REGULATION OF PLATELET SHAPE CHANGES

描述：(改编自申请者摘要)血小板在体内循环 血液作为小圆盘，通过改变其形式对刺激做出反应。 这种形状的变化是通过丝状孢子的生长和 在依赖肌动蛋白的过程中，细胞表面的片状脂体 集合。超过98%的带刺(快速交换)肌动蛋白 静息状态下的血小板纤丝末端被阻断。激活 凝血酶作用下的血小板导致肌动蛋白快速组装 细胞骨架肌动蛋白细丝的休眠细胞 支离破碎，然后又不封顶。凝血酶导致一连串的 产生肌动蛋白组装和形状改变的细胞内信号 在15-75秒内。为了了解细胞内的信号系统 从凝血酶受体到带刺细丝的快速肌动蛋白组装 最后，申请者开发了一种渗透性系统，它可以响应 通过去顶刺捕获(凝血酶受体激活肽) 灯丝末端。使用这个系统，申请者已经建立了 途径如下：TRAP--&gt；racGTP酶--&gt；PI-激酶-- 磷脂酰肌醇4，5-二磷酸(PI4，5P2)--&gt；灯丝脱帽。 该提案的第一个目标是定义PI-4和/或PI-5 激酶被TRAP激活，在通透性中产生PI4，5P2 血小板。功能激酶及其产物PI4，5P2将被 位于血小板中，以及这一途径的上游中间体 导致该PI-激酶和肌动蛋白封顶蛋白的激活 由PPI和上游信令中间体发布 已确认身份。 这项提案的第二个目标将集中在肌动蛋白在 从激活蛋白质开始的信号转导途径 蛋白激酶C(PKC)。佛波酯(PMA)激活PKC 产生缓慢的形态变化和肌动蛋白组装。这 途径与凝血酶激活的途径有很大的不同，因为 只需几分钟即可启动，不依赖于钙，并且具有PI-3 KK及其产物PI3，4P2为必需组分。 凝血酶诱导的肌动蛋白组装不需要PI3，4P2。PMA- 然而，诱导途径也通过以下方式启动肌动蛋白组装 解帽的带刺肌动蛋白细丝结束，尽管解帽的程度 与凝血酶相比很小。申请者将定义PI3，4P2是否 对于细丝脱帽是必不可少的，如果GPIIb/IIIa受体， 它以其连接和活性的形式引起形状变化， 参与这一途径的。信号转导的位置 静息和活动中的蛋白质、PI-3激酶、GPIIb/IIIa和紧张素 将测定血小板细胞骨架是否为复合体 形成过程发生在激活过程中。

DESCRIPTION: (Adapted from Applicant's Abstract) Platelets circulate in blood as small discs which respond to stimuli by changing their forms. This shape transformation occurs by the growth of filopodia and lamellipodia from the cell surface in a process dependent on actin assembly. Greater than 98% of the barbed (fast exchanging) actin filament ends are blocked in resting platelets. Activation of platelets by thrombin leads to rapid actin assembly by causing the cytoskeletal actin filaments of the resting cell to become fragmented and then uncapped. Thrombin results in a cascade of intracellular signals that produces actin assembly and shape change in 15-75 sec. To understand the intracellular signaling system for fast actin assembly from the thrombin receptor to barbed filament ends, the Applicant developed a permeabilized system that responds to TRAP (thrombin receptor activating peptide) by uncapping barbed filament ends. Using this system, the Applicant has established the following pathway: TRAP-->racGTPase-->PI-kinases -- >phosphatidylinositol 4,5-bisphosphate (PI4,5P2)-->filament uncapping. The first goal of this proposal is to define if PI-4 and/or PI-5 kinase are activated by TRAP to produce PI4,5P2 in the permeabilized platelets. The functional kinase and its product, PI4,5P2, will be located in the platelet, and upstream intermediates of this pathway lead to activation of this PI- kinase and actin capping proteins released by ppis and upstream signaling intermediates will be identified. The second goal of this proposal will focus on actin assembly in a signal transduction pathway that begins by activating protein kinase C (PKC). Phorbol myristate acetate (PMA) activates PKC to generate a slow morphological transformation and actin assembly. This pathway differs dramatically from that activated by thrombin as it requires minutes to initiate, is calcium independent, and has PI-3 kinase and its product, PI3,4P2, as essential components. Thrombin-induced actin assembly does not require PI3,4P2. The PMA- induced pathway, however, also initiates actin assembly by uncapping barbed actin filament ends, although the extent of uncapping is small compared with thrombin. The Applicant will define if PI3,4P2 is essential for filament uncapping and if the GPIIb/IIIa receptor, which in its ligated and active form causes a shape change, is involved in this pathway. The location of the signal transduction proteins, PI-3 kinase, GPIIb/IIIa, and tensin in resting and active platelet cytoskeleton will be determined to see if complex formation occurs during activation.