Studio dell'attivazione piastrinica ed endoteliale in pazienti con sindrome da anticorpi antifosfolipidi ad alto rischio in trattamento aferetico

Antiphospholipid syndrome (APS) is a systemic, autoimmune disease characterized by thromboembolic manifestations and/or obstetric morbidity in association with medium to high titres of antiphospholipid antibodies (aPL), such as anticardiolipin (aCL), anti-β2glycoprotein I (aβ2GPI) antibodies and Lupus anticoagulant activity. A small percentage (1%) of APS patients develop a life-threatening variant of the disease, the Catastrophic Antiphospholipid Syndrome (CAPS), which is defined as multiorgan thrombosis over a one-week time affecting at least three organs, systems and/or tissues, with histopathologic confirmation of small vessel occlusion in at least one organ or tissue. CAPS is associated with a high mortality rate (~50%) mostly due to cerebral and cardiac thrombotic involvement, infections and multiorgan failure. Conventional therapy with aspirin and/or heparin is at times incapable of preventing complications in high risk patients with APS. In those cases, in addition to conventional therapy strategies, the so-called second-line treatment protocols, including aphaeretic techniques, are employed. The first part of the study is a report on three APS pregnant patients who were successfully treated with plasma exchange (PE) (two cases) or with immunoadsorption (IA) (one case) as a second-line treatment strategy. The efficacy of these procedures in removing IgG aCL and IgG aβ2GPI antibodies from blood was evaluated. Serum samples were collected before and after apheretic treatment sessions. Serum aCL and aβ2GPI antibodies were determined using an “in-house” enzyme-linked immunosorbent assay (ELISA) and showed that before pregnancy all three patients had medium/high IgG aCL and IgG aβ2GPI titres. In all three patients, a significant decrease in IgG aCL (p=0.00, p=0.00, p=0.00, respectively) and IgG aβ2GPI (p=0.00, p=0.00, p=0.00, respectively) antibody titres were observed after PE and IA sessions. Moreover, there was a significant, steady fall in serum IgG aCL pretreatment levels during the course of all three pregnancies (p=0.00, p=0.00, p=0.001, respectively). The fall in IgG aβ2GPI was significant in two of the patients (p=0.00, p=0.00) both with high antibody titres, but not in one with medium antibody titres. In the second part of this work, we evaluated by in vitro studies, the effect on platelet activation of anti-β2GPI antibodies removed by aphaeretic sessions. Anti-β2GPI antibodies were isolated from plasma of the patient n.3 (previous work), in two different stages of APS (quiescent and catastrophic, respectively). Platelet P-selectin (P-sel) expression was assessed by flow cytometry. The results showed that anti-β2GPI antibodies induced significant platelet P-sel expression only in presence of a platelet agonist at a subthreshold concentration. Notably, anti-β2GPI antibodies from the patient with catastrophic APS enhanced platelet P-sel expression more than those from the same patient in quiescent stage of disease. Following previous results, in the third part of the study ex vivo platelet and endothelium activation in APS was investigated. In plasma samples from six patients with quiescent APS, four with catastrophic APS and nine healthy controls, the main markers of platelet and endothelium activation were measured. The data showed that APS patients had significantly higher levels of the most investigated markers than control subjects. Moreover, soluble P-sel significantly prevailed in catastrophic APS in comparison with quiescent APS.