Biomedical and Functional Studies of CD39

冠状动脉、脑动脉或外周动脉的损伤会引起局部的血小板活化、再聚集和 血栓闭塞。预防和治疗血小板驱动的血栓形成是一种治疗方法 这是一项具有重大公共卫生影响的挑战。在内皮细胞存在的情况下，血小板 对激动剂无反应。这种无反应是由于内皮NTPDase1/CD39/ecto-ADPase， 它能迅速代谢活化的血小板释放的ATP和ADP。这取消了聚合和 招聘。重组可溶性人CD39(SolCD39)在体外阻断人血小板聚集，并 体外抑制猪和小鼠的血小板聚集。CD39缺失的小鼠表现出潜在的血栓前状态 表型对中风、心脏和肺血栓的易感性增加。这一点得到了缓解 在野生型和CD39缺失的小鼠中，通过输注solCD39，证明了它在 血栓调节。本项目的长期目标和具体目标包括：一、产生大型 用于生化和功能研究的重组可溶性CD39的数量，包括小鼠模型 血管损伤和闭塞，使用我们的CD39缺失小鼠。这些模型包括：a)经光速导线 在小鼠股动脉损伤模型中，我们将确定CD39在初始血小板-血小板活化中的作用。 依赖阶段，以及这种血管损伤的晚期增殖期；以及b)小鼠LAD结扎 导致心肌缺血/再灌注损伤。这将勾勒出solCD39在 短期内心肌缺血/再灌注(与solCD39缩小面积的效果相当 CD39基因缺失小鼠的脑梗塞)，以及长期状况(左心室重构和充血心脏 失败)。心脏和外周组织中嘌呤能和肾上腺素能神经传递的L调节 缺血时NTPDase1/CD39对交感神经系统的影响。这些研究将阐明 CD39在心脏交感神经传递中的作用，并证明了CD39在 周围交感神经-效应器连接。此外，他们将核实ATP和NE的溢出，以及 再灌注性心律失常的严重程度可以减轻。因此，CD39提供的心脏保护作用可以 在CD39缺失的动物体内恢复。在……里面。利用圆二色谱和核磁共振仪对可溶性apyrase的结构进行分析 蛋白质-核苷酸相互作用，结合动力学，并描绘了活性部位的重要方面 SolCD39--主要的血栓调节因子。拟议的研究代表了一项多学科和 了解CD39作为血液流动性和血液流动性主要调节因子的关键作用的协作方法 血小板介导的闭塞性血栓形成。这些研究基于令人信服的可行性数据和 历史性的合作成功，并将促进对NTPDase生物学和 血栓调节。这最终将成为治疗血栓性疾病的独特而新颖的治疗手段。

Injury to coronary, cerebral, or peripheral arteries evokes local platelet activation, recruitment, and thrombotic occlusion. Prevention and treatment of platelet-driven thrombus formation is a therapeutic challenge, with major public health implications. In the presence of endothelial cells, platelets are unresponsive to agonists. This unresponsiveness is due to endothelial NTPDase1/CD39/ecto-ADPase, which rapidly metabolizes ATP and ADP released from activated platelets. This abolishes aggregation and recruitment. Recombinant, soluble human CD39 (solCD39) blocks human platelet aggregation in vitro, and inhibits porcine and murine platelet aggregation ex vivo. CD39 null mice exhibit a latent prothrombotic phenotype with increased susceptibility to stroke and cardiac and pulmonary thrombosis. This is alleviated in both wild-type and CD39 null mice by infusion of solCD39, demonstrating its critical role in thromboregulation. Long-term objectives and specific aims of this project include: I. Generation of large quantities of recombinant soluble CD39 for biochemical and functional studies, including mouse models of vascular injury and occlusion, employing our CD39 null mice. These models include: a) Transluminal wire injury of mouse femoral artery, in which we will ascertain the role of CD39 in both the initial platelet- dependent stage, as well as the late proliferative stage of this vascular injury; and b} murine LAD ligation leading to myocardial ischemia/reperfusion injury. This will delineate the effectiveness of solCD39 in myocardial ischemia/reperfusion in short-term (comparable to effectiveness of solCD39 in reducing the size of brain infarct in CD39 null mice), as well as long-term conditions (LV remodeling and congestive heart failure). [L Modulation of purinergic and adrenergic neurotransmission in the heart and peripheral sympathetic nervous system by NTPDase1/CD39 during ischemia. These studies will elucidate the role of CD39 in sympathetic transmission in the heart and demonstrate the crucial modulatory role of CD39 at peripheral sympathetic neuro-effector junctions. Moreover, they will verify that overflow of ATP and NE, and severity of reperfusion arrhythmias, can be attenuated. Thus, cardioprotection afforded by CD39 can be restored in CD39 null animals. IN. Structural analyses of soluble apyrases using CD and NMR will establish protein-nucleotide interactions, kinetics of binding, and delineate important aspects of the active site of solCD39 - the principal thromboregulator. The proposed research represents a multidisciplinary and collaborative approach to understanding the critical role of CD39 as the prime regulator of blood fluidity and of platelet-mediated occlusive thrombosis. The studies are based on compelling feasibility data and historical collaborative success, and will advance the understanding of NTPDase biology and thromborequlation. This should culminate in a unique and novel therapeutic aqent for thrombotic diatheses.