Antiphospholipid antibodies and lupus: new molecular targets for treatment

描述（由申请人提供）：抗磷脂（aPL）抗体（Abs）与系统性红斑狼疮（SLE）和抗磷脂综合征（APS）患者的血栓形成和妊娠丢失相关。血栓形成是APS和SLE患者发病和死亡的重要原因。aPL抗体识别22GPI（22糖蛋白I）结构域I （DI）。22GPI通过结构域V结合靶细胞[即内皮细胞（EC）、血小板、单核细胞]，触发细胞内信号转导和亲凝、促炎表型[即组织因子（TF）、细胞间细胞粘附分子-1 （ICAM-1）、血管细胞粘附分子-1 （VCAM-1）的表达，上调细胞因子[IL1b、IL6、IL8、TNF-a、血管内皮生长因子（VEGF）]。有强有力的证据表明，膜联蛋白A2（组织纤溶酶原激活物（tPA）和纤溶酶原受体）和toll样受体4 (TLR-4)（细菌脂多糖（LPS）和载脂蛋白E受体2' （apoER2'）受体）可能结合22GPI并触发靶细胞内信号传导。因此，靶细胞中的22GPI受体可能涉及不止一种蛋白质，这些蛋白质最终会与aPL/a22GPI抗体聚集或交联，并启动细胞内信号转导事件，导致促血栓形成。我们假设，通过抑制aPL/a22GPI抗体与22GPI的DI特异性结合或阻断22GPI与靶细胞上22GPI识别的受体蛋白的相互作用，可以消除aPL/a22GPI的致病作用。我们将利用各种体外和体内方法来研究这个问题。我们将首先研究TLR-4配体拮抗剂、抗TLR-4抗体、抗膜联蛋白A2抗体或apoER2'的可溶性结合域1 (BD1)，或LDL受体家族成员受体相关蛋白（RAP）的共同拮抗剂是否会影响apl介导的EC中TF、ICAM-1、细胞因子和p38丝裂原活化蛋白激酶（p38 MAPK）磷酸化的上调以及单核细胞和血小板的活化。然后，我们将检测aPL/a22GPI抗体对小鼠主动脉血栓形成、VCAM-1和TF表达的影响（使用量子点纳米晶体和双光子激发激光扫描显微镜）、细胞因子上调（使用Multiplex/Luminex平台系统）、TF在颈动脉均质液和单核腹膜细胞中的功能、血小板聚集、膜联蛋白A2、髓样分化因子（MyD）88 （TLR-4下游的细胞内蛋白）、在apoER2'缺陷小鼠和用特异性抗体/拮抗剂和aPL/a22GPI抗体治疗的正常小鼠中。此外，我们将研究聚乙二醇化的野生型22GPI DI及其一些突变体在体外各种靶细胞和小鼠中影响aPL/a22GPI抗体致病作用的能力，这些突变体已被证明能以不同的亲和力结合aPL/a22GPI并抑制一些aPL介导的作用。这些研究将提供22GPI /aPL/a22GPI复合物与靶细胞体外和体内相互作用性质的重要信息，并将有助于设计新的靶向模式，用于治疗/预防aPL/a22GPI抗体SLE患者的血栓形成。

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.