Dynamic regulation of thrombosis by the platelet proteome

项目摘要 血栓性心血管疾病是发达国家死亡和残疾的普遍原因。 目前的抗血小板疗法尚未征服这些疾病，而且我们缺乏新抗血小板疗法的合适靶点。 血栓形成方法和药剂。血小板是由磷酸化调节的，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泛素化它们的蛋白质组，并不断重塑这些内收链。 通过广泛作用的去泛素化酶抑制剂抑制这种重塑抑制了响应于 几分钟内几种激动剂中的任何一种，并在微流体中的高剪切流动下阻断血小板粘附。 渠道细胞去泛素化酶的抑制也能显著抑制受损血管中的闭塞性血栓形成。 体内颈动脉。我们的数据表明，抑制特异性去泛素化酶可以抑制血小板聚集， activation.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： 抑制独特的去泛素化酶USP7（疱疹病毒相关的泛素特异性蛋白酶，HAUSP）。我们 还发现仅选择性抑制两种蛋白酶体相关的去泛素化酶USP14和UCHL5， Trim多聚泛素也抑制血小板功能。这使我们提出去泛素化酶串联作用， 使激动剂信号和血栓形成。泛素重塑的重要作用也存在于 巨核细胞细胞系允许遗传干预，以确认我们的药理学方法。 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地 在体内干预这种代谢以抑制动脉血栓形成。此外，两种特异性去泛素化酶 每一种都在血小板作用中具有重要作用。这些酶的特异性抑制剂是可用的，并且在一种情况下， 可以安全地长期服用。这使我们能够通过它们的作用迅速抑制血小板的反应性。 在血栓形成中的作用目前还未得到重视。 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 识别刺激后被选择性去泛素化酶选择性去泛素化的蛋白质，并将识别 信号级联中的蛋白质，由泛素重塑调节。 目标2.阐明泛素重构在体内血栓形成中的作用。在这里，我们将确定目前 可用的特异性抑制剂抑制血管内血栓形成。

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.