Thromboregulation by Endothelial Cells: Role of CD39

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患者护理使命的主要挑战。血小板过度活化和募集是血小板诱导的闭塞性血管病发病机制的基础。我们的建议侧重于开发一种新的方法来预防和治疗这些疾病，即使用血栓调节性外腺苷三磷酸双磷酸酶ATPDase/CD 39/NTPDase 1。CD 39通过代谢从活化的血小板和其它细胞释放的促血栓形成ATP和ADP来作为血液流动性的主要调节剂起作用，以防止血栓形成。我们以前曾报道过经证实的冠状动脉疾病患者中CD 39活性的血栓前改变。随后，我们检查了中风患者的白细胞CD 39活性，发现与对照组相比，这些受试者的淋巴细胞显示出ADDRESS/ATP酶活性比值的变化。这一观察结果导致我们的假设，即CD 39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以前的研究，并集中在三个主要领域。首先，我们将定义CD 39表达和活性调节的分子生物学机制，以及由此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CD 39酶活性需要形成更高的分子量的复合物定位在富含胆固醇的膜亚结构域通过一个过程，需要裂解的蛋白质。我们计划充分证实和扩展这些观察结果，包括阐明CD 39中的切割位点和切割事件的亚细胞位置。我们还确定了我们最近发现的CD 39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CD 39选择性剪接变异体在调节CD 39细胞内运输和切割中的作用。我们还将确定CD 39剪接变体的产生和翻译是否受组织和刺激特异性调节。CD 39剪接变体调节质膜中酶活性CD 39物质的形成。这决定了细胞的促血栓形成或抗血栓形成表型。其次，我们将进一步建立CD 39活性、血小板反应性和卒中之间的关系。W将我们的卒中研究扩展到3个不同的患者组：（1）隐源性卒中患者（无临床病因的年轻人群中的卒中）;（2）经证实为阻塞性睡眠呼吸暂停的“卒中前”患者;（3）经典动脉粥样硬化血栓性卒中患者。我们已经确定了隐源性卒中和卒中倾向患者，他们对血小板激动剂的反应性增加，血小板和白细胞活化标志物的表达增加。将处理这些患者的去识别血液样本，以分离血小板、淋巴细胞和中性粒细胞。然后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宽范围血小板聚集系统。根据血小板反应性阈值对这些患者进行深入分类将增加我们对卒中发病机制的理解。这也将使我们能够验证我们的假设，即隐源性卒中可能在很大程度上代表了血小板反应性增强的疾病。第三，我们将通过流式细胞术、实时定量PCR和DNA片段分析来确定中风患者与对照组白细胞中的CD 39表面表达和剪接变体谱。这些研究将建立卒中病因与CD 39变异谱之间的关系。这将进一步验证我们的假设，即改变的CD 39活性在隐源性卒中中发挥作用，并为这种作用提供机制基础。我们提出的研究代表了缺血性卒中发病机制、预防和治疗以及我们新发现的CD 39剪接变异体的调控方面的多学科方法。这项研究将提高我们对CD 39作用机制的认识，并最终导致开发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。

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.