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粘附ATP受损：一种新的输血损伤红细胞（rbc）的输血是一种常用且昂贵的治疗方法。然而，在一些病人群体中，例如危重病人，输血后的临床结果令人失望，并且可能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变化已经得到了很好的证实，但很少或根本没有与输血接受者的不良临床结果可靠地联系在一起。我们已经证明，储存逐渐损害了人类红细胞释放ATP的能力，并且这种损害促进了体外红细胞与内皮细胞（ECs）的粘附，以及体内红细胞在肺中的隔离，并在小鼠输血的新模型中导致氧合恶化。这种新型的促粘附性红细胞“储存损伤”是由红细胞表面粘附受体ICAM-4 （LW）和内皮整合素介导的，并通过共输注真正的ATP类似物来预防。我们假设，输血的人红细胞中储存诱导的ATP释放不足，通过红细胞内皮粘附的局部损伤后遗症，损害O2的摄取和传递，有助于宿主的病理生理。我们将通过实现以下具体目标来验证这一假设：1)确定释放的ATP抑制红细胞粘附内皮细胞的机制。我们将通过研究panx1缺陷红细胞输出ATP的能力及其粘附内皮的倾向来扩展我们的新发现，即药物抑制panx1（阻断红细胞ATP释放）也促进红细胞粘附。我们还将通过测试特异性嘌呤能拮抗剂的影响以及ATP敏感激活相关粘附受体的作用，来确定红细胞来源的ATP抗粘附作用的机制。2)确定a)在储存的红细胞中补充ATP和b)在体外和体内输血时促进或模拟ATP释放的策略的有效性。我们将在体外内皮细胞粘附试验和我们最近开发的小鼠人红细胞输血模型中实现这一目标。我们将在体外和体内研究储存后对红细胞ATP补充（“返青”）的影响，并研究共输注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粘附是如何被ATP的释放调节的初步理解，ATP是一种可能具有更广泛意义的新功能，如镰状、糖尿病和疟疾感染的红细胞内皮粘附增加。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研究结果在输血医学临床研究中的转化。这些研究的结果预计将告知策略的合理设计，以改善风险-效益平衡在关键的红细胞输血

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.