Mechanisms of platelet stimulation in hemolysis

摘要 而镰状细胞病(SCD)是由一种突变的血红蛋白引起的，这种突变的血红蛋白在低氧条件下聚合而导致 溶血和血管闭塞、炎症在这一发病机制中起着基础性作用。SCD 患者患血栓的风险要高得多，血栓会导致严重的危及生命的并发症。 比如肺动脉高压和中风。在细胞水平上，已经确定在SCD患者中，血小板 活性升高，并与溶血相关。然而，炎症信号对 血小板活化及其与溶血的串扰仍不清楚。高移动性组盒1(HMGB1)、 由细胞释放的炎症介质，已被证明在SCD患者的血液中升高，并 刺激血小板活化。值得注意的是，HMGB1介导血小板活化的能力依赖于 蛋白质中关键半胱氨酸残基的氧化状态。初步数据显示，HMGB1介导的血小板 在存在无细胞血红蛋白(Hb)的情况下，激活作用显著增强，Hb是 溶血。此外，Hb和HMGB1各自独立地增加了血小板线粒体的活性氧物种 (MtROS)生成，这是一种已知的刺激血小板激活的因素。基于这些数据，我假设在SCD中， Hb氧化HMGB1增加血小板mtROS的产生，导致血小板活化和二次释放 其他血栓刺激剂，包括来自血小板的HMGB1。目标1将确定Hb是否氧化 HMGB1体外利用人健康、镰刀状Hb和血小板增强其对血小板的激活作用 实验模型。目标2将确定HMGB1诱导的mtROS刺激血小板的机制 激活。目标3将利用小鼠溶血模型来确定中和HMGB1是否会减弱 血小板活化和血栓形成。这些目标的成功实现将阐释一部小说 溶血和炎症信号在增强血栓形成中的协同作用机制。这项工作将 揭示治疗SCD血栓形成的新靶点和潜在治疗方法。此外, 这个项目，以及我的多学科指导团队的指导，职业发展计划，以及 匹兹堡大学血管医学研究所的尖端研究环境将为 作为一名专注于血栓形成机制的研究人员，我作为一个强大的培训工具来促进我的独立性 在溶血性疾病中。

ABSTRACT While sickle cell disease (SCD) is instigated by a mutant hemoglobin that polymerizes in hypoxia to cause hemolysis and vaso-occlusion, inflammation plays a fundamental role in propagating this pathogenesis. SCD patients are at significantly higher risk of thrombosis, which contributes to severe life-threatening complications such as pulmonary hypertension and stroke. On a cellular level, it is well established that in SCD patients, platelet activation is elevated and correlates with hemolysis. However, the contribution of inflammatory signaling to platelet activation and its cross-talk with hemolysis remain unknown. High-mobility group box 1 (HMGB1), an inflammatory mediator released by cells, has been shown to be elevated in the blood of SCD patients and to stimulate platelet activation. Notably, the ability of HMGB1 to mediate platelet activation is dependent on the oxidation state of critical cysteine residues in the protein. Preliminary data shows that HMGB1-mediated platelet activation is significantly enhanced in the presence of cell-free hemoglobin (Hb), a major component of hemolysis. Further, Hb and HMGB1 each independently increase platelet mitochondrial reactive oxygen species (mtROS) generation, a known stimulus for platelet activation. Based on these data, I hypothesize that in SCD, Hb oxidizes HMGB1 to increase platelet mtROS production leading to platelet activation and secondary release of additional thrombotic stimulants including HMGB1 from platelets. Aim 1 will determine whether Hb oxidizes HMGB1 to potentiate its activation of platelets using human healthy and sickle Hb and platelets in ex vivo experimental models. Aim 2 will determine the mechanism by which HMGB1-induced mtROS stimulates platelet activation. Aim 3 will utilize murine models of hemolysis to determine whether neutralizing HMGB1 attenuates platelet activation and thrombus formation. Successful completion of these aims will elucidate a novel mechanism of synergy between hemolysis and inflammatory signaling in potentiating thrombosis. This work will reveal novel therapeutic targets and potential therapeutics for the treatment of thrombosis in SCD. In addition, this project, along with the guidance of my multi-disciplinary mentorship team, career development plan, and cutting-edge research environment at the Vascular Medicine Institute at the University of Pittsburgh, will serve as a strong training vehicle to facilitate my independence as a researcher focused on mechanisms of thrombosis in hemolytic disease.