Biochemical and functional studies of CD39

冠状动脉和脑动脉的损伤促进了血小板的激活、重新聚集和血栓闭塞。预防和逆转血小板血栓形成的信号是一项重大的治疗挑战，对公众健康具有重要影响。在内皮细胞(EC)存在的情况下，即使当二十烷类化合物和一氧化氮的产生被阻断时，血小板对激动剂也没有反应。这是由于EC CD39/ecto-ADPase能迅速代谢活化的血小板释放的ADP，从而消除血小板聚集和募集的信号。重组可溶性人CD39(SolCD39)在体外能有效阻断激动剂诱导的人血小板聚集，并能延长猪和小鼠的体内出血时间。我们的CD39小鼠表现出潜在的血栓前表型，增加了对损伤诱导的血栓形成的敏感性，这可以通过输注solCD39来缓解。因此，CD39在血栓调节中起着关键作用。具体目标包括：[1]利用我们的回交CD39缺失小鼠在体内鉴定CD39的抗血栓形成特性，并研究一种新型的、可能更具促血栓作用的CD39-Annexin II双敲除。检测CD39和其他EC血栓调节因子(前列环素和一氧化氮)在正常EC和经人端粒酶逆转录酶(HTERT)转基因永生化EC体外老化过程中的表达。[2]我们在豚鼠心脏突触小体中发现的胞外核苷酸酶活性的表征，并检测了缺血/再灌注对其表达的影响。核苷酸酶活性降低是否导致去甲肾上腺素释放增加？添加solCD39是否影响正常和缺血豚鼠心脏的突触体信号？[3]solCD39的构象分析，包括翻译后修饰对其稳定性和蛋白水解性的影响，以及CD和核磁共振研究，以确定solCD39与核苷酸的相互作用，结合动力学以及关于solCD39活性部位的构象信息。我们合作的体内和体外实验的最终健康相关目标是开发CD39作为一种治疗剂，用于阻断因血小板过度激活而导致的冠状动脉和脑血管疾病患者的血小板激活和募集信号。

Injury to coronary and cerebral arteries promotes platelet activation, recruitment and thrombotic occlusion. Prevention and reversal of the signals for platelet thrombus formation is a major therapeutic challenge with important implications for public health. In the presence of endothelial cells (EC), platelets are unresponsive to agonists, even when eicosanoid and nitric oxide production are blocked. This is due to EC CD39/ecto-ADPase, which rapidly metabolizes ADP released from activated platelets, thereby deleting the signal for platelet aggregation and recruitment. Recombinant soluble human CD39 (solCD39) potently blocks agonist-induced human platelet aggregation in vitro, and prolongs porcine and murine bleeding times in vivo. Our CD39 mice exhibits exhibit a latent pro-thrombotic phenotype with increased susceptibility to injury-induced thrombosis, which is alleviated by infusion of solCD39. Thus CD39 plays a critical role in thromboregulation. Specific aims include: [1] Characterization of in vivo anti-thrombotic properties of CD39 using our backcrossed CD39 null mice, combined with studies of a novel, potentially even more pro-thrombotic CD39-annexin II double knockout. Expression of CD39 and other EC thromboregulators (prostacyclin and nitric oxide) will be examined during in vitro aging of normal EC, and EC immortalized by transfection with human telomerase reverse transcriptase (hTERT). [2] Characterization of the ecto- nucleotidase activity we identified in guinea-pig cardiac synapotosomes, and examination of the effects of ischemia/reperfusion on its expression. Does decreased nucleotidase activity lead to enhanced norepinephrine release? Does added solCD39 affect synaptosomal signaling in normal and ischemic guinea-pig heart? [3] Conformational analyses of solCD39, including effects of post-translational modifications on its stability and resistance to proteolysis, as well as CD and NMR studies to determine solCD39- nucleotide interactions, kinetics of binding as well as conformational information concerning the active site of solCD39. The ultimate health-related goal of our collaborative in vivo and in vitro experiments is to develop CD39 as a therapeutic agent that will block signals for platelet activation and recruitment in patients with coronary and cerebral vascular disorders induced by excessive platelet activation