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，其逆转提出了重大的治疗挑战。在内皮细胞 (EC) 存在的情况下，即使没有类二十烷酸和一氧化氮，血小板始终对激动剂无反应。这一观察最终导致我们将内皮细胞 CD39/ecto-ADPase 作为主要的血栓调节剂进行表征。CD39 快速代谢从活化血小板中释放的 ADP，从而消除聚集和募集。开发了人 CD39 的重组可溶形式 solCD39，它可以在体外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-/-小鼠表现出潜在的促血栓形成表型，对缺血性脑血栓形成和损伤的易感性增加，表明CD39在脑血栓调节中的关键作用。 此次合作将破译内源性 CD39 在抑制缺血性血栓形成中的关键生物学作用，并将 CD39 开发为新型抗血栓剂。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 中有助于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，以了解核苷酸去磷酸化的机制；生成 CD39 胞外结构域的多价衍生物，以定义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CD39-/-小鼠中进行研究，这些小鼠在没有用solCD39重建的情况下遭受缺血性中风。 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。使用来自对照小鼠和 CD39-/- 小鼠的内皮细胞，以及缺血性小鼠和狒狒脑组织，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方法，以了解 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。此次合作基于令人信服的可行性数据和历史合作成功，将增进对 CD39 血栓调节的理解，并开发出一种治疗血栓素质的新型治疗剂。

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 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.