Thromboregulation by Endothelial Cells: Role of CD39

内皮细胞的血栓调节：CD39 的作用

• 批准号: 8540643
• 负责人: Aaron Jacob Marcus
• 金额: --
• 依托单位: VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-10-01 至 2017-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8540643
• 关键词: ATP phosphohydrolase; Accounting; Agonist; Alternative Splicing; Animal Model; Apyrase; Area; Biological; Blood; Blood Cells; Blood Platelets; Blood Vessels; Blood specimen; Cause of Death; Cell membrane; Cell physiology; Cell surface; Cells; Cerebrovascular Circulation; Cessation of life; Cholesterol; Classification; Clinical; Complex; Comprehension; Coronary Arteriosclerosis; Coronary Circulation; DNA; Data; Development; Diagnosis; Disease; Endothelial Cells; Enzymes; Etiology; Event; Family suidae; Flow Cytometry; Generations; Goals; Inflammation Mediators; Injury; Ischemic Stroke; Knowledge; Lead; Leukocytes; Life; Location; Lymphocyte; Measures; Membrane; Membrane Proteins; Metabolism; Methods; Mission; Modality; Modeling; Molecular; Molecular Profiling; Molecular Weight; Morbidity - disease rate; Mus; Myocardial Infarction; Obstruction; Obstructive Sleep Apnea; Pathogenesis; Patient Care; Patients; Pattern; Peripheral Vascular Diseases; Phenotype; Platelet Activation; Platelet Inhibitors; Play; Population; Predisposition; Prevention; Process; Proteolysis; RNA Splicing; Rattus; Recruitment Activity; Recurrence; Regulation; Reporting; Research; Role; Site; Sleep Apnea Syndromes; Stimulus; Stroke; Surface; System; Testing; Therapeutic; Thrombosis; Thrombus; Time; Tissues; Translations; Variant; Vascular Diseases; atherothrombosis; base; cell type; ectoADPase; effective therapy; enzyme activity; fluidity; in vivo; injured; interdisciplinary approach; leukocyte activation; mortality; multidisciplinary; nervous system disorder; neutrophil; novel strategies; prevent; programs; public health relevance; response; trafficking

DESCRIPTION (provided by applicant): The diagnosis, treatment, and prevention of platelet-induced occlusive diseases of the coronary and cerebral circulation are a major challenge to the VA patient care mission. Excessive platelet activation and recruitment underlies the pathogenesis of platelet-induced occlusive vascular disease. Our proposal focuses on development of a novel approach toward prevention and treatment of these disorders, namely the use of the thromboregulatory ecto-apyrase ATPDase/CD39/NTPDase1. CD39 functions as the principal regulator of blood fluidity by metabolizing prothrombotic ATP and ADP released from activated platelets and other cells to prevent thrombus formation. We previously reported prothrombotic alterations in CD39 activity in patients with verified coronary artery disease. Subsequently, we examined leukocyte CD39 activity in stroke patients and found that lymphocytes from these subjects displayed changes in the ADPase/ATPase activity ratio, as compared to controls. This observation led to our hypothesis that alterations in CD39 activity can result in a predisposition for stroke. This application extends our previous research and focuses on three main areas. First, we will define the molecular biological mechanisms underlying regulation of expression and activity of CD39, and alterations therefrom that influence cellular function in vascular disease. Our preliminary data indicate that maximal CD39 enzyme activity requires formation of higher molecular weight complexes localized in cholesterol-rich membrane subdomains via a process requiring cleavage of the protein. We plan to fully confirm and extend these observations, including elucidation of the site of cleavage in CD39 and the subcellular location of the cleavage event. We have also determined that our recently discovered CD39 splice variants (V1-4) regulate apyrase activity at the cell surface. We will elucidate the role of these CD39 alternative splice variants in regulatio of CD39 intracellular trafficking and cleavage. We will also determine whether CD39 splice variant generation and translation is under tissue- and stimulus-specific regulation. CD39 splice variants modulate formation of enzymatically active CD39 species in the plasma membrane. This determines the pro- or antithrombotic phenotype of the cell. Second, we will further establish the relationship between alterations in CD39 activity, platelet reactivity, and stroke. W will extend our stroke research to 3 different patient groups: (1) Cryptogenic stroke patients (stroke in a young population without clinical etiology); (2) "pre-stroke" patients who have verified obstructive sleep apnea; and (3) classical atherothrombotic stroke patients. We have identified cryptogenic stroke and stroke-prone patients who have increased responsiveness to platelet agonists and increased expression of platelet and leukocyte activation markers. De-identified blood samples from these patients will be processed for isolation of platelets, lymphocytes and neutrophils. These will then be analyzed by several methods, including flow cytometry and our newly developed Broad Range Platelet Aggregometry System. In depth classification of these patients with regard to their threshold for platelet reactivity will increae our comprehension of the pathogenesis of stroke. This will also allow us to test our hypothesis that cryptogenic stroke may, in large part, represent a disorder of enhanced platelet reactivity. Third, we will determine CD39 surface expression and splice variant profiles in leukocytes of stroke patients versus controls by flow cytometry, quantitative real-time PCR, and DNA Fragment analyses. These studies will establish the relationship between stroke etiology and CD39 variant profile. This will further verify our hypothesis that altered CD39 activities play a role in cryptogenic stroke and provide a mechanistic basis for this role. Our proposed studies represent a multidisciplinary approach to the pathogenesis, prevention and treatment of ischemic stroke as well as to the regulatory aspects of our newly discovered CD39 splice variants. This research will enhance our knowledge of the mechanisms of action of CD39 and ultimately lead to development of a safe and effective treatment of platelet-driven occlusive vascular disorders in patients.

描述（由申请人提供）： 血小板诱发的冠状动脉和脑循环闭塞性疾病的诊断、治疗和预防是 VA 患者护理任务的主要挑战。血小板过度活化和募集是血小板诱发的闭塞性血管疾病的发病机制的基础。我们的提案侧重于开发一种预防和治疗这些疾病的新方法，即使用血栓调节性外切腺苷三磷酸双磷酸酶 ATPDase/CD39/NTPDase1。 CD39 通过代谢活化的血小板和其他细胞释放的促血栓 ATP 和 ADP 来防止血栓形成，从而发挥血液流动性的主要调节作用。我们之前报道过经证实的冠状动脉疾病患者中 CD39 活性的促血栓改变。随后，我们检查了中风患者的白细胞 CD39 活性，发现与对照组相比，这些受试者的淋巴细胞显示 ADPase/ATPase 活性比值发生变化。这一观察结果得出我们的假设：CD39 活性的改变可能导致中风的易感性。该应用程序扩展了我们之前的研究，重点关注三个主要领域。首先，我们将定义 CD39 表达和活性调节的分子生物学机制，以及影响血管疾病中细胞功能的改变。我们的初步数据表明，最大的 CD39 酶活性需要通过需要裂解蛋白质的过程形成位于富含胆固醇的膜子结构域中的较高分子量复合物。我们计划充分确认并扩展这些观察结果，包括阐明 CD39 中的裂解位点和裂解事件的亚细胞位置。我们还确定，我们最近发现的 CD39 剪接变体 (V1-4) 调节细胞表面的腺苷三磷酸双磷酸酶活性。我们将阐明这些 CD39 选择性剪接变体在调节 CD39 细胞内运输和切割中的作用。我们还将确定 CD39 剪接变体的生成和翻译是否受到组织和刺激特异性的调节。 CD39 剪接变体调节质膜中具有酶活性的 CD39 物质的形成。这决定了细胞的促血栓表型或抗血栓表型。其次，我们将进一步建立CD39活性、血小板反应性和中风之间的关系。 W 将把我们的中风研究扩展到 3 个不同的患者群体：（1）隐源性中风患者（无临床病因的年轻人群中风）； (2) 已证实患有阻塞性睡眠呼吸暂停的“中风前期”患者； (3)典型的动脉粥样硬化血栓性中风患者。我们已经确定了隐源性中风和易发生中风的患者对血小板激动剂的反应性增加以及血小板和白细胞激活标记物的表达增加。来自这些患者的去识别化血液样本将被处理以分离血小板、淋巴细胞和中性粒细胞。然后将通过多种方法进行分析，包括流式细胞术和我们新开发的宽范围血小板聚集测量系统。根据血小板反应阈值对这些患者进行深入分类将增加我们对中风发病机制的理解。这也将使我们能够检验我们的假设，即隐源性中风可能在很大程度上代表血小板反应性增强的疾病。第三，我们将通过流式细胞术、定量实时 PCR 和 DNA 片段分析来确定中风患者与对照白细胞中 CD39 表面表达和剪接变异谱。这些研究将建立中风病因与 CD39 变异谱之间的关系。这将进一步验证我们的假设，即改变的 CD39 活性在隐源性中风中发挥作用，并为该作用提供机制基础。我们提出的研究代表了缺血性中风发病机制、预防和治疗以及我们新发现的 CD39 剪接变体的调控方面的多学科方法。这项研究将增强我们对 CD39 作用机制的了解，并最终开发出一种安全有效的治疗血小板驱动的闭塞性血管疾病的方法。