Subcellular Mechanisms pf Platelet Activation

血小板活化开始于血小板在受损血管壁上的初始沉积，然后继续， 额外的血小板被募集并彼此粘附。这些事件使血小板稳定接触 在相邻血小板之间可发生蛋白质：蛋白质相互作用的地方形成连接。 这个项目的长期目标是了解血小板连接处的事件如何有助于血小板 对伤害的反应。我们的假设是：1）相关信号在血小板聚集后继续存在， 开始，2）一些信号传导来自分子之间的相互作用，而不是整合素上的整合素。 3）这些连接处的接触依赖性相互作用可以用作 正调节剂，促进止血块的生长和稳定以防止再出血，或作为 负调节因子，限制生长和稳定性，从而避免血管闭塞。在他㆒生㆗最容易 在最近的资助期间，我们在血小板表面鉴定了肝配蛋白B1和脑信号蛋白4D，并显示 这些配体与它们各自的受体（EphB 1和EphA 4用于肝配蛋白BI; CD 72和CD 74用于肝配蛋白BI）的结合， 对于sema 4D，丛蛋白B1）促进血栓生长。我们还确定了ESAM，一种假定的细胞， 当血小板被激活时，CTX家族中的粘附分子易位到连接处，然后作为 负调节因子，因此ESAM表达的丧失促进而不是损害细胞的延伸。 血小板量该提案中描述的研究分为四个具体目标，重点是血小板 连接和接触依赖的相互作用。目标1将测试我们当前的模型，即ESAM是阴性的 血小板调节因子：血小板相互作用，并探讨ESAM功能丧失对 使用现有的ESAM敲除小鼠系进行血小板活化。目标#2将集中在分子基础上， ESAM的贡献，从我们最近鉴定的两种支架蛋白NHERF-1和CAL开始， 与ESAM胞质结构域结合。目的#3是对其他三种CTX家族的比较分析 血小板中表达的成员（JAM-A，JAM-C和CD 226），以确定它们的作用是否与 ESAM。用JAM-A敲除小鼠获得的初步结果表明，情况可能就是这样。目标#4 致力于血小板中额外的连接分子的表征，从肝配蛋白A1开始， 血小板，并继续无偏见地寻找新的蛋白质。

Platelet activation begins with the initial deposition of platelets on a damaged vessel wall, then continues as additional platelets are recruited and adhere to each other. These events bring platelets into stable contact with each other, forming junctions where protein:protein interactions can occur between adjacent platelets. The long term goal of this project is to understand how events at platelet junctions contribute to the platelet response to injury. Our hypothesis is that 1) relevant signaling continues after platelet aggregation has begun, 2) some of the signaling arises from interactions between molecules other than integrins on the surface of adjacent platelets, and 3) these contact-dependent interactions at junctions can serve either as positive regulators, promoting the growth and stability of the hemostatic mass to prevent re-bleeding, or as negative regulators, limiting growth and stability so that vascular occlusion is avoided. During the most recent funding period we have identified ephrin B1 and semaphorin 4D on the platelet surface, and shown that the binding of these ligands to their respective receptors (EphB1 and EphA4 for ephrinBI; CD72 and plexin B1 for sema4D) promotes thrombus growth. We have also determined that ESAM, a putative cell adhesion molecule in the CTX family, translocates to junctions when platelets are activated and then acts as a negative regulator, so that loss of ESAM expression promotes, rather than impairs, extension of the platelet mass. The studies described in this proposal are divided into four specific aims focusing on platelet junctions and contact-dependent interactions. Aim #1 will test our current model that ESAM is a negative regulator of platelet:platelet interactions and explore the consequences of a loss of ESAM function on platelet activation using an existing line of ESAM knockout mice. Aim #2 will focus on the molecular basis for ESAM's contribution, starting with our recent identification of two scaffold proteins, NHERF-1 and CAL, that bind to the ESAM cytoplasmic domain. Aim #3 is a comparative analysis of the three other CTX family members expressed in platelets (JAM-A, JAM-C and CD226) to determine whether their role is the same as ESAM. Initial results obtained with JAM-A knockout mice, suggest that this may be the case. Aim #4 is devoted to the characterization of additional junction molecules in platelets, starting with ephrin A1 on platelets and continuing with an unbiased search for novel proteins.