Investigation of ligand-mediated regulation of the ITIM-containing receptor G6b-B

在血管损伤时，血小板迅速粘附到新暴露的细胞外基质（ECM）的成分上，被激活并形成密封伤口的塞。然而，过度的血小板活化也可能阻塞血管，从而引起危及生命的疾病，如心肌梗死或缺血性中风。血小板来源于骨髓中的前体细胞，称为巨核细胞（mk）。一个关键的问题是，尽管mk表达了与血小板相同的细胞表面受体，但它如何在骨髓中富含ecm的环境中保持相对难降解。最近对敲除小鼠的研究发现，含有免疫受体酪氨酸基抑制基序（ITIM）的受体G6b-B是调节MK功能和血小板产生的重要抑制受体，因为它的缺失会导致严重的巨血小板减少症和血小板功能异常。尽管G6b-B具有核心作用，但人们对其调控知之甚少。目前项目的主要目的是解决关于G6b- b的两个关键问题：(i)其生理配体的鉴定和（ii） G6b剪接异构体的生物学功能的研究。这些目标并非相互排斥；由于G6b-A和-E与G6b-B具有相同的外结构域，它们必须结合相同的配体，并可能通过共聚或作为诱饵受体分别调节G6b-B信号转导。为了解决这些问题，广泛的生化和分子生物学技术以及体外细胞培养系统和小鼠模型将被使用。该研究项目的发现将有助于我们对血小板产生和反应性如何被控制的基本理解，并为开发针对该受体的新型抗血栓药物奠定基础。

Upon vascular injury platelets rapidly adhere to components of the newly exposed extracellular matrix (ECM), become activated and form a plug that seals the wound. However, excessive platelet activation may also occlude vessels, thereby causing life-threatening diseases like myocardial infarction or ischemic stroke. Platelets are derived from precursor cells in the bone marrow, termed megakaryocytes (MKs). A key question is how MKs, despite expressing the same repertoire of cell-surface receptors as platelets remain relatively refractory in the ECM-rich environment of the bone marrow. Recent studies in knockout-mice established the immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B as an essential inhibitory receptor in the regulation of MK function and platelet production, since its loss results in severe macrothrombocytopenia and aberrant platelet function. Despite this central role of G6b-B little is known about its regulation. The primary aim of the current project is to address two key questions regarding G6b-B: (i) the identification of its physiological ligand and (ii) the investigation of the biological functions of the G6b splice isoforms. These aims are not mutually exclusive; since G6b-A and -E have the same ectodomain as G6b-B, they must bind the same ligand, and potentially regulate G6b-B signaling by co-clustering or acting as decoy receptors, respectively. To address these questions a wide range of biochemical and molecular biological techniques as well as in vitro cell culture systems and mouse models will be used. Findings from this research project will contribute to our fundamental understanding of how platelet production and reactivity are controlled, and lay the foundation for development of novel anti-thrombotic drugs targeting this receptor.