VASCULAR CONTROL OF BLOOD CELL REACTIVITY IN THROMBOSIS

冠状动脉、脑动脉和外周动脉的血管损伤引起局部血小板活化、募集和血栓闭塞，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，血小板始终对激动剂无反应，即使在没有类二十烷和一氧化氮的情况下也是如此。这一观察结果在内皮细胞CD39/ecto-ADPase作为主要血栓调节剂的表征中达到顶峰。CD39迅速代谢活化血小板释放的ADP，从而消除聚集和募集。一种重组的、可溶的人CD39， solCD39，在体外能有效阻断激动剂诱导的人血小板聚集，并在体内延长小鼠出血时间。CD39-/-小鼠表现出潜在的促血栓表型，对缺血性脑血栓形成和损伤的易感性增加，表明CD39在脑血栓调节中起关键作用。这项合作将破译内源性CD39在抑制缺血驱动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活性位点的具体信息，以确定酶催化所需的氨基酸；绘制CD39中与三级结构相关的关键区域；确定糖基化对CD39酶活性的贡献；确定solCD39的生物物理和结构，以了解核苷酸去磷酸化的机制；生成CD39细胞外结构域的多价衍生物，以确定寡聚化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CD39-/-小鼠中进行研究，这些小鼠遭受缺血性卒中，没有用solCD39重建。SolCD39还将作为一种潜在的治疗药物在已建立的狒狒缺血性中风模型中进行研究。使用来自对照和CD39-/-小鼠的内皮细胞，以及缺血小鼠和狒狒的脑组织，将研究缺血或缺氧驱动的CD39表达调节。该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剂的关键作用。此次合作，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，并导致一种新的治疗血栓形成的药物。

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 pro-thrombotic 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 ischemia-driven thrombosis, and will develop CD39 as a novel anti-thrombotic 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 structure of solCD39 to comprehend mechanisms of nucleotide dephosphorylation; Generate multivalent derivatives of the extracellular domain of CD39 to define the structure 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 our 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 multi-disciplinary 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 diathesis.