VASCULAR CONTROL OF BLOOD CELL REACTIVITY IN THROMBOSIS

冠状动脉、脑动脉和外周动脉中的血管损伤引起局部血小板活化、募集和血栓性闭塞，其逆转提出了主要的治疗挑战。在内皮细胞（EC）存在下，即使在类花生酸和一氧化氮不存在的情况下，血小板对激动剂也始终无反应。这一观察结果最终导致我们将内皮细胞CD 39/外膜ADP作为主要的血栓调节剂，CD 39快速代谢活化血小板释放的ADP，从而消除聚集和募集。开发了一种重组可溶形式的人CD 39，solCD 39，其在体外有效地阻断激动剂诱导的人血小板聚集，并在体内缩短小鼠的出血时间。CD 39-/-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，证明了CD 39在脑血栓调节中的关键作用。 这项合作将破译内源性CD 39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CD 39作为一种新的抗血栓形成剂。结构-功能研究将：开发关于CD 39活性位点的具体信息，以确定酶催化所需的氨基酸;绘制CD 39中有助于其三级结构的关键区域;确定糖基化对CD 39酶活性的贡献;确定solCD 39的生物物理和结构，以理解核苷酸去磷酸化的机制;产生CD 39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。将在⑶ 39-/-小鼠中研究内源性⑶ 39在微血管血栓形成中的作用，所述小鼠经受缺血性中风，用或不用solCD 39重建。还将在已建立的缺血性中风狒狒模型中研究SolCD 39作为潜在治疗剂。使用来自对照和CD 39-/-小鼠的内皮细胞，以及缺血小鼠和狒狒脑组织，将研究缺血或缺氧驱动的CD 39表达调节。该研究代表了一种多学科方法来理解CD 39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。这项合作基于令人信服的可行性数据和历史合作成功，将促进对CD 39血栓调节的理解，并导致血栓素质的新型治疗药物。

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.