FUNCTIONAL ANALYSIS OF VASCULAR NTPDASES

CD39是三磷酸腺苷/核苷三磷酸的原型 二磷酸水解酶(ATPDase/NTPDase；EC 3.6.1.5)是E-型家族的成员 胞外核苷酸酶。CD39调节嘌呤能受体介导的信号转导， 在ATP和ADP水解酶之后，还促进了 细胞外腺嘌呤核苷酸在细胞内转运的腺苷。 尽管CD39似乎在体内受到调控并广泛表达 血管系统，胞外酶表达的功能意义 血管内皮细胞尚不清楚。胞外核苷酸酶可能参与了这一调控。 影响血小板微血栓的血管炎症反应 形成、内皮细胞激活或凋亡与全身性嘌呤 动态平衡。为了研究血管NTPDase的功能意义， 研究人员已经开发出反义试剂、重组腺病毒和 通过同源重组获得CD39缺陷和缺失小鼠。这些 突变小鼠已被证明是完全存活的，但具有出血的素质。 选择性嘌呤能P2Y1受体继发的血小板功能减退 脱敏，可以证明。与预计的损失保持一致 这种血管血栓调节机制，纤维蛋白沉积已被观察到 在多个位置的血管系统内；突变小鼠也会对 不利的血管损伤和他们的心脏异种移植迅速失败。 CD39在调节不同内皮细胞嘌呤受体中的作用 体外介导性效应和随之而来的体内血栓反应 在这项提案中进一步研究。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排斥反应 在部分高表达NTPDase的血管化小鼠组织中的评价 CD39基因缺失突变小鼠的生化活性。重建 使用可溶的NTPDase的实验将并行进行。嘌呤能 CD39影响血小板和细胞活化的机制似乎 不同于内皮细胞构成的一氧化氮 合成酶(ENOS)，第二个阿司匹林不敏感的血栓调节系统。这些 途径可能是相加的或协同的，并将通过体外系统进行研究 并通过调节CD39小鼠体内的NO活性。缺铁性贫血小鼠的产生 ENOS和CD39也将被承接。这些双淘汰赛可能是 可行；然而，致命的表型也将提供信息。这些实验 应提示血管NTPDase/CD39的参与，以及 阐明在某些血管炎症中与一氧化氮合酶系统的相互作用 在人类疾病状态下观察到的紊乱。

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.