Mechanism of a novel approach for platelet cold storage

摘要 冷藏会缩短血小板寿命，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 依赖性活性氧（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以下情况： 创伤患者治疗，不用于预防或治疗干细胞移植受体， 血液学/肿瘤学患者。Rho家族GTP酶RHOA和RAC 1是细胞凋亡的中心调节因子。 细胞骨架重排，并已显示控制脂筏的形成和组成; Rho GT3活性的变化可能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 和凋亡活性。我们的初步，提交和发表的数据使用遗传和 药理学手段表明，可逆的RHOA GT3抑制导致肌球蛋白的抑制， 活性和预防富含的脂质筏的网格蛋白非依赖性形成和内化 活性糖基转移酶（GT）和GPIb。RHOA GT3抑制可防止代谢性 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，当在存在铅的情况下在冷藏条件下储存长达14天时 RHOA抑制剂G 04可使细胞的存活功能保持在与室温储存的细胞相似的水平 血小板和保留止血活性在体内，和抗氧化剂phenocopies的一些影响， G04。我们假设，RHOA控制过程中的GP集群在冷藏通过 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、GP聚集和内吞作用的机制， 冷冻后的血小板。我们还将确定RHOA的药理学抑制的结果 在防止长期冷藏血小板的代谢和线粒体损伤方面， 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。 掌握代谢调节因子AMPK对血小板代谢和线粒体自噬的调节作用。我们的研究将 为新方法的转化价值提供了机制和严格的原理证明 到冷藏血小板储存

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.