FUNCTIONAL ANALYSIS OF VASCULAR NTPDASES

CD 39是ATP/核苷三磷酸的原型 二磷酸水解酶（ATPDase/NTPDase; EC 3.6.1.5）E型的家族成员 外核苷酸酶CD 39调节嘌呤能受体介导的信号传导， 在ATP和ADP水解之后，还促进 腺苷用于从细胞外腺嘌呤核苷酸进行细胞内转运。 尽管CD 39在细胞内受到调控并广泛表达， 血管外酶表达的功能意义， 内皮细胞不清楚。外核苷酸酶可能参与调控 通过影响血小板微血栓， 形成、内皮细胞活化或凋亡和全身嘌呤 体内平衡为了研究血管NTPD酶的功能意义， 研究人员已经开发了反义试剂、重组腺病毒和 通过同源重组产生CD 39缺陷型和无效型小鼠。这些 突变小鼠已被证明是完全可行的，但具有出血素质。 血小板功能减退，继发于选择性嘌呤能P2 Y1受体 脱敏，可以证明。与预计损失的 已经观察到这种血管血栓调节机制，即纤维蛋白沉积， 在血管系统的多个部位;突变小鼠也响应 对血管损伤不利，并且它们的心脏异种移植迅速失败。 CD 39在调节不同内皮细胞嘌呤受体中的作用 体外介导的作用和随后的体内血栓形成反应将是 在这个提案中进一步研究。血管对全身血小板的反应性 活化、缺血-再灌注损伤和异种移植物排斥将是 在选择的过表达NTPD酶的血管化鼠组织中评价 生化活性和CD 39缺陷的突变小鼠。复溶 使用可溶性NTPD酶的实验将平行进行。嘌呤能 CD 39影响血小板和细胞活化的机制似乎 与内皮组织型一氧化氮相关的那些不同 合成酶（eNOS），第二个阿司匹林不敏感的血栓调节系统。这些 途径可以是相加的或协同的，并将通过体外系统进行研究。 并通过调节CD 39小鼠中的NO活性。缺乏以下物质的小鼠的一代 eNOS和CD 39也将被进行。这些双淘汰赛可能是 存活的;然而，致死表型也将提供信息。这些实验 应该表明血管NTPD酶/CD 39的参与， 阐明与NOS系统的相互作用，在某些血管炎性 在人类疾病状态下观察到的疾病。

CD39 is the prototype ATP/ nucleoside triphosphate diphosphohydrolase (ATPDase/NTPDase; EC 3.6.1.5) member of a family of E-type ectonucleotidases. CD39 modulates purinergic receptor-mediated signaling, following the hydrolysis of ATP and ADP, but also facilitates generation of adenosine for intracellular transport from extracellular adenine nucleotides. Although CD39 appears to be regulated and widely expressed within the vasculature, the functional significance of ectoenzyme expression by the endothelium is unclear. Ectonucleotidases could be involved in the regulation of vascular inflammatory reactions by influencing platelet microthrombi formation, endothelial cell activation or apoptosis and systemic purine homeostasis. To study the functional significance of the vascular NTPDase, the investigators have developed antisense reagents, recombinant adenoviruses and generated CD39-deficient and null mice by homologous recombination. These mutant mice have been shown to be fully viable but have a bleeding diathesis. Platelet hypofunction, secondary to selective purinergic P2Y1 receptor desensitization, can be demonstrated. In keeping with the predicted loss of this vascular thromboregulatory mechanism, fibrin deposition has been observed within the vasculature at multiple sites; the mutant mice also respond adversely to vascular insults and their cardiac xenotransplants rapidly fail. The role of CD39 in modulating differing endothelial cell purinoreceptor mediated effects in vitro and consequent thrombotic reactions in vivo will be further examined in this proposal. Vascular responsiveness to systemic platelet activation, ischemia-reperfusion injury and xenograft rejection will be evaluated in selected vascularized murine tissues over-expressing NTPDase biochemical activtity and in mutant mice deficient in CD39. Reconstitution experiments, using soluble NTPDases, will be performed in parallel. Purinergic mechanisms by which CD39 influences platelet and cellular activation appear to differ from those associated with endothelial constitutive nitric oxide synthase (eNOS), a second aspirin-insensitive thromboregulatory system. These pathways may be additive or synergistic and will be studied by in vitro systems and by modulating NO-activity in CD39 mice. The generation of mice deficient in both eNOS and CD39 will also be undertaken. These double knock-outs may be viable; however, a lethal phenotype will be also informative. These experiments should indicate the involvement of the vascular NTPDase/CD39, and also elucidate interactions with NOS systems, in certain vascular inflammatory disorders observed in human disease states.