Antiphospholipid antibodies and lupus: new molecular targets for treatment

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DESCRIPTION (provided by applicant): Antiphospholipid (aPL) antibodies (Abs) are associated with thrombosis and pregnancy loss in patients with systemic lupus erythematosus (SLE) and antiphospholipid syndrome (APS). Thrombosis is an important cause of morbidity and mortality in APS and SLE patients with aPL Abs. APL Abs antibodies recognize domain I (DI) of 22glycoprotein I (22GPI). 22GPI binds to target cells [i.e.: endothelial cells (EC), platelets, monocytes] through domain V and trigger an intracellular signaling and a pro-coagulant and pro-inflammatory phenotype [i e.: expression of tissue factor (TF), intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), upregulation of cytokines [IL1b, IL6, IL8, TNF-a, vascular endothelial growth factor (VEGF)]. There is strong evidence that annexin A2, a receptor for tissue plasminogen activator (tPA) and plasminogen, and toll-like receptor 4 (TLR-4), a receptor for bacterial lipopolysaccharide (LPS) and apolipoprotein E receptor 2' (apoER2') may bind 22GPI and trigger intracellular signaling in target cells. Hence, the receptor(s) for 22GPI in target cells may involve more than one protein that would ultimately cluster or cross-link with aPL/a22GPI Abs and initiate intracellular signaling events, leading to a pro-thrombotic diathesis. We hypothesize that aPL/anti-22GPI pathogenic effects may be abrogated by inhibiting the specific binding of aPL/a22GPI Abs to DI of 22GPI or by blocking the interaction of 22GPI with the receptor(s) proteins recognized by 22GPI on target cells. We will examine this question utilizing various in vitro and in vivo approaches. We will first examine whether a TLR-4 ligand antagonist, anti-TLR-4 antibodies, anti-annexin A2 Abs or soluble binding domain 1 (BD1) of apoER2', or a common antagonist to members of the LDL receptor family named receptor associated protein (RAP) affect aPL-mediated upregulation of TF, ICAM-1, cytokines and p38 mitogen activated protein kinase (p38 MAPK) phosphorylation in EC and activation of monocytes and platelets. Then, we will examine the effects aPL/a22GPI Abs on thrombus formation, VCAM-1 and TF expression in aortas of mice (using quantum dot nano crystals and two-photon excitation laser scanning microscopy), cytokine upregulation (using a Multiplex/Luminex platform system), TF function in carotid artery homogenates and mononuclear peritoneal cells and platelet aggregation, in annexin A2, in myeloid differentiation factor (MyD)88 - an intracellular protein downstream from TLR-4, in apoER2' deficient mice and in normal mice treated with the specific abs/antagonists and aPL/a22GPI antibodies. In addition, we will study the ability of pegylated wild-type DI of 22GPI and some of its mutants - that have been shown to bind aPL/a22GPI with various affinities and inhibit some aPL-mediated effects- to affect the pathogenic effects of aPL/a22GPI Abs in vitro in various target cells and in mice. These studies will provide significant information on the nature of the interactions of 22GPI /aPL/a22GPI complexes with target cells in vitro and in vivo and will help to devise new targeted modalities for treatment/prevention of thrombosis in SLE patients with aPL/a22GPI Abs. PUBLIC HEALTH RELEVANCE: Antiphospholipid (aPL) antibodies (Abs) are associated with thrombosis and pregnancy loss in patients with lupus and the antiphospholipid syndrome (APS) and those clinical manifestations are an important cause of morbidity and mortality in individuals affected. Here we propose to target the interactions of aPL Abs with their antigen on various types of cells as a means to ameliorate aPL Abs-pathogenic effects in vitro and in animal models. These studies may help to devise new more specific and less harmful modalities than the anticoagulation or general immunosuppression currently used to treat and prevent thrombosis in patients with aPL Abs.