Glycoprotein Ib in vascular biology and host defense

项目总结 我们的目标是了解血小板膜糖蛋白(GP)Ib-IX-V复合体的新功能，它是 除其他外，几种配体介导粘附性相互作用(主要通过von Willebrand因子)、细胞-细胞 接触(与白细胞整合素、α、M、β2和P-选择素相互作用)和凝血(结合几种凝血 凝血酶等因素)。通过这个项目，我们打算扩大对GPIB生理病理学的理解 超越巨核细胞/血小板区域，更坚定地进入先天免疫机制领域- 尼斯主义。尽管如此，血管性血友病因子(VWF)和凝血酶，GPIB在止血和血栓中的关键合作伙伴- Bosis，仍然是拟议的新研究的主要感兴趣的配体。在我们近30年的工作中，我们 为理解vWF、凝血酶和GPIBα是如何在体内的主要亚基 GPIbIX-V复合体，相互作用。关于凝血酶，即使具有GPIBα-凝血酶复合体的结构- Plex在原子水平的细节上揭开面纱，阐明依赖功能仍然难以捉摸。有了这样的知识 通过过去的研究和下一代缺乏凝血酶-GPIB结合的小鼠获得的， 并受到使用这些模型获得的令人惊讶的初步结果的启发，我们提出了两个铰接式空间。 CICIC的目标是开发一个解决意想不到的新功能的项目。在目标1中，我们将继续努力定义 凝血酶与血小板GPIBα结合的生理病理意义首先， 我们将探索新发现的高和低亲和力凝血酶-GPIBα结合模式的功能作用， 使用表达突变型人GPIBα(Y279F)的小鼠品系，在该突变株中， 结合丢失，但保留低亲和力。在第二部分中，我们将讨论Se-Se带来的问题。 小鼠和人GPIBα在凝血酶结合区的序列差异。我们已经确定了 研究了凝血酶结合所需的小鼠GPIBα残基，并建议建立一种小鼠模型。 小鼠GPIBα在凝血酶结合中选择性缺陷，但保留所有其他配体与 完整的物种专一性。这将是确定人类生理病理学价值的重要工具 凝血酶诱导不同PAR表达的小鼠血小板活化机制的研究 在血小板上。在目标2中，我们将研究肥大细胞上GPIBα的表达和功能及其功能 在过敏反应中的作用。我们将制定两个子目标。首先，我们将描述肥大细胞的结构。 按下与GPIB-IX-V复合体的其他组件相关的GPIB。在第二节中，我们将探索 表达GPIBα的肥大细胞功能评估在细胞黏附和对凝血酶刺激的反应中的作用 提顿。我们已经产生了几个肥大细胞表达GPIB缺陷的新小鼠模型。 这些研究的结果将促进对血小板功能联系机制的理解。 对炎症、感染和先天免疫等相关过程的止血作用。

Project summary Our goal is to understand novel functions of the platelet glycoprotein (GP) Ib-IX-V complex, a key receptor for several ligands mediating, among others, adhesive interactions (mostly through von Willebrand factor), cell-cell contacts (interacting with leukocyte integrin αMβ2 and P-selectin) and coagulation (binding several coagulation factors including thrombin). With this project we intend to extend the understanding of GPIb physiopathologic relevance beyond megakaryocyte/platelet domains and more firmly into the area of innate immune mecha- nisms. Nonetheless, von Willebrand factor (VWF) and thrombin, key GPIb partners in hemostasis and throm- bosis, remain the main ligands of interest for the proposed new studies. In our work over nearly 30 years we have contributed to establish the bases for understanding how VWF, thrombin and GPIbα, the main subunit in the GPIbIX-V complex, interact. With respect to thrombin, even with the structure of the GPIbα-thrombin com- plex unveiled at atomic level detail, elucidating dependent functions has remained elusive. With the knowledge acquired through past studies and the ensuing generation of mice lacking thrombin-GPIb binding on platelets, and stimulated by surprising preliminary results obtained using these models, we propose two articulated spe- cific aims to develop a project addressing unexpected new functions. In aim 1 we will extend the effort to define the physiopathological significance of thrombin binding to platelet GPIbα developing two sub-aims. In the first, we will explore the functional roles of the newly identified high and low affinity thrombin-GPIbα binding modes, using for the purpose a mouse strain expressing a mutant human GPIbα (Y279F) in which high affinity throm- bin binding is lost but low affinity is retained. In the second, we will address the problem posed by the se- quence divergence between mouse and human GPIbα in the region of thrombin binding. We have identified the residues of mouse GPIbα required for thrombin binding and propose to generate a mouse model with en- dogenous mouse GPIbα selectively defective in thrombin binding but retaining all other ligand interactions with intact species specificity. This will be an essential tool to ascertain the value for human physiopathology of studying mechanisms of thrombin-induced platelet activation in mouse models with different PAR expression on platelets. In aim 2 we will characterize expression and function of GPIbα on mast cells and its functional role in anaphylaxis. We will develop two sub-aims. In the first we will characterize the structure of mast cell ex- pressed GPIb in relation to the other components of the GPIb-IX-V complex. In the second we will explore functions of mast cell expressed GPIbα evaluating roles in cell adhesion and in response to thrombin stimula- tion. We have already generated several new mouse models with defective GPIb expression on mast cells. The results of these studies will advance the understanding of mechanisms linking the function of platelets in hemostasis to relevant processes in inflammation, infection and innate immunity.