TREATMENT STRATEGIES FOR THE ANTI PHOSPHOLIPID SYNDROME

抗磷脂综合征（APS），也称为休斯综合征，是一种多器官血管疾病，与心肌梗死、卒中血栓形成和复发性胎儿丢失相关。在50岁以下的中风患者中，45%的人也表现出磷脂抗体水平升高。我们的假设是，循环中抗磷脂抗体的存在导致血管疾病，而结合破坏性抗体的药物可以减轻不断升级的组织损伤。为了验证这一假设，有必要创造一种遗传上确定的APS小鼠，它可以分泌抗磷脂抗体到血液中。我们制备并鉴定了一种特异性识别磷脂酰丝氨酸的小鼠单克隆抗体。初步数据表明，我们的单克隆抗体结合特异性与APS患者存在的抗体相似；因此，这种单克隆抗体适合开发一种治疗模型。我们的单克隆抗磷脂抗体的重链和轻链将通过RT-PCR克隆。这些cdna将包括内源性分泌肽信号序列。双基因小鼠将被用于直接配体诱导的肝脏表达。每个单克隆免疫球蛋白链将被HA或FLAG表位不同地标记。转基因动物将被表征，并与正常动物比较APS的标志性症状，包括体外凝血时间延长、复发性胎儿丢失和血管疾病。本提案的另一个目标是制定APS的治疗策略。我们的方法是中和致病抗体。体外结合和阻断单克隆抗磷脂抗体的肽可以从随机肽的组合噬菌体展示文库中选择。将评估分离的肽是否能够减轻这种转基因小鼠显示的APS症状。这项研究将产生一种遗传定义的APS小鼠模型，这将证明对开发治疗策略以改善APS相关症状是有用的。

Antiphospholipid Syndrome (APS), also known as Hughes Syndrome, is a multiorgan vascular disease associated with myocardial infarction, stroke thrombosis and recurrent fetal loss. Forty- five percent of the people under the age of fifty who suffer strokes also demonstrate elevated levels of phospholipid antibody. Our hypothesis is that the presence of antiphospholipid antibodies in the circulation leads to vascular disorders, and that the escalating tissue damage can be abated with agents that bind the destructive antibodies. In order to test this hypothesis, it is necessary to create a genetically defined APS mouse that secretes antiphospholipid antibodies into the blood. We have produced and characterized a mouse monoclonal antibody which specifically recognizes phosphatidylserine. Preliminary data demonstrates that our monoclonal antibody binding specificity is similar to antibodies present in APS patients; this monoclonal antibody is therefore appropriate to develop a treatment model. The heavy and light chain of our monoclonal antiphospholipid antibody will be cloned by RT-PCR. These cDNAs will include the endogenous secretory peptide signal sequence. Bigenic mice will be used to direct ligand induced expression in the liver. Each monoclonal immunoglobulin chain will be differentially tagged with HA or FLAG epitopes. The transgenic animals will be characterized and compared with normal animals for the hallmark symptoms of APS, including prolonged in vitro coagulation times, recurrent fetal loss and vascular disease. An additional goal of this proposal is to develop treatment strategies for APS. Our approach is to neutralize the disease-causing antibodies. Peptides which bind and block the monoclonal antiphospholipid antibody in vitro can be selected from a combinatorial phage-display library of random peptides. Isolated peptides will be assessed for the ability to reduce the symptoms of APS displayed by this transgenic mouse. This study will produce a genetically-defined mouse model of APS which will prove useful for developing treatment strategies to ameliorate the symptoms associated with APS.