Tissue factor over-expression in platelets of patients with anti-phospholipid syndrome: induction role of anti-ß2-GPI antibodies.

Anti-phospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis and pregnancy morbidity. It is well known that in these patients thrombosis may be the result of a hypercoagulable state related to anti-ß2-glycoprotein I (ß2-GPI) antibodies. Moreover, platelets may play a role in thrombotic manifestations by binding of anti-ß2-GPI antibodies. Platelets express tissue factor (TF), the major initiator of the clotting cascade, after activation. We primarily analyzed whether anti-ß2-GPI antibodies may trigger a signal transduction pathway leading to TF expression in human platelets. Platelets from healthy donors were incubated with affinity purified anti-ß2-GPI antibodies for different times. Platelet lysates were analyzed for phospho-interleukin-1 receptor-associated kinase 1 (IRAK), phospho-p65 nuclear factor kappaB (NF-κB) and TF by Western blot. IRAK phosphorylation was observed as early as 10 min of anti-ß2-GPI treatment, with consequent NF-κB activation, whereas TF expression, detectable at 45 min, was significantly increased after 4 h of anti-ß2-GPI treatment. Virtually no activation was observed following treatment with control immunoglobulin IgG. We then analyzed TF expression in platelets from 20 APS patients and 20 healthy donors. We observed a significant increase of TF in APS patients versus control subjects (P < 0·0001). This work demonstrates that anti-ß2-GPI antibodies may trigger in vitro a signal transduction pathway in human platelets, which involves IRAK phosphorylation and NF-κB activation, followed by TF expression. Furthermore, ex vivo, platelets of APS patients showed a significantly increased expression of TF. These findings support the view that platelets may play a role in the pathogenesis of APS, with consequent release of different procoagulant mediators, including TF.

Antibodies to age-ß2 glycoprotein I in patients with anti-phospholipid antibody syndrome.

Anti-phospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized clinically by arterial and/or venous thromboses, recurrent abortions or fetal loss and serologically by the presence of 'anti-phospholipid antibodies' (aPL). The main target antigen of the antibodies is ß2 glycoprotein I (ß2 GPI). Post-translational oxidative modifications of the protein have been widely described. In this study we aimed to analyse sera reactivity to glucose-modified ß2 GPI (G-ß2 GPI). Sera collected from 43 patients with APS [15 primary APS (PAPS) and 28 APS associated with systemic lupus erythematosus (SLE) (SAPS)], 30 with SLE, 30 with rheumatoid arthritis (RA) and 40 healthy subjects were analysed by an enzyme-linked immunosorbent assay (ELISA) using a G-ß2 GPI. Nine of 15 consecutive PAPS out-patients (60%) and 16 of 28 SAPS (57.1%) showed serum antibodies [immunoglobulin (Ig)G class] against G-ß2 GPI (anti-G-ß2 GPI) by ELISA. The occurrence of anti-G-ß2 GPI was significantly higher in APS patients compared to patients suffering from SLE. No RA patients or control healthy subjects resulted positive for anti-G-ß2 GPI. Of note, aG-ß2 GPI prompted to identify some APS patients (four PAPS and seven SAPS), who were negative in the classical anti-ß2 GPI test. Moreover, in APS patients, anti-G-ß2 GPI titre was associated significantly with venous thrombosis and seizure in APS patients. This study demonstrates that G-ß2 GPI is a target antigen of humoral immune response in patients with APS, suggesting that ß2 GPI glycation products may contain additional epitopes for anti-ß2 GPI reactivity. Searching for these antibodies may be useful for evaluating the risk of clinical manifestations.