), and healthy controls. (C) Plots showing mean absorbance values ± error bars (95% CI) stratified by sex (females vs males) within each cohort. (D) Differences observed in anti-EPCR absorbance values in males as determined with lipidated (gray) and delipidated (black) EPCR in UC, CD, and controls. The y-axis represents absorbance units (450 nm). Statistical significance between groups: p < 0 . 001: ***; p < 0 . 01: **; p < 0 . 05: *; p < 0 . 1 . (E) Differences in anti-EPCR titres were calculated by subtracting the values obtained with delipidated EPCR from those obtained with native EPCR. The bar plots highlight the patients with the highest difference values, all of whom fall within the UC cohort. The table below details clinical characteristics of these patients. EPCR is a transmembrane protein characterized by the presence of a bound phospholipid in a CD1-like groove that shapes the receptor’s structure 7 , 8 . The lipid hydrophobic region is buried in this pocket, while the polar head group is exposed to the outside, being potentially accessible for molecular recognition 9 , 10 . Thus, we explored whether the anti-EPCR antibodies could target, to a greater or lesser extent, the bound lipid. To this end, we analysed the patient samples using delipidated EPCR. Under this modified protocol, and relative to controls, both UC and CD patients still exhibited increased levels of anti-EPCR autoantibodies. However, while the UC vs. control comparison remained statistically significant (p=0.014) ( Figure 1D and Supplementary Table 2 ) , supporting the robustness of the UC-associated signal, the modified ELISA yield lowered anti-EPCR titres, particularly in UC males (1.09 vs 0.62), resulting in a reduced stratification between UC and CD patients. CD patients showed a borderline elevation compared to controls (p=0.077), indicating that the altered assay may be more sensitive to lower-level responses or potentially less specific. However, the discrepancy between the anti-EPCR levels in UC and CD patients was no longer significant (p=0.784), suggesting that the new assay had a reduced discriminatory power. By comparing the differences in anti-EPCR levels when using the ELISA assay with native or delipidated EPCR, we detected several high outliers that highlight a subset of UC patients with strong reactivity towards the lipid moiety ( Figure 1E ) . These patients are characterized by being all males with UC, either left-sided colitis (E2) or extensive colitis (E3), consistent with broader epithelial disruption and possibly more systemic immune exposure to EPCR. The fact that these patients show mild to negligible activity suggest they are not an active signal of inflammation, or that they reflect immune imprinting from previous mucosal injury or inflammation. Overall, these observations suggest that a significant fraction of the anti-EPCR autoantibodies are directed against the bound lipid or a motif that involves both a fraction of EPCR’s and its bound lipid. Removal or alteration of the bound lipid results in a potential loss in diagnostic accuracy, particularly in distinguishing UC from CD, and with a significant relevance in males. The drop in the discriminatory power underscores the importance of the assay design in preserving clinically and mechanistically relevant motifs that are critical for recognition by circulating anti-EPCR antibodies. Our findings add two elements that had not yet been explored in the context of anti-EPCR levels in IBD. On the one hand, this study provides evidence for a role of the bound lipid in driving antigen recognition by a significant fraction of the anti-EPCR antibodies found in UC patients. On the other hand, we observed a statistically higher proportion of anti-EPCR antibodies in males with UC compared to females. These findings unlock features relevant for the clinical application of these antibodies in IBD diagnostics. Moreover, this study encourages further research to address the question of whether this lipid-driven reactivity associates with a specific gut microbiota that underlies UC. Funding Jacinto López Sagaseta is a Ramón y Cajal Investigator (grant RYC-2017-21683), Ministry of Science and Innovation of Spain. Nerea Ugidos-Damboriena is a recipient of a María Zambrano contract funded by UPNA and the Ministry of Universities of Spain within the Plan of Recovery, Transformation and Resilience and the European Recovery Instrument Next Generation EU. Llùcia Jaime-Gómez was funded by Gobierno de Navarra, Departamento de Innovación y Transformacion Digital /Servicio de I+D+i Programa MRR Investigo (grant 0011-4001-2022-000070). Author contributions Conceptualization: JLS; Methodology: NUD, LLJG, CRG, LZ, ON, MGDR and JLS; Manuscript writing: JLS. Competing interests JLS, MGDR and LLJG are inventors and declare that a patent application has been filed concerning the ELISA method described in this study for detecting anti-EPCR autoantibodies recognizing both lipid-dependent and lipid-independent epitopes. Institutional Review Board Statement This study was carried out according to the Declaration of Helsinki and was approved by the Drug Research Ethics Committee of Navarra (PI_2021/11). Informed Consent Statement Informed consent was obtained from all subjects involved in the study. Data availability Due to an active patent application, proprietary details regarding of the ELISA protocol are not disclosed in this preprint. Additional technical information is available from the corresponding author (J.L.S.) upon reasonable request for academic, clinical, and non-commercial use. Supplementary Material View this table: View inline View popup Download powerpoint Supplementary Table 1. Characteristics of the three cohorts analyzed in this study. View this table: View inline View popup Download powerpoint Supplementary Table 2. Effect of EPCR lipidation on ELISA absorbance. Preservation of EPCR lipidation (Native EPCR) leads to higher absorbance values and UC/CD stratification only in males. In contrast, delipidated EPCR results in a reduced detection power and loss of disease discrimination. The upper values (Native EPCR) indicate male-only data, while the values in the lower section (Delipidated EPCR) applies to all patients. Significant results are indicated with asterisks: p < 0.001: ***; p < 0.01: **; p < 0.05: *; p < 0.1; UC: Ulcerative colitis; CD: Crohn’s disease. Acknowledgements We thank Sergio Morales-Hernández for technical contributions. We also thank Eduardo Albéniz for initial discussions concerning the focus of the study. References 1. ↵ Hurtado V , Montes R , Gris J-C , et al. Autoantibodies against EPCR are found in antiphospholipid syndrome and are a risk factor for fetal death . Blood 2004 ; 104 : 1369 – 1374 . OpenUrl Abstract / FREE Full Text 2. ↵ Müller-Calleja N , Hollerbach A , Royce J , et al. Lipid presentation by the protein C receptor links coagulation with autoimmunity . Science 2021 ; 371 . 3. ↵ Bouwens EAM , Stavenuiter F , Mosnier LO . Mechanisms of anticoagulant and cytoprotective actions of the protein C pathway . Journal of Thrombosis and Haemostasis 2013 ; 11 : 242 – 253 . OpenUrl 4. ↵ Mutoh T , Shirai T , Ishii T , et al. Identification of two major autoantigens negatively regulating endothelial activation in Takayasu arteritis . Nat Commun 2020 ; 11 : 1253 . OpenUrl PubMed 5. ↵ Kakuta Y , Shirai T , McGovern DPB , et al. Novel Diagnostic Autoantibodies Against Endothelial Protein C Receptor in Patients With Ulcerative Colitis . Clinical Gastroenterology and Hepatology 2023 ; 21 : 844 – 846 . OpenUrl 6. ↵ Sawahashi M , Kakuta Y , Naito T , et al. Autoantibodies against endothelial protein C receptor and integrin αvβ6 predict the development of ulcerative colitis . J Gastroenterol 2025 . 7. ↵ Oganesyan V , Oganesyan N , Terzyan S , et al. The crystal structure of the endothelial protein C receptor and a bound phospholipid . J Biol Chem 2002 ; 277 : 24851 – 24854 . OpenUrl Abstract / FREE Full Text 8. ↵ Adams EJ , Lopez-Sagaseta J. The immutable recognition of CD1d . Immunity 2011 ; 34 : 281 – 283 . OpenUrl CrossRef PubMed Web of Science 9. ↵ Oganesyan V , Oganesyan N , Terzyan S , et al. The crystal structure of the endothelial protein C receptor and a bound phospholipid . J Biol Chem 2002 ; 277 : 24851 – 24854 . OpenUrl Abstract / FREE Full Text 10. ↵ Erausquin E , Morán-Garrido M , Sáiz J , et al. Identification of a broad lipid repertoire associated to the endothelial cell protein C receptor (EPCR) . Sci Rep 2022 ; 12 : 15127 . OpenUrl PubMed View the discussion thread. Back to top Previous Next Posted October 20, 2025. Download PDF Email Thank you for your interest in spreading the word about medRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Anti-EPCR autoantibodies in ulcerative colitis show sex- and lipid-dependent patterns Message Subject (Your Name) has forwarded a page to you from medRxiv Message Body (Your Name) thought you would like to see this page from the medRxiv website. 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