Antiphospholipid antibodies and lupus: new molecular targets for treatment

描述(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有关。血栓形成是急性早幼粒细胞白血病和系统性红斑狼疮患者发病和死亡的重要原因。APL抗体识别22GPI的I区(DI)。22GPI通过结构域V与靶细胞[即：内皮细胞、血小板、单核细胞]结合，触发细胞内信号传导和促凝、促炎表型[即：组织因子(TF)、细胞间黏附分子-1(ICAM-1)、血管细胞黏附分子-1(VCAM-1)的表达，细胞因子[IL1b、IL6、IL8、TNF-α、血管内皮生长因子(VEGF)]的表达。有强有力的证据表明，组织纤溶酶原激活物(TPA)和纤溶酶原受体Annexin A2和细菌脂多糖(LPS)受体Toll样受体4(TLR-4)和载脂蛋白E受体2‘(ApoER2’)可能与22GPI结合并在靶细胞内触发细胞内信号转导。因此，靶细胞中22GPI的受体(S)可能涉及多个蛋白，最终与APL/a22GPI抗体聚集或交联，启动细胞内信号事件，导致促血栓形成。我们推测，通过抑制APL/a22GPI抗体与22GPI的DI的特异性结合或阻断22GPI与靶细胞上22GPI识别的受体(S)的相互作用，可以消除APL/抗22GPI的致病作用。我们将利用不同的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Annexin A2抗体或ApoER2‘的可溶性结合域1(BD1)，或低密度脂蛋白受体家族成员常见的受体相关蛋白(RAP)拮抗剂是否影响APL介导的内皮细胞TF、ICAM-1、细胞因子和p38丝裂原激活蛋白激酶(P38 MAPK)的磷酸化以及单核细胞和血小板的激活。然后，我们将检测APL/a22GPI抗体对血栓形成、VCAM-1和Tf在小鼠主动脉中的表达(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功能和血小板聚集、Annexin A2、TLR-4下游的细胞内蛋白髓样分化因子(Myd)88、ApoER2缺陷小鼠以及用特定的abs/拮抗剂和APL/a22GPI抗体治疗的正常小鼠的影响。此外，我们还将研究22GPI的聚乙二醇化野生型DI及其一些突变体--已被证明与APL/a22GPI与各种亲和力结合并抑制某些APL介导的效应--在体外影响APL/a22GPI抗体在各种靶细胞和小鼠中的致病效应的能力。这些研究将提供体内和体外22GPI/APL/a22GPI复合体与靶细胞相互作用的重要信息，并将有助于设计新的靶向治疗/预防APL/a22GPI抗体治疗/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)患者的血栓形成和妊娠丢失有关，这些临床表现是受影响个体发病率和死亡率的重要原因。在此，我们建议以APL抗体与其抗原在不同类型细胞上的相互作用为靶点，作为一种在体外和动物模型中改善APL抗体致病效应的手段。这些研究可能有助于设计出比目前用于治疗和预防APL Abs患者血栓形成的抗凝或全身免疫抑制更具特异性和更少伤害的新方法。

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.