Biomedical and Functional Studies of CD39

冠状动脉、脑动脉或外周动脉损伤会引起局部血小板活化、募集和 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 挑战，对公共卫生产生重大影响。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 造成的， 它快速代谢从活化的血小板中释放的 ATP 和 ADP。这废除了聚合并且 招聘。重组可溶性人 CD39 (solCD39) 可在体外阻断人血小板聚集，并且 离体抑制猪和鼠血小板聚集。 CD39缺失小鼠表现出潜在的促血栓形成 中风、心肺血栓形成的易感性增加的表型。这样就缓解了 通过输注 solCD39 在野生型和 CD39 缺失小鼠中发挥作用，证明了其在 血栓调节。本项目的长期目标和具体目标包括： 一、产生大型 大量重组可溶性 CD39 用于生化和功能研究，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) 腔内导线 小鼠股动脉损伤，其中我们将确定 CD39 在初始血小板- 依赖性阶段，以及这种血管损伤的增殖后期；和b}鼠LAD结扎 导致心肌缺血/再灌注损伤。这将描述 solCD39 在 短期心肌缺血/再灌注（与 solCD39 减少心肌大小的有效性相当） CD39 缺失小鼠的脑梗塞），以及长期病症（左心室重塑和充血性心脏 失败）。 [L 调节心脏和外周的嘌呤能和肾上腺素能神经传递 缺血期间 NTPDase1/CD39 影响交感神经系统。这些研究将阐明其作用 CD39 在心脏交感神经传递中的作用，并证明 CD39 在 外周交感神经效应器连接。此外，他们将验证 ATP 和 NE 的溢出，以及 再灌注心律失常的严重程度可以减弱。因此，CD39 提供的心脏保护作用可以是 在 CD39 缺失的动物中恢复。在。使用 CD 和 NMR 对可溶性腺苷三磷酸双磷酸酶进行结构分析将建立 蛋白质-核苷酸相互作用、结合动力学，并描绘了活性位点的重要方面 solCD39 - 主要的血栓调节剂。拟议的研究代表了多学科和 合作方法来了解 CD39 作为血液流动性的主要调节剂的关键作用 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，并将增进对 NTPDase 生物学和 血栓调节。这应该最终产生一种独特且新颖的血栓素质治疗剂。

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.