Subcellular Mechanisms pf Platelet Activation

血小板激活始于血小板在受损的血管壁上的初始沉积，然后继续 更多的血小板被招募，并相互粘连。这些事件使血小板保持稳定的接触 彼此之间形成连接，在那里蛋白质：蛋白质相互作用可以发生在相邻的血小板之间。 这个项目的长期目标是了解在血小板连接处发生的事件对血小板的影响。 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 开始，2)一些信号来自分子之间的相互作用，而不是整合素 3)在交界处的这些依赖于接触的相互作用可以用作 积极的调节剂，促进止血肿块的生长和稳定，以防止再次出血，或AS 负调节，限制生长和稳定性，从而避免血管闭塞。在最多的时候 最近的资助期，我们发现了血小板表面的ePhrin B1和信号素4D，并显示了 这些配体与它们各自的受体(EphB1和EphA4对ePhinBI；CD72和 Sema4D的Plexin B1)促进血栓生长。我们还确定了ESAM，一个假定的细胞 CTX家族中的黏附分子，当血小板被激活时移位到连接处，然后起到 负调控，因此ESAM表达的丧失促进而不是损害ESAM的延伸 血小板块。这项提案中描述的研究分为四个具体目标，重点是血小板 结点和依赖于接触的相互作用。目标1将测试我们当前的模型，即ESAM是负面的 血小板调节因子：血小板相互作用和探讨ESAM功能丧失的后果 利用现有的ESAM基因敲除小鼠品系激活血小板。目标2将重点放在分子基础上 ESAM的贡献，首先是我们最近鉴定的两种支架蛋白，NHERF-1和CAL，即 与ESAM细胞质结构域结合。目标3是对其他三个CTX家族的比较分析 在血小板(JAM-A、JAM-C和CD226)中表达的成员，以确定它们的作用是否与 伊萨姆。在JAM-A基因敲除小鼠身上获得的初步结果表明，情况可能是这样的。目标4是 致力于研究血小板中其他连接分子的特征，从Ephin A1开始 并继续不偏不倚地寻找新的蛋白质。

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.