Dynamic regulation of thrombosis by the platelet proteome

血小板蛋白质组对血栓形成的动态调节

• 批准号: 9336334
• 负责人: Thomas M McIntyre
• 金额: $ 39.63万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2020-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9336334
• 关键词: Ablation; Acute; Adhesions; Affect; Affinity; Agonist; Animals; Antiplatelet Drugs; Aspirin; Binding Proteins; Blood Chemical Analysis; Blood Platelets; Bortezomib; Calcium Channel; Cardiovascular Diseases; Carotid Arteries; Carotid Artery Thrombosis; Cause of Death; Cell Line; Cells; Collagen; Data; Deubiquitination; Disease; Dose; Embolism; Embryonic Development; Enzyme Inhibitor Drugs; Face; Fibrinolytic Agents; Genetic; Herpesviridae; Human; Human Genome; Infarction; Injection of therapeutic agent; Intervention; Lysosomes; MG132; Malignant - descriptor; Megakaryocytes; Metabolism; Microfluidics; Movement; Pharmacology; Phenocopy; Phosphorylation; Platelet Activation; Polyubiquitin; Process; Proteasome Inhibitor; Proteins; Proteolysis; Proteome; Publishing; Recombinants; Regulation; Renal function; Role; Scheme; Signal Transduction; System; Thrombin; Thrombosis; Thromboxanes; Thrombus; Time; Tumorigenicity; Ubiquitin; Ubiquitination; Velcade; Work; clopidogrel; disability; exosome; gene therapy; genetic approach; in vivo; inhibitor/antagonist; migration; multicatalytic endopeptidase complex; response; tumorigenesis; tumorigenic; ubiquitin-specific protease

Project Summary Thrombotic cardiovascular diseases are prevalent causes of death and disability in the developed world. Current anti-platelet therapies have not conquered these diseases, and we lack suitable targets for new anti- thrombotic approaches and agents. Platelets are regulated by phosphorylation, but we now find also by remodeling of ubiquitinated proteins. We find platelets continuously ubiquitinate their proteome and continuously remodel these adducted chains. Inhibition of this remodeling by broadly acting deubiquitinase inhibitors suppresses activation in response to any of several agonists within minutes, and blocks platelet adhesion under high shear flow in microfluidic channels. Inhibition of cellular deubiquitinases also sharply suppresses occlusive thrombosis in damaged carotid arteries in vivo. Our data now show that inhibition of specific deubiquitinases suppresses platelet activation. We can inhibit aggregation, adhesion, spreading and migration of washed human platelets by inhibiting the unique deubiquitinase USP7 (Herpes virus-associated ubiquitin specific protease, HAUSP). We also find selective inhibition of just the two proteasome-associated deubiquitinases USP14 and UCHL5, which trim poly-ubiquitin, also inhibited platelet function. This leads us to propose deubiquitinases act in tandem to enable agonist signaling and thrombosis. The essential role of ubiquitin remodeling is also present in megakaryocytes cell lines allowing genetic interventions to confirm our pharmacologic approach. This work shows ubiquitin metabolism regulates platelet reactivity, and shows platelet ubiquitin metabolism can be manipulated. This not only defines new anti-thrombotic targets, our work shows we can successfully intervene in this metabolism in vivo to suppress arterial thrombosis. Moreover, two specific deubiquitinases each have essential roles in platelet action. Specific inhibitors of these enzyme are available and in one case can be safely administered over long times. This allows us to rapidly suppress platelet reactivity through their currently unappreciated roles in thrombosis. Aim 1. Establish the role of targeted deubiquitination in platelet activation and thrombosis. We will identify proteins selectively deubiquitinated by the selective deubiquitinase after stimulation, and will identify proteins in signaling cascades that are regulated by ubiquitin remodeling. Aim 2. Elucidate the role of ubiquitin remodeling in in vivo thrombosis. Here we will determine currently available specific inhibitors suppress intravascular thrombosis.

项目摘要 血栓性心血管疾病是发达国家常见的死亡和残疾原因。 目前的抗血小板疗法还没有克服这些疾病，我们缺乏合适的靶点来寻找新的抗血小板药物。 血栓形成方法和制剂。血小板是通过磷酸化来调节的，但我们现在发现， 泛素化蛋白质的重塑。 我们发现，血小板不断地泛化他们的蛋白质组，并不断地重塑这些加成链。 通过广泛作用的去泛素酶抑制剂抑制这种重塑，抑制了对 几种激动剂中的任何一种，在几分钟内，并在微流控高剪切流下阻止血小板粘连 频道。抑制细胞脱泛素酶也能显著抑制受损的闭塞性血栓形成 活体颈动脉。我们的数据现在表明，抑制特定的去泛素酶会抑制血小板 激活。我们可以抑制洗涤过的人血小板的聚集、黏附、扩散和迁移 抑制独特的脱泛素酶USP7(疱疹病毒相关泛素特异性蛋白酶，Hausp)。我们 还发现选择性抑制只有两个与蛋白酶体相关的去泛素酶USP14和UCHL5，这两个酶 修剪多泛素，也抑制了血小板功能。这导致我们建议去泛素酶的行为与 启用激动剂信号和血栓形成。泛素重塑的重要作用也存在于 巨核细胞系允许基因干预来证实我们的药理学方法。 这项工作表明泛素代谢调节血小板的反应性，并显示了血小板泛素代谢。 可以被操控。这不仅定义了新的抗血栓靶点，我们的工作表明我们可以成功地 在体内干预这种代谢，以抑制动脉血栓形成。此外，两种特定的脱泛素酶 每一个都在血小板的作用中起着至关重要的作用。这些酶的特定抑制剂是可用的，在一种情况下 可以安全地长时间服用。这使我们能够通过其自身的功能快速抑制血小板的反应性 目前未被认识到在血栓形成中的作用。 目的1.确定靶向去泛素化在血小板活化和血栓形成中的作用。我们会 鉴定刺激后被选择性脱泛素酶选择性脱泛素化的蛋白质，并将鉴定 受泛素重塑调控的信号级联蛋白。 目的2.阐明泛素重塑在体内血栓形成中的作用。在这里我们将确定目前 可用的特定抑制剂可抑制血管内血栓形成。