Antiphospholipid antibodies and lupus: new molecular targets for treatment

描述（由申请方提供）：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伴aPL Ab患者发病和死亡的重要原因。APL抗体识别22糖蛋白I（22 GPI）的结构域I（DI）。22 GPI结合靶细胞[即：内皮细胞（EC）、血小板、单核细胞]，并触发细胞内信号传导以及促凝血和促炎表型[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，细胞因子[IL 1b、IL 6、IL 8、TNF-α、血管内皮生长因子（VEGF）]的上调。有强有力的证据表明，膜联蛋白A2（组织纤溶酶原激活物（tPA）和纤溶酶原的受体）和toll样受体4（TLR-4）（细菌脂多糖（LPS）和载脂蛋白E受体2'（apoER 2'）的受体）可以结合22 GPI并触发靶细胞中的细胞内信号传导。因此，靶细胞中22 GPI的受体可能涉及一种以上的蛋白质，其最终将与aPL/a22 GPI Ab聚集或交联并引发细胞内信号传导事件，导致促血栓形成素质。我们推测aPL/抗22 GPI的致病作用可以通过抑制aPL/a22 GPI Abs与22 GPI的DI的特异性结合或通过阻断22 GPI与靶细胞上22 GPI识别的受体蛋白的相互作用来消除。我们将利用各种体外和体内方法来研究这个问题。我们将首先检查TLR-4配体拮抗剂、抗TLR-4抗体、抗膜联蛋白A2 Ab或apoER 2 ′的可溶性结合结构域1（BD 1）或称为受体相关蛋白（RAP）的LDL受体家族成员的共同拮抗剂是否影响aPL介导的TF、ICAM-1、EC中的细胞因子和p38丝裂原活化蛋白激酶（p38 MAPK）磷酸化以及单核细胞和血小板的活化。然后，我们将检测aPL/a22 GPI Abs对小鼠血栓形成、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），颈动脉匀浆和腹膜单核细胞中的TF功能和血小板聚集，膜联蛋白A2，在骨髓分化因子（MyD）88 -TLR-4下游的细胞内蛋白中，在apoER 2-缺陷小鼠和用特异性abs/拮抗剂和aPL/a22 GPI抗体处理的正常小鼠中。此外，我们将研究22 GPI的聚乙二醇化野生型DI及其一些突变体（已显示其以各种亲和力结合aPL/a22 GPI并抑制一些aPL介导的作用）在体外影响aPL/a22 GPI Ab在各种靶细胞和小鼠中的致病作用的能力。这些研究将提供关于22 GPI/aPL/a22 GPI复合物与靶细胞在体外和体内的相互作用的性质的重要信息，并将有助于设计用于治疗/预防具有aPL/a22 GPI Ab的SLE患者中血栓形成的新靶向模式。

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.