Impaired release of antiadhesive ATP from stored RBCs: a novel transfusion lesion

描述(由申请人提供)：从储存的红细胞中释放抗黏附的ATP受阻：一种新的输血损伤红细胞输注(RBCs)是一种经常使用且昂贵的治疗方法。然而，在一些患者群体中，例如危重患者，输血后的临床结果令人失望，而且可能会随着红细胞储存时间的延长而恶化。储存红细胞的生化和功能改变已得到很好的证实，但很少或没有可靠的证据与输血接受者的不良临床结局有关。我们已经证明，储存逐渐损害了人RBC释放ATP的能力，这种损害在体外促进了RBC与内皮细胞(ECs)的黏附，并在体内促进了RBC在肺内的隔离，并在一种新的小鼠输血模型中加剧了氧合。这种新的前黏附性红细胞“储存损伤”是由红细胞表面黏附受体ICAM-4(LW)和内皮细胞整合素介导的，并通过共同输注真正的ATP类似物来预防。我们假设，储存诱导的人红细胞ATP释放不足通过红细胞内皮细胞黏附的局部损伤后遗症，影响氧的摄取和输送，参与宿主的病理生理学。我们将通过实现这些特定的目标来验证这一假设：1)确定释放的ATP抑制红细胞与内皮细胞黏附的机制。我们将通过研究Panx1缺陷的红细胞输出ATP的能力及其与内皮的黏附倾向来扩展我们的新发现，即药物抑制Panx1(阻断RBC ATP释放)也促进RBC黏附。我们还将通过检测特定的嘌呤能拮抗剂的影响和ATP敏感激活相关黏附受体的作用来确定RBC来源的ATP的抗黏附作用的机制。2)确定a)在储存的红细胞中补充ATP和b)在体外和体内输注时促进或模拟ATP释放的策略的有效性。我们将在体外的内皮细胞黏附试验和我们最近开发的小鼠人-红细胞输注模型中实现这一目标。将在体外和体内确定保存后红细胞的ATP补充(“复壮”)的影响，也将确定共输注ATP类似物的影响。这些新的研究有望加速我们对正常红细胞的黏附如何受ATP释放的调节的初步理解，这一新的功能可能具有更广泛的意义，如在镰刀形红细胞、糖尿病和疟疾感染的红细胞增加内皮黏附方面。在小鼠模型中检测人(和小鼠)红细胞粘附性的创新方法有望加速我们的发现转化为输血医学的临床研究。这些研究的结果有望为合理设计策略以改善危重患者红细胞输注的风险-收益平衡提供参考。 疾病患者和其他贫血患者。 公共卫生相关性：输注红细胞(RBC)治疗贫血在许多患者群体中并不能带来好处，可能会增加一些死亡和其他问题(如呼吸衰竭)的风险。这个项目的重点是储存引起的红细胞释放信号分子ATP的能力下降，以及这些渐进变化的功能后果。特别是，我们调查了储存的红细胞损害ATP输出与红细胞粘在血管上而不是顺畅流过的有害倾向之间的联系，以便在肺中摄取氧气并将其输送到身体。然后，我们研究这些缺陷是否可以在人红细胞和输血小鼠身上得到纠正。

DESCRIPTION (provided by applicant): Impaired release of anti-adhesive ATP from stored RBCs: a novel transfusion lesion Transfusion of red blood cells (RBCs) is a frequently administered and costly therapy. Yet in some patient populations - the critically ill, for example clinical outcomes after transfusion are disappointing, and may worsen as a function of the duration of RBC storage. Biochemical and functional changes in banked RBCs are well established, but few or none are credibly linked to the adverse clinical outcomes in transfusion recipients. We have demonstrated that storage progressively impairs the ability of human RBC to release ATP, and that this impairment promotes the adhesion of RBCs to endothelial cells (ECs) in vitro, and sequestration of RBCs in the lung in vivo, with worsened oxygenation in a novel model of transfusion in mice. This novel proadhesive RBC "storage lesion" is mediated by the RBC-surface adhesion receptor ICAM-4 (LW) and endothelial ¿v¿3 integrin, and is prevented by co-infusion of an authentic ATP analog. We hypothesize that the storage- induced deficiency in ATP release from transfused human RBCs contributes to pathophysiology in the host through the locally injurious sequelae of endothelial adhesion of RBCs, compromising O2 uptake and delivery. We will test this hypothesis by accomplishing these Specific Aims: 1) Determine the mechanism by which released ATP inhibits RBC adhesion to endothelial cells. We will extend our novel findings that pharmacological inhibition of panx1 (that blocks RBC ATP release) also promotes RBC adhesion by studying the ability of panx1-deficient RBCs to export ATP and their propensity to adhere to endothelium. We will also determine the mechanism of anti-adhesive effects of RBC-derived ATP by testing the influence of specific purinergic antagonists and the role of ATP-sensitive activation of the relevant adhesion receptors. 2) Determine the effectiveness of strategies to a) replete ATP in stored RBCs and b) promote or mimic ATP-release in vitro and upon transfusion in vivo. We will achieve this aim in endothelial cell adhesion assays in vitro and in the murine human-RBC transfusion model we have recently developed. The influence of post-storage ATP repletion ("rejuvenation") of RBCs will be determined in vitro and in vivo, and the influence of co-infusion of ATP analogs will also be determined. These novel studies are expected to accelerate our nascent understanding of how the adhesion of normal RBCs is modulated by the release of ATP, a novel function likely to have broader significance, as in the increased endothelial adhesion of sickle, diabetic and malaria-infected RBCs. The innovative approach of examining the adhesion of both human (and mouse) RBCs in a murine model is expected to accelerate the translation of our findings toward clinical investigation in transfusion medicine. The results of these studies are expected to inform the rational design of strategies to improve the risk-benefit balance for RBC transfusion in critically ill and other anemic patients. PUBLIC HEALTH RELEVANCE: Transfusion of red blood cells (RBCs) for anemia fails to provide a benefit in many patient groups, and may raise the risk in some of death and other problems such as respiratory failure. This project focuses on storage- induced declines in the RBC's ability to release the signaling molecule ATP, and the functional consequences of these progressive changes. In particular, we investigate the link between impaired ATP export by stored RBCs and the harmful tendency for RBCs to stick to blood vessels rather than flowing through smoothly in order to pick up oxygen in the lungs and deliver it to the body. We then investigate whether these deficiencies can be corrected in human RBCs, and in transfused mice.