Target discovery in platelets by in situ proteome reactivity profiling

描述（由申请人提供）： 当暴露于细胞外基质蛋白和可溶性激动剂时，循环血小板产生细胞内信号，导致粘附受体如整联蛋白β 1 b-β 3的快速活化、血小板聚集、颗粒状内容物的分泌和促凝血活性增加。这些变化不仅有助于正常止血，而且还有助于血栓形成事件，如心肌梗死和中风。血小板是无核的，因此不适合直接的分子方法，从而限制了我们对这些活化依赖性信号通路的特定组分的理解。Parise实验室已经精通在巨核细胞（血小板前体）中使用敲低和过表达方法来剖析与血小板功能相关的信号通路。在这里，我们建议与Scripps研究所的Benjamin Cravatt博士合作，他开发了一种新的天然产物灵感螺环氧化物探针库，以前曾用于识别转化细胞中重要的酶。来自该文库的探针在基于细胞的筛选中共价结合到靶酶，从而影响特定的细胞读数。通过使用称为原位蛋白质组分析的新技术，然后通过使用“点击化学”用生物素和荧光标签标记共价结合的探针，从而便于通过质谱法快速鉴定靶标。然而，该文库从未应用于血小板。因此，我们建议1）通过用螺环氧化物探针文库筛选人血小板并进行原位蛋白质组反应性分析来鉴定必需的血小板功能所需的分子靶标，以及2）通过使用涉及过表达和敲低的独立计数器筛选以及候选靶标的药理学抑制和酶活性测定（如可能）来验证巨核细胞和血小板中的分子靶标。基于血小板的筛选和蛋白质组反应性分析与该库一起，以及调节巨核细胞中蛋白质表达水平的能力，提供了血小板领域中用于快速鉴定和验证用于调节血小板功能的新治疗靶点的方法的有力组合。

DESCRIPTION (provided by applicant): Circulating platelets generate intracellular signals when exposed to extracellular matrix proteins and soluble agonists, resulting in rapid activation of adhesion receptors such as integrin alphallb-beta3, platelet aggregation, secretion of granular contents and increased procoagulant activity. These changes contribute not only to normal hemostasis but also to thrombotic events such as myocardial infarction and stroke. Platelets are anucleate and therefore not amenable to direct molecular approaches, thus limiting our understanding of the specific components of these activation-dependent signaling pathways. The Parise laboratory has become proficient in the use of knockdown and overexpression approaches in megakaryocytes, which are platelet precursors, to dissect signaling pathways relevant to platelet function. Here we propose to join forces with Dr. Benjamin Cravatt of The Scripps Research Institute, who has developed a novel natural products inspired spiroepoxide probe library that has been used previously to identify enzymes of importance in transformed cells. Probes from this library bind covalently to target enzymes in cell-based screens, thus affecting specific cellular readouts. By use of new technology termed in situ proteome profiling, covalently bound probes are then labeled with a biotin and fluorescent tag by use of "click chemistry", thus facilitating rapid identification of targets via mass spectrometry. However, this library has never been applied to platelets. Therefore, we propose to 1) identify molecular targets necessary for essential platelet functions by screening human platelets with the spiroepoxide probe library and performing in situ proteome reactivity profiling, and 2) validate molecular targets in megakaryocytes and platelets by use of independent counter screens involving overexpression and knock down, as well as pharmacological inhibition and enzyme activity assays, when possible, of candidate targets. Platelet-based screening and proteome reactivity profiling with this library, together with the ability to modulate protein expression levels in megakaryocytes provides a powerful combination of approaches in the platelet field for rapidly identifying and verifying new therapeutic targets for modulating platelet function.