title: MOLECULAR DETERMINANTS OF STEC-HUS: FROM COMPLEMENT ACTIVATION TO MICROVASCULAR THROMBOSIS
Creators: name: Santarsiero, Donata
Publisher: name: The Open University
Year: 2026

description: Shiga-like toxin producing <i>E.coli</i>-induced haemolytic uraemic syndrome (STEC-HUS) is a rare disease encompassing microangiopathic haemolysis, thrombocytopenia and renal failure. Increasing evidence has suggested activation of complement system as a possible mechanism in the development of STEC-HUS. However, the specific role of activation of each complement pathway on microvascular endothelium in STEC-HUS remains undefined.<br><br>
This project investigated the contribution of activation of the three complement pathways in STEC-HUS and evaluated the role of complement in STEC-HUS-associated microvascular thrombosis. A large cohort of patients with diagnosis of STEC-HUS were studied, including patients in acute phase and in remission.<br><br> 
To reproduce <i>in vitro</i> the complex interaction between complement and endothelium, which constitutes the main site of damage in STEC-HUS, we used the <i>ex vivo</i> test developed in our laboratory and based on serum and cultured microvascular endothelial cells. Our <i>ex vivo</i> test documented that all sera from STEC-HUS patients in acute phase induced abnormal C3 and C5b-9 deposition on resting and activated endothelial cells. Addition to patients’ serum of C1-inhibitor or anti-MASP2 did not prevent C3 and C5b-9 deposits on endothelial cells. On the other hand, a CFB inhibitor successfully inhibited both C3 and C5b-9 deposition, indicating AP as the main complement pathway that is activated at endothelial cell level in acute STEC-HUS.<br><br> 
We also documented massive formation of platelet aggregates on endothelial cells exposed to acute STEC-HUS serum and then perfused with whole blood. Data showing that thrombus formation was blocked in the presence of the CFB inhibitor, whereas no effect was observed with either a C1-inhibitor or an anti-MASP2 blocking antibody, point to AP as the key pathway involved in the thrombotic process. In addition, acute STEC-HUS serum induced an increased expression of P-selectin and vWF as well as the loss of THBD from cell surface, determining a pro-thrombotic phenotype of endothelial cells.<br><br> 
Altogether, these results provide evidence on the role of AP in acute STEC-HUS and indicate that CFB inhibitors could represent possible therapeutic tools to control this disease.<br><br> 
Through our <i>ex vivo</i> test, we found that sera from a substantial fraction of patients at remission induced abnormal C3 and C5b-9 deposition both on unstimulated and on activated endothelial cells, indicating that some patients had persistent complement activation at endothelial cell level also after the resolution of the acute episode.<br><br> 
A more in-depth analysis of patients’ clinical data revealed that 67% of patients with elevated C5b-9 formation both on unstimulated and on activated endothelial cells, had abnormalities in at least one haematological parameter, indicating that those patients were not in full remission. The positive correlation between serum-induced C5b-9 formation and LDH levels in patients at remission suggests that patients who did not completely recover from the disease had circulating factors, possibly derived from an underlying status of tissue injury and/or haemolysis, which could be responsible for the persistent complement activation at endothelial level.<br><br> 
These results would suggest that our <i>ex vivo</i> test may be able to detect complement activation on endothelial cells in the fraction of patients who had not achieved a status of complete remission and are in line with evidence documenting that STEC-HUS is a complex disease that may not fully resolve after the recovery from the acute phase.<br><br>