Mechanism of a novel approach for platelet cold storage

血小板冷藏新方法的机制

基本信息

批准号：
10682608
负责人：
Jose A Cancelas
金额：
$ 60.74万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-08-15 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10682608
关键词：
Actomyosin Antioxidants Apoptosis Apoptotic Asialoglycoproteins Aspirin Biochemical Blood Banks Blood Platelets Blood Transfusion Clathrin Complex Congenic Mice Cryopreservation Cytoskeletal Modeling Cytoskeleton Data Development Dreams Effectiveness Endocytosis Exocytosis Family Generations Genetic Glycoprotein Ib Glycoproteins Goals Guanosine Triphosphate Phosphohydrolases Hematology Hematopoietic stem cells Hemorrhage Hemostatic Agents Hepatocyte Human Inflammatory Inflammatory Response Lead Lesion Long-Term Effects Longevity Macaca mulatta Macrophage Maintenance Membrane Membrane Glycoproteins Membrane Lipids Membrane Microdomains Metabolic Metabolism Methods Mitochondria Molecular Mus Myosin ATPase Oncology Outcome Oxidative Phosphorylation Oxygen Consumption Patients Phagocytosis Phenocopy Platelet Transfusion Play Post-Translational Protein Processing Prevention Process Production Prophylactic treatment Protons Publishing RHOA gene Reactive Oxygen Species Refrigeration Regulation Respiration Role Signal Pathway Signal Transduction Stem cell transplant Stress Supportive care Temperature Transferase Transfusion Transplant Recipients Transplantation Trauma patient Vesicle cold temperature conditioning cytokine glycosyltransferase humanized mouse improved in vivo inhibitor mitochondrial dysfunction novel strategies patient population pharmacologic platelet storage platelet storage lesion preservation prevent receptor response rho rho GTP-Binding Proteins small molecule inhibitor stem cells trafficking transfusion medicine vesicle transport

项目摘要

ABSTRACT Refrigerated storage reduces platelet life-span because it causes cytoskeletal rearrangements, de- sialylated glycoprotein-Ib (GPIb) to cluster, form microdomains, shed and induces mitochondrial dependent reactive oxygen species (ROS) and apoptosis, which may result in inflammatory response in vulnerable patient populations. Recognition by host of clustered glycoproteins (GP) results in platelet phagocytosis and clearance. As a consequence, cold stored platelets are only allowed for use in trauma patient therapy and not for prophylaxis or treatment of stem cell transplant recipients and hematology/oncology patients. The Rho family GTPases RHOA and RAC1 are central regulators of cytoskeletal rearrangements, and have been shown to control lipid raft formation and composition; changes in Rho GTPase activities may influence platelet membrane lipid raft assembly, post- translational modifications of membrane glycoproteins, included GpIb and increased mitochondrial ROS and apoptotic activity. Our preliminary, submitted and published data using genetic and pharmacological means show that reversible RHOA GTPase inhibition results in an inhibition of myosin activity and prevention of clathrin-independent formation and internalization of lipid rafts enriched in active glycosyl-transferases (GT) and GPIb. RHOA GTPase inhibition prevents the metabolic reprogramming effect and allows the maintenance of glycolytic flux and mitochondrial dependent respiration and ROS production. Importantly, we further demonstrate that murine, human and Rhesus- macaque platelets, when stored in refrigerated conditions for up to 14 days in the presence of a lead RHOA inhibitor, G04, can retain survival function at a level similar to that of room-temperature stored platelets and retain hemostatic activity in vivo, and an antioxidant phenocopies some of the effects of G04. We hypothesize that RHOA controls the process of GP clustering during cold storage through the regulation of actomyosin activity, vesicle trafficking and mitochondrial respiration. We will first identify the mechanism by which RHOA regulates lipid raft formation, GP clustering and endocytosis in platelets upon refrigeration. We will also determine the outcomes of pharmacologic inhibition of RHOA in preventing the metabolic and mitochondrial damage of long-term cold stored platelets by analyzing the effect of long-term storage on mitochondrial activity and the crosstalk between RHOA and the master metabolic regulator AMPK in regulating platelet metabolism and mitophagy. Our studies will provide the mechanism and a stringent proof-of-principle for the translational value of a novel approach to refrigerated platelet storage.

抽象的冷藏会缩短血小板的寿命，因为它会导致细胞骨架重排，唾液酸化糖蛋白-Ib (GPIb) 聚集、形成微结构域、脱落并诱导线粒体依赖的活性氧（ROS）和细胞凋亡，这可能导致炎症反应弱势患者群体。宿主对聚集糖蛋白 (GP) 的识别导致血小板吞噬作用和清除作用。因此，冷藏血小板仅允许用于创伤患者治疗，不适用于干细胞移植接受者的预防或治疗，以及血液学/肿瘤学患者。 Rho 家族 GTPases RHOA 和 RAC1 是细胞骨架重排，并已被证明可以控制脂筏的形成和组成； Rho GTPase 活性的变化可能会影响血小板膜脂筏的组装，膜糖蛋白的翻译修饰，包括 GpIb 和增加的线粒体 ROS 和细胞凋亡活性。我们使用遗传和药理学方法表明，可逆的 RHOA GTPase 抑制会导致肌球蛋白的抑制富含网格蛋白的脂质筏的形成和内化的活性和预防活性糖基转移酶 (GT) 和 GPIb。 RHOA GTPase 抑制可防止代谢重编程效应并允许维持糖酵解通量和线粒体依赖性呼吸和 ROS 产生。重要的是，我们进一步证明小鼠、人类和恒河猴- 猕猴血小板，在存在铅的情况下冷藏保存长达 14 天 RHOA抑制剂G04可以将存活功能保留在与室温保存相似的水平血小板并在体内保留止血活性，并且抗氧化剂表型具有某些作用 G04。我们假设 RHOA 通过以下方式控制冷存储期间 GP 聚类的过程：肌动球蛋白活性、囊泡运输和线粒体呼吸的调节。我们首先会识别 RHOA 调节脂筏形成、GP 聚集和内吞作用的机制冷藏后的血小板。我们还将确定 RHOA 药物抑制的结果通过分析防止长期冷藏血小板的代谢和线粒体损伤长期储存对线粒体活性的影响以及RHOA和线粒体之间的串扰主要代谢调节因子 AMPK 调节血小板代谢和线粒体自噬。我们的研究将为新方法的转化价值提供机制和严格的原理验证冷藏血小板储存。