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.

项目概要 血栓性心血管疾病是发达国家死亡和残疾的常见原因。 目前的抗血小板疗法尚未征服这些疾病，并且我们缺乏新的抗血小板药物的合适靶点 血栓形成方法和药物。血小板受磷酸化调节，但我们现在发现也受磷酸化调节 泛素化蛋白质的重塑。 我们发现血小板不断泛素化其蛋白质组并不断重塑这些加合链。 通过广泛作用的去泛素酶抑制剂抑制这种重塑，抑制响应的激活 几分钟内即可使用几种激动剂中的任何一种，并在微流体的高剪切流下阻止血小板粘附 渠道。细胞去泛素酶的抑制也能显着抑制受损组织中的闭塞性血栓形成 体内颈动脉。我们的数据现在表明，抑制特定的去泛素酶可以抑制血小板 激活。我们可以通过以下方式抑制洗涤后的人血小板的聚集、粘附、扩散和迁移： 抑制独特的去泛素酶 USP7（疱疹病毒相关泛素特异性蛋白酶，HAUSP）。我们 还发现选择性抑制两种蛋白酶体相关的去泛素化酶 USP14 和 UCHL5， 修剪多聚泛素，还抑制血小板功能。这导致我们建议去泛素酶与 促进激动剂信号传导和血栓形成。泛素重塑的重要作用也存在于 巨核细胞系允许遗传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反应性，并显示血小板泛素代谢 可以被操纵。这不仅定义了新的抗血栓目标，我们的工作表明我们可以成功 干预体内这种代谢以抑制动脉血栓形成。此外，两种特定的去泛素酶 每种都在血小板作用中发挥重要作用。这些酶的特定抑制剂是可用的，并且在一种情况下 可以长期安全地给药。这使我们能够通过其快速抑制血小板反应性 目前尚未认识到其在血栓形成中的作用。 目标 1. 确定靶向去泛素化在血小板活化和血栓形成中的作用。我们将 识别刺激后选择性去泛素酶选择性去泛素化的蛋白质，并将识别 信号级联中受泛素重塑调节的蛋白质。 目标 2. 阐明泛素重塑在体内血栓形成中的作用。在这里我们将确定当前 可用的特异性抑制剂可抑制血管内血栓形成。