Dysregulation of the complement system in immune thrombocytopenia

Abstract

Abstract Background Primary immune thrombocytopenia (ITP) is a rare acquired autoimmune disease characterised by a destruction and impaired production of platelets (PLT), mediated by autoantibodies and autoreactive T cells, resulting in bleeding tendency. Previous research also indicates complement involvement. This is supported by promising results of recent trials of sutimlimab, a monoclonal antibody targeting C1s, in chronic ITP. However, the complement system's exact role in the pathogenesis of ITP remains uncertain. Methods Twelve acute ITP patients (newly diagnosed or acute relapses, median (IQR) age 33 (26-36) years, 3 women, median (IQR) PLT count 24 (11-81) G/L), eleven chronic ITP patients (median (IQR) age 38 (26-52) years, 3 women, median (IQR) PLT count 26 (12-76) G/L), and 30 healthy controls (HC, median (IQR) age 38 (30-53) years, 12 women) were selected from the Vienna ITP Biobank, a prospective cohort study of adult primary ITP patients. Exclusion criteria were secondary ITP, hereditary thrombocytopenia, pregnancy, active malignancy, and PLT counts ≥100 without treatment. Acute ITP patients with available samples were included. Chronic patients and HC were selected to ensure comparable age and sex distributions across groups. ELISA (Quidel, San Diego, CA, USA; Hycult Biotech Inc., Uden, Netherlands) was performed with serum and EDTA-plasma samples, to obtain detailed biomarker profiles, focussing on markers of the classical pathway and terminal pathway components, regulators, and activation markers (C1s-C1-inhibitor, C4a, C4d, C5a, Bb, TCC, MASP-1-C1-inhibitor). Differences between all ITP patients and HC were assessed using Mann-Whitney U test, while acute ITP, chronic ITP, and HC were compared using Kruskal-Wallis test with post-hoc Dunn's test, adjusted for multiple testing using Bonferoni-Holm. Results Compared with HC, ITP patients showed significantly higher median levels of C1s-C1-inhibitor (1506 vs 1371 ng/mL; p=0.041), C4d (2.66 vs 1.43μg/mL; p=0.015), and TCC (184 vs 120ng/mL; p<0.001). The alternative pathway marker Bb was significantly lower in patients than HC (0.96 vs 1.08μg/mL; p=0.031). C3a levels were higher in ITP patients than HC (56 vs 44ng/mL), though this difference was non-significant. MASP-1-C1-inhibitor concentrations did not differ between patients and controls. According to disease duration, median levels of classical pathway marker C4a (1532 vs 1082 ng/mL; p=0.0208) were significantly higher and terminal pathway marker were non-significantly higher (10.03 vs 6.1 ng/mL; p=0.12) in chronic than in acute patients. Furthermore, compared with acute patients, C1s-C1-inhibitor levels were significantly elevated in chronic patients (1303 vs 2158 ng/mL; p=0.0028). Compared with HC, median levels of C1s-C1-inhibitor (1371 vs 2158 ng/mL; p<0.001) and C4a (1137 vs 1532ng/mL; p<0.0196). Though not significant, C5a levels were highest in chronic and C4d levels were highest in acute patients. TCC levels were substantially higher in acute patients than in HC (205 vs 120ng/mL; p<0.002). Contrary, complement factor Bb was significantly lower in acute patients than HC (0.75 vs 1.08μg/mL; p= 0.0144) but not chronic patients (0.99μg/mL; p=0.14). No differences were observed in levels of lectin pathway marker MASP1-C1-inhibitor between groups. Complement marker levels did not differ in ITP patients when stratified by the presence or absence of platelet-bound anti-platelet antibodies. Conclusions In our cohort of adult primary ITP patients, complement biomarker profile was significantly different from HC. We observed notable differences between disease phases: Chronic ITP patients exhibited substantially increased levels of classical pathway activation, confirming previous research. The finding of significantly elevated C1s-C1-inhibitor complex levels in chronic compared to acute patients, is a novel observation. These results suggest possible variations in the underlying pathways and pathophysiology of different ITP disease phases.In summary, our results corroborate existing data and introduce novel insights into specific complement components and distinct pathway mechanisms across ITP disease phases. These observations could offer new tools for patient stratification in future clinical trials and treatments.