Cytoskeletal Signaling IN Platelets

这一建议的总体假设是，血小板肌动蛋白重组在动脉粥样硬化中起着关键作用。 止血和血栓形成。丝状肌动蛋白的重组是血小板的最早期事件之一 activation.我们最近的数据集中在血小板肌动蛋白动力学的两个关键方面，磷脂 信号传导和翻译后肌动蛋白修饰。磷脂酰肌醇二磷酸（PIP 2）对于 血小板细胞骨架的调节，是肌动蛋白组装的几个步骤所必需的。这包括 作为磷酸肌醇第二信使形成的底物，以及直接结合和 从而调节肌动蛋白结合蛋白。磷脂酰肌醇具有β和γ两种亚型 磷酸5-激酶I（PIP 5 KI），它们各自能够将PI 4P转化为PI4.5P2（PIP 2），因此尚不清楚 为什么血小板需要一种以上的亚型来完成这一单一的生化反应。测试 假设这些同种型具有不重叠的功能，我们已经产生了含有null 在PIP 5 KI 3或PIPSKIy基因中的突变，其编码PIP 5 KI的显性同种型， 血小板我们对来自这些小鼠的血小板的初步研究表明，PIP 5 KI 3产生PIP 2是一个重要的机制。 需要产生第二信使，而PIP 5 Kly合成PIP 2需要维持第二信使。 膜骨架的完整性。我们最近还报道，在迁移的前沿， 在成纤维细胞中，肌动蛋白必须具有在其N-末端上后修饰添加的精氨酸。没有这种 修饰损害成纤维细胞的粘附和迁移，证明了这种修饰的重要作用 在肌动蛋白装配中。我们的初步研究表明，在对激动剂刺激的反应中， 也是乙酰化的。我们推测，这种最近发现的翻译后修饰对于 血小板肌动蛋白动力学本申请的目的是在分子基础上了解体内血小板 肌动蛋白的变化，并确定是否肌动蛋白重组是至关重要的稳定血小板粘附。在目标1中， 我们将使用我们最近开发的鼠模型来确定PIP 5 KI在血小板中的生物学作用。在目标2中， 我们将通过生物化学，鼠遗传学， 和血小板粘附模型。在目标3中，我们将讨论肌动蛋白乙酰化对血小板的作用。 粘附使用生物化学和小鼠遗传改变，缺乏能力，使血小板肌动蛋白。

The overall hypothesis of this proposal is that platelet actin reorganization plays a critical role in arterial hemostasis and thrombosis. The reorganization of filamentous actin is one of the earliest events of platelet activation. Our recent data has focused on two critical aspects of platelet actin dynamics, phospholipid signaling and post-translational actin modifications. Phosphatidylinositol bisphosphate (PIP2) is critical for the regulation of the platelet cytoskeleton and is required for several steps of actin assembly. This includes serving as a substrate for phosphoinositide second messenger formation, as well as directly binding to and thereby regulating actin-binding proteins. Platelets have both the (3 and y isoforms of phosphatidylinositol phosphate 5-kinase I (PIP5KI) that are each capable of converting PI4P to PI4.5P2 (PIP2), so it is unclear why platelets require more than one isoform to perform this single biochemical reaction. To test the hypothesis that these isoforms have non-overlapping functions, we have generated mice containing null mutations in either the PIP5KI3 or the PIPSKIy genes, which encode for the dominant PIP5KI isoforms in platelets. Our preliminary studies of platelets from these mice suggest that production of PIP2 by PIP5KI3 is required to generate second messengers, while synthesis of PIP2 by PIP5Kly is required to maintain the integrity of the membrane skeleton. We have also recently reported that at the leading edge of migrating fibroblasts, actin must have an arginine post-translationally added onto its N-terminus. Absence of this modification impairs fibroblast adhesion and migration, demonstrating the essential role of this modification in actin assembly. Our preliminary studies indicate that in response to agonist stimulation, platelet actin is also arginylated. We hypothesize that this recently discovered post-translational modification is critical for platelet actin dynamics. The goal of this application is to understand on a molecular basis the in vivo platelet actin changes, and to determine whether actin reorganization is vital for stable platelet adhesion. In Aim 1, we will define the biologic roles of PIP5KI in platelets using our recently developed murine models. In Aim 2, we will determine how integrins regulate PIP2 production by a combination of biochemistry, murine genetics, and platelet adhesion models. In Aim 3, we will address the contribution of actin arginylation on platelet adhesion using biochemistry and mice genetically altered to lack the ability to arginylate platelet actin.