Introduction and aims: Immune Thrombotic Thrombocytopenic Purpura (iTTP) is a rare, life-threatening thrombotic microangiopathy caused by severe ADAMTS13 deficiency due to autoantibodies against ADAMTS13. Current treatment with therapeutic plasma exchange (TPE), immunosuppression, and the nanobody caplacizumab has dramatically reduced mortality to <10%, although the clinical severity of each acute episode remains unpredictable. While ADAMTS13 deficiency is central to pathogenesis, growing evidence implicates systemic inflammation and NETosis in disease progression. In the frame of a large longitudinal observational study involving iTTP patients, we aim to investigate the role of biomarkers of NETosis (citrullinated H3 histone, H3Cit) and inflammation (tumor necrosis factor-alpha, TNF-α, and interleukin-6, IL-6).

Methods: Six patients (67% female; median age: 57 years, range 20–79) diagnosed with acute iTTP at the Hospitals of Bergamo and Brescia, Italy, between Feb 2024 and Apr 2025 were evaluated. Plasma samples were collected at baseline (D0), and on days 6 (D6), 9 (D9), and 15 (D15) following diagnosis, during treatment. ADAMTS13 activity, anti-ADAMTS13 antibodies, and ADAMTS13 inhibitory activity were assessed by ELISA (Technozym). Levels of H3Cit were quantified by ELISA (Cayman Chemical), and TNF-α and IL-6 levels by multiplex assay (Millipore Luminex). The analyses were performed at the Hemostasis and Thrombosis Center of Bergamo Hospital. Concurrent laboratory assessments comprised schistocyte detection, complete blood counts, and general biochemistry, including hemolysis parameters (reticulocytes, lactate dehydrogenase (LDH), bilirubin, haptoglobin). Clinical data and administered treatments were documented. Statistical analyses were conducted using SPSS v21.0 (IBM, Armonk, NY, USA) and R v4.2.2 (R Foundation, Vienna, Austria).

Results: At onset, five patients presented neurological symptoms, ranging from mild headache to aphasia and acute ischemic cerebellar lesions. HIV infection, myelodysplastic syndrome (MDS), and autoimmune disorders were the most significant comorbidities. As first-line treatment, all patients received daily TPE, immunosuppression (including rituximab), and caplacizumab. Hematological remission (i.e., platelet count >150×10⁹/L and LDH<250 mg/dL) was achieved in a median time of 3 days, except for the patient with MDS who reached a stable platelet count >20 ×10⁹/L by day 9. Platelets' rise correlated with LDH reduction (p=0.001). Median time to ADAMTS13 >20% was 8 days; from D0 to D15, ADAMTS13 activity increased (1–43%; p=0.03), antibody titers declined (50 to 5 U/mL), as well as inhibitory activity (73 to 33%; p=0.045).

During treatment, a significant decrease in H3Cit (p=0.0018) and TNF-α (p=0.015) plasma levels was observed over the different time points, while no significant changes were seen in the levels of IL-6. Specifically, H3Cit levels markedly decreased from D0 [5.90 ng/mL (IQR: 4.0–13.2)] to D6 [1.32 (0.9–2.9), p=0.017], D9 [0.55 (0.3–2.1), p=0.009], and D15 [0.69 (0.4–2.1), p=0.016]. TNF-α also significantly (p=0.035) declined from baseline to D9 [14.83 pg/mL (9.9–32.0) vs 4.90 (3.2–11.8) pg/mL]. Furthermore, a significant positive correlation was found between TNF-α levels and anti-ADAMTS13 antibodies (r=0.783; p<0.001). A subgroup analysis revealed that patients who received caplacizumab within 2 days of diagnosis (n=3) had significantly lower post-treatment TNF-α levels [4.6 (3.3–5.3) vs 7.2 (5.2–25.4), p=0.009] and showed a trend toward lower IL-6 levels [2.4 (1.2–6.1) vs 7.3 (2.9–16.5)] compared to those who initiated treatment later (n=3).

Conclusions: Our data show significant reductions in H3Cit and TNF-α during treatment, paralleling hematologic and biochemical recovery. This suggests that such biomarkers may serve as indicators of NET-driven inflammation and immune activation. The positive correlation between TNF-α and anti-ADAMTS13 antibodies further supports the link between inflammation and the autoimmune process in iTTP. Additionally, our preliminary data suggest that early initiation of caplacizumab may be associated with reduced inflammatory markers. Despite the small sample size, these results highlight the potential for using NETosis and inflammation markers to monitor disease activity and optimize therapeutic strategies in iTTP.

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2025
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