LUPUS ANTICOAGULANTS AND THE PROTEIN C PATHWAY

狼疮抗凝剂和蛋白质 C 途径

• 批准号: 6273073
• 负责人: NAOMI L ESMON
• 金额: $ 22.16万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-02-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6273073
• 关键词: antiantibody; anticoagulants; chemical binding; chimeric proteins; clinical research; coagulation factor V; disease /disorder proneness /risk; enzyme linked immunosorbent assay; human tissue; liposomes; molecular pathology; monoclonal antibody; phosphatidylethanolamines; platelets; polymerase chain reaction; protein C; prothrombin; systemic lupus erythematosus; thrombomodulin; venous thrombosis

Lupus anticoagulants and antiphospholipid antibodies (APAs) are associated with an increased incidence of thrombosis. Although a causal relationship has not been established, the correlation suggests that these antibodies contribute directly to the thrombotic process. The protein C pathway has been proposed by many, including ourselves, as a possible target for involvement of these APAs. Using a newly developed ELISA assay, we have found that a relatively high percentage of APA patients (20/61) and patients with unexplained thrombosis (20/200) have an antithrombomodulin (TM) antibody at least some of which inhibit protein C activation. Furthermore, our recent finding that optimal activated protein C (APC) anticoagulant activity, but not procoagulant activity, requires the presence of phosphatidylethanolamine (PE) in the membranes provides a potential mechanism to explain selective reduction in APC activity by antibodies that bind to membrane surfaces or protein- membrane complexes. Patients with APAs have been identified whose plasma inhibits APC activity to a greater extent than prothrombin activation and do so in a PE dependent fashion. Preliminary screening suggests that high levels of anti APC activity in APA patients is associated with a history of thrombosis. The goals of the present application are to determine the prevalence of anti-APC and anti-TM activity in APA patients with and without histories of thrombosis and to obtain a molecular understanding of how PE facilitates APC function and participates in APA inhibition of APC activity. This will aid in the design of better assays for detecting APAs with anti-APC activity. The molecular basis of PE function will be approached by determining the nature of PE involvement in facilitating APC binding interactions, by mapping the sites in APC responsible for PE dependent membrane interaction and by determining the mechanisms of inhibition (i.e., direct binding to membranes and the influence of membrane composition vs. binding to protein C or protein S-membrane complexes and whether the antibodies selectively displace APC or protein S), and by using chimeric proteins to map the antigenic determinants. The ability of APAs to inhibit platelet mediated factor Va inactivation vs. prothrombin activation will be examined. Monoclonal antibodies from APA patient B cells will be prepared to study their mechanism of action and potential thrombogenicity. These studies should provide information on the mechanisms by which APAs function, may allow initial insights into which specificities contribute directly to thrombogenicity, and may provide improved assays to monitor the effectiveness of therapeutic approaches.

狼疮抗凝剂和抗磷脂抗体（APA）是 与血栓形成的发生率增加有关。虽然因果 关系尚未建立，相关性表明， 这些抗体直接促进血栓形成过程。 的 蛋白C通路已经被许多人提出，包括我们自己， 可能是这些APA参与的目标。使用新开发的 ELISA检测，我们发现阿帕的百分比相对较高， 患者（20/61）和不明原因血栓形成患者（20/200） 抗血栓调节蛋白（TM）抗体，其中至少一些抑制 蛋白C激活。此外，我们最近发现， 活化蛋白C（APC）抗凝活性，但无促凝活性 活性，需要磷脂酰乙醇胺（PE）的存在下， 膜提供了一个潜在的机制来解释选择性还原 在APC活性中，抗体与膜表面或蛋白质结合， 膜复合物已确定APA患者的血浆 抑制APC活性的程度大于凝血酶原活化， 以PE依赖的方式这样做。初步筛查显示， 阿帕患者的抗APC活性水平与病史相关 血栓形成本申请的目标是确定所述方法的实施例。 阿帕患者中抗APC和抗TM活性的患病率， 没有血栓形成史， PE如何促进APC功能并参与阿帕抑制， APC活动。这将有助于设计更好的检测方法， 具有抗APC活性的APA。PE功能的分子基础将是 通过确定PE参与促进的性质来接近 APC结合相互作用，通过绘制APC中负责PE的位点 依赖的膜相互作用，并通过确定的机制， 抑制（即，直接与细胞膜结合， 膜组成与蛋白C或蛋白S-膜结合 复合物以及抗体是否选择性地置换APC或蛋白质 S），并通过使用嵌合蛋白质来绘制抗原决定簇。的 APA抑制血小板介导的因子Va失活的能力与 将检查凝血酶原活化。阿帕单克隆抗体 制备患者B细胞以研究其作用机制， 潜在血栓形成性。这些研究应提供以下信息： APA发挥作用的机制，可以让我们初步了解 这些特异性直接导致血栓形成，并且可以 提供了改进的测定以监测治疗有效性 接近。