THROMBOREGULATORY ROLE OF CD39 (ECTOADPASE) IN STROKE

冠状动脉、脑动脉和外周动脉的血管损伤会引起局部的血小板活化、重新聚集和血栓闭塞，其逆转是一个主要的治疗挑战。在内皮细胞(EC)存在的情况下，即使在没有二十烷类化合物和一氧化氮的情况下，血小板也始终对激动剂没有反应。我们将内皮细胞CD39/ECTO-ADPase定性为主要的血栓调节因子。CD39迅速代谢活化的血小板释放的ADP，从而取消聚集和募集。人CD39的一种重组可溶性形式solCD39在体外能有效阻断激动剂诱导的人血小板聚集，并在体内延长小鼠的出血时间。CD39-/-小鼠表现出潜在的血栓前表型，增加了对缺血性脑血栓形成和损伤的易感性，显示了CD39在脑血栓调节中的关键作用。这一合作将破译内源性CD39在抑制缺血驱动的血栓形成中的关键生物学作用，并将开发CD39作为一种新的抗血栓药物。结构功能研究将：开发有关CD39活性位点的特定信息，以确定酶催化所需的氨基酸；绘制CD39中影响其三级结构的关键区域；确定糖基化对CD39酶活性的贡献；确定solCD39的生物物理和结构性质，以了解核苷酸去磷酸化的机制；产生CD39胞外域的多价衍生物，以确定寡聚介导的酶活性促进的结构基础。内源性CD39在微血管血栓形成中的作用将在接受或不接受solCD39重建的缺血性中风的CD39-/-小鼠中进行研究。SolCD39还将作为一种潜在的治疗剂在已建立的狒狒缺血性中风模型中进行研究。利用对照组和CD39-/-小鼠的内皮细胞，以及缺血小鼠和恒河猴脑组织，将研究缺血或低氧驱动的CD39表达的调节。这项研究代表了一种多学科的方法来理解CD39作为血小板介导的闭塞性动脉血栓形成的主要调节因子的关键作用。这一合作基于令人信服的可行性数据和历史上的合作成功，将促进对CD39血栓调节的理解，并导致一种新的血栓疾病治疗剂。

Vascular injury in coronary, cerebral, and peripheral arteries evokes local platelet activation, recruitment and thrombotic occlusion, reversal of which presents a major therapeutic challenge. Platelets are consistently unresponsive to agonists in the presence of endothelial cells (EC), even in the absence of eicosanoids and nitric oxide. This observation culminated in our characterization of endothelial cell CD39/ecto-ADPase as the prime thromboregulator. CD39 rapidly metabolizes ADP released from activated platelets, thereby abolishing aggregation and recruitment. A recombinant, soluble form of human CD39, solCD39, was developed which potently blocked agonist-induced human platelet aggregation in vitro, and prolongs bleeding time in mice in vivo. CD39 -/- mice exhibited a latent prothrombotic phenotype with increased susceptibility to ischemic cerebral thrombosis and injury, demonstrating a critical role for CD39 in cerebral thromboregulation. This collaboration will decipher the pivotal biological role of endogenous CD39 in inhibiting ischemic-driven thrombosis, and will develop CD39 as a novel antithrombotic agent. Structure-function studies will: Develop specific information about the CD39 active site, to identify which amino acids are required for enzyme catalysis; Map critical regions in CD39 which contribute to its tertiary structure; Establish the contribution of glycosylation to CD39 enzymatic activity; Determine biophysical and structural properties of solCD39 to comprehend mechanisms of nucleotide dephosphorylation; Generate multivalent derivatives of the extracellular domain of CD39 to define the structural basis for oligomerization-mediated promotion of enzymatic activity. The role of endogenous CD39 in microvascular thrombosis will be studied in CD39 -/- mice, subjected to ischemic stroke with or without reconstitution with solCD39. SolCD39 will also be investigated as a potential therapeutic agent in an established baboon model of ischemic stroke. Using endothelial cells from control and CD39 -/- mice, as well as ischemic murine and baboon brain tissue, ischemia- or hypoxia-driven modulation of CD39 expression will be studied. The research represents a multidisciplinary approach to understanding the critical role of CD39 as the prime regulator of platelet-mediated occlusive arterial thrombosis. This collaboration, based on compelling feasibility data and historical collaborative success, will advance the understanding of CD39 thromboregulation, and lead to a novel therapeutic agent for thrombotic diatheses.