Cytoskeletal Signaling IN Platelets

这一提议的总体假设是，血小板肌动蛋白重组在动脉粥样硬化中起关键作用。 止血和血栓形成。丝状肌动蛋白的重组是血小板最早发生的事件之一 激活。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 信号转导和翻译后肌动蛋白修饰。磷脂酰肌醇二磷酸(PIP2)是 调节血小板细胞骨架，是肌动蛋白组装的几个步骤所必需的。这包括 作为磷脂酰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。血小板同时具有磷脂酰肌醇的(3和y)亚型 磷酸5-激酶I(PIP5KI)，每个都能将PI4P转化为PI4.5P2(PIP2)，因此尚不清楚 为什么血小板需要不止一种异构体来执行这一单一的生化反应。要测试 假设这些异构体具有不重叠的功能，我们产生了含有Null的小鼠 PIP5KI3或PIPSKIy基因突变，它们编码PIP5KI亚型 血小板。我们对这些小鼠的血小板的初步研究表明，PIP5KI3产生的PIP2是 需要产生第二信使，而PIP5Kly合成PIP2是维持 膜骨架的完整性。我们最近还报道说，在移民的前沿 对于成纤维细胞，肌动蛋白的N-末端必须含有翻译后的精氨酸。没有这一点 修饰损害成纤维细胞的黏附和迁移，证明了这种修饰的基本作用。 在肌动蛋白组装中。我们的初步研究表明，对激动剂刺激的反应是，血小板肌动蛋白 也是精氨酸化。我们假设，最近发现的翻译后修饰对 血小板肌动蛋白动力学。这一应用的目标是在分子基础上了解体内的血小板 肌动蛋白的变化，并确定肌动蛋白重组是否对稳定的血小板黏附至关重要。在目标1中， 我们将使用我们最近开发的小鼠模型来确定PIP5KI在血小板中的生物学作用。在目标2中， 我们将结合生物化学、小鼠遗传学、 和血小板黏附模型。在目标3中，我们将讨论肌动蛋白精氨酸化对血小板的作用。 使用生物化学的黏附和基因改变的小鼠缺乏精氨酸化血小板肌动蛋白的能力。

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.