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）患者的血栓形成和流产相关。血栓形成是具有 aPL 抗体的 APS 和 SLE 患者发病和死亡的重要原因。 APL Abs 抗体可识别 22 糖蛋白 I (22GPI) 的结构域 I (DI)。 22GPI 通过结构域 V 与靶细胞 [即：内皮细胞 (EC)、血小板、单核细胞] 结合，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、细胞因子的上调 [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（组织纤溶酶原激活剂 (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' (apoER2') 的受体）可能结合 22GPI 并触发靶细胞中的细胞内信号传导。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 Ab 聚集或交联，并启动细胞内信号传导事件，导致促血栓形成素质。我们假设aPL/抗22GPI致病作用可以通过抑制aPL/a22GPI抗体与22GPI的DI的特异性结合或通过阻断22GPI与靶细胞上22GPI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 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) 的常见拮抗剂是否影响 EC 中 aPL 介导的 TF、ICAM-1、细胞因子和 p38 丝裂原激活蛋白激酶 (p38 MAPK) 磷酸化以及单核细胞和单核细胞的激活。血小板。然后，我们将检查 aPL/a22GPI Abs 对血栓形成、小鼠主动脉中的 VCAM-1 和 TF 表达（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）、颈动脉匀浆和单核腹膜细胞中的 TF 功能以及血小板聚集、膜联蛋白 A2 和骨髓分化因子 (MyD)88 的影响- 在apoER2'缺陷小鼠和用特定abs/拮抗剂和aPL/a22GPI抗体处理的正常小鼠中，TLR-4下游的细胞内蛋白。此外，我们将研究 22GPI 的聚乙二醇化野生型 DI 及其一些突变体（已被证明能够以各种亲和力结合 aPL/a22GPI 并抑制某些 aPL 介导的效应）在体外影响 aPL/a22GPI Abs 在各种靶细胞和小鼠中的致病作用的能力。这些研究将提供有关 22GPI /aPL/a22GPI 复合物与靶细胞在体外和体内相互作用的性质的重要信息，并将有助于设计新的靶向方式，用于使用 aPL/a22GPI Abs 治疗/预防 SLE 患者的血栓形成。 公共卫生相关性：抗磷脂 (aPL) 抗体 (Abs) 与狼疮和抗磷脂综合征 (APS) 患者的血栓形成和流产有关，这些临床表现是受影响个体发病和死亡的重要原因。在这里，我们建议将 aPL Abs 与其抗原在各种类型细胞上的相互作用作为目标，作为改善体外和动物模型中 aPL Abs 致病效应的手段。这些研究可能有助于设计出比目前用于治疗和预防 aPL 抗体患者血栓形成的抗凝或一般免疫抑制更具体、危害更小的新方法。

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.