Mechanism of a novel approach for platelet cold storage

血小板冷藏新方法的机制

• 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
• 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

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.

摘要 冷藏缩短了血小板的寿命，因为它会导致细胞骨架重排、解体和死亡。 唾液酸化糖蛋白Ib(GPIB)聚集、形成微区、脱落和诱导线粒体 依赖活性氧簇(ROS)和细胞凋亡，可能导致炎症反应 脆弱的患者群体。宿主对聚集性糖蛋白(GP)的识别导致血小板 吞噬和清除。因此，冷藏的血小板仅被允许用于 创伤患者的治疗，而不是干细胞移植接受者的预防或治疗 血液科/肿瘤科患者。Rho家族GTP酶RHOA和RAC1是 细胞骨架重排，并已被证明控制脂筏的形成和组成； Rho GTP酶活性变化可能影响血小板膜脂筏组装 膜糖蛋白的翻译修饰，包括GPIB和线粒体ROS增加 和细胞凋亡活性。我们的初步、提交和发布的数据使用遗传和 药理学手段表明可逆的RHOA GTP酶抑制导致肌球蛋白的抑制 富含蛋白质的脂筏非蛋白依赖的形成和内化的活性和预防 活性糖基转移酶(GT)和GPIB。抑制RhoA GTP酶可阻止代谢 重编程效应，并允许维持糖酵解通量和线粒体依赖 呼吸作用和ROS的产生。重要的是，我们进一步证明了小鼠、人类和恒河猴- 猕猴血小板，在有铅存在的冷藏条件下保存长达14天 RhoA抑制剂G04可以将存活功能保持在与室温保存相似的水平 并在体内保留了血小板的止血活性，并具有抗氧化剂的部分作用 G04。我们假设RHOA在冷藏期间通过 肌动球蛋白活性、囊泡运输和线粒体呼吸的调节。我们将首先确定 RHOA调节脂筏形成、糖蛋白聚集和内吞作用的机制 冷藏后的血小板。我们还将确定药物抑制RHOA的结果 分析在预防长期冷藏血小板代谢和线粒体损伤中的作用 长期储存对线粒体活性的影响及其与RHOA之间的串扰 掌握代谢调节剂AMPK在调节血小板代谢和吞丝分裂中的作用。我们的研究将 为新方法的翻译价值提供机制和严格的原则性证明 到冷藏的血小板储存。