LUPUS ANTICOAGULANTS AND THE PROTEIN C PATHWAY

狼疮抗凝剂和蛋白质 C 途径

基本信息

批准号：
6302361
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
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-02-15 至 2001-01-31
项目状态：
已结题

项目摘要

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)，并使用嵌合蛋白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。