Thromboregulation by Endothelial Cells: Role of CD39

内皮细胞的血栓调节：CD39 的作用

• 批准号: 8966657
• 负责人: Joan H. F. Drosopoulos
• 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/8966657
• 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; Health; 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; response; stroke treatment; 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）： 冠状动脉和脑循环的血小板引起的闭塞性疾病的诊断，治疗和预防是VA患者护理任务的主要挑战。过度的血小板激活和招募是血小板诱导的闭塞性血管疾病的发病机理的基础。我们的建议重点是开发一种新的预防和治疗这些疾病的方法，即使用血小板调节性型 - 兴奋酶ATPDase/CD39/NTPDase1。 CD39通过代谢血栓形成的ATP和ADP从活化的血小板和其他细胞中释放出血液流动性的主要调节剂，以防止血栓形成。我们此前曾报道过验证冠状动脉疾病患者CD39活性的促血栓性改变。随后，我们检查了中风患者的白细胞CD39活性，发现这些受试者的淋巴细胞与对照组相比显示出ADPase/ATPase活性比的变化。这一观察结果导致了我们的假设，即CD39活性的改变可能导致中风易感性。该应用程序扩展了我们以前的研究，并专注于三个主要领域。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酶活性需要形成较高的分子量复合物，该酶通过需要裂解蛋白质的过程来形成在富含胆固醇的膜子域中局部局部的。我们计划充分确认和扩展这些观察结果，包括阐明CD39中的裂解部位以及裂解事件的亚细胞位置。我们还确定了我们最近发现的CD39剪接变体（V1-4）调节细胞表面的Apyrase活性。我们将阐明这些CD39替代剪接变体在CD39细胞内运输和裂解中的作用。我们还将确定CD39剪接变异的产生和翻译是否在组织和刺激特异性调节下。 CD39剪接变体调节质膜中酶活性CD39种的形成。这确定了细胞的副或抗血栓表型。其次，我们将进一步建立CD39活性，血小板反应性和中风的改变之间的关系。 w将把我们的中风研究扩展到3个不同的患者组：（1）隐态性中风患者（在没有临床病因学的年轻人群中中风）； （2）已验证阻塞性睡眠呼吸暂停的“中风前”患者； （3）经典的动脉粥样硬化中风患者。我们已经确定了对血小板激动剂的反应性提高以及血小板和白细胞激活标记的表达增加的隐性中风和中风的患者。这些患者的去鉴定的血液样本将进行处理，以分离血小板，淋巴细胞和中性粒细胞。然后，这些方法将通过几种方法进行分析，包括流式细胞术和我们新开发的广泛范围血小板聚集系统。在这些患者的血小板反应性阈值方面的深入分类中，我们对中风的发病机理的理解会增加。这也将使我们能够检验我们的假设，即隐源性中风在很大程度上可能代表了增强的血小板反应性障碍。第三，我们将通过流式细胞仪，定量实时PCR和DNA片段分析来确定中风患者白细胞中的CD39表面表达和剪接变异谱。这些研究将确定中风病因与CD39变体谱之间的关系。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起作用，并为此作用提供机械基础。我们提出的研究代表了一种多学科的方法，用于缺血性中风的发病机理，预防和治疗以及我们新发现的CD39剪接变体的调节方面。这项研究将增强我们对CD39作用机制的了解，并最终导致对患者血小板驱动的闭塞血管疾病的安全有效治疗的发展。