Effect of Heparanase inhibitor on Tissue Factor overexpression in platelets and endothelial cells induced by anti-β2-GPI antibodies

Anti-phospholipid syndrome (APS) is characterized by arterial and/or venous thrombosis and pregnancy morbidity associated with the presence of “anti-phospholipid antibodies”. Thrombosis may be the result of a hypercoagulable state related to activation of endothelial cells and platelets by anti-2-glycoprotein I (β2-GPI) antibodies. Anti-β2-GPI antibodies induce a proinflammatory and procoagulant phenotype in these cells that, after activation, express Tissue Factor (TF), the major initiator of the clotting cascade, playing a role in thrombotic manifestations. Moreover, TF expression may also be induced by Heparanase, an endo--D-glucuronidase, that generates heparan sulfate fragments, regulating inflammatory responses. Objectives: In this study we analyzed, in human platelets and endothelial cells, the effect of a new symmetrical 2-aminophenyl-benzazolyl-5-acetate derivative (RDS3337), able to inhibit Heparanase activity, on signal transduction pathway leading to TF expression triggered by anti-β2- GPI. Methods: Platelets and endothelial cells were incubated with affinity purified anti-β2-GPI after pretreatment with RDS3337. Cell lysates were analyzed for phospho-interleukin-1 receptor- associated kinase 1 (IRAK1), phospho-p65 nuclear factor kappa B (NF-κB) and TF by Western blot. In addition, platelet activation and secretion by ATP release dosage were evaluated. Results: IRAK phosphorylation and consequent NF-κB activation, as well as TF expression, triggered by anti-β2-GPI treatment were significantly prevented by previous pretreatment with RDS3337. In the same vein, pretreatment with RDS3337 prevented platelet aggregation and ATP release triggered by anti-β2-GPI antibodies. Conclusion: These findings support the view of Heparanase involvement in a prothrombotic state related to APS syndrome, suggesting a novel target to regulate overexpression of procoagulant protein(s).