LUPUS ANTICOAGULANTS AND THE PROTEIN C PATHWAY

狼疮抗凝剂和蛋白质 C 途径

• 批准号: 6110489
• 负责人: NAOMI L ESMON
• 金额: $ 22.97万
• 依托单位: UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-02-01 至 2000-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6110489
• 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) 与血栓发生率增加有关。虽然有因果关系 关系尚未建立，相关性表明 这些抗体直接参与血栓形成过程。 这 包括我们自己在内的许多人已提出将蛋白 C 途径作为一种 这些预约定价安排参与的可能目标。使用新开发的 ELISA检测，我们发现APA的比例相对较高 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，至少其中一些抗体抑制 蛋白C激活。此外，我们最近发现最优 活化蛋白 C (APC) 具有抗凝活性，但不具有促凝活性 活性，需要磷脂酰乙醇胺（PE）的存在 膜提供了解释选择性还原的潜在机制 通过与膜表面或蛋白质结合的抗体来影响 APC 活性 膜复合物。已确定 APA 患者的血浆 比凝血酶原激活更大程度地抑制 APC 活性， 以依赖 PE 的方式执行此操作。初步筛选表明，高 APA 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 APA 患者中抗 APC 和抗 TM 活性的患病率 无血栓形成史并获得分子了解 PE 如何促进 APC 功能并参与 APA 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，通过映射 APC 中负责 PE 的位点 依赖的膜相互作用并通过确定 抑制（即直接与膜结合以及 膜组成与蛋白 C 或蛋白 S 膜的结合 复合物以及抗体是否选择性取代 APC 或蛋白质 S)，并通过使用嵌合蛋白来绘制抗原决定簇图谱。这 APA 抑制血小板介导的因子 Va 失活的能力与其他药物的比较 将检查凝血酶原激活。 APA 的单克隆抗体 患者 B 细胞将准备好研究其作用机制并 潜在的血栓形成性。这些研究应提供以下信息： 预约定价安排发挥作用的机制可能有助于初步了解 哪些特异性直接导致血栓形成，并且可能 提供改进的检测方法来监测治疗的有效性 接近。