The Biological Roles of Phoshadylinositol Transfer Proteins in Platelets

说明(申请人提供)：磷酸化磷脂酰肌醇(磷脂酰肌醇)是一种膜结合磷脂，在血小板激活过程中参与多种不同的过程。磷脂酰肌醇转移蛋白(PITPs)是一个小的蛋白质家族，在体外已被证明在囊泡运输和磷脂信号转导过程中以能量不依赖的方式结合并将磷脂酰肌醇单体从一个细胞室转移到另一个细胞室。尽管还没有关于PITPs在造血细胞中的作用的研究，但在酵母细胞中有证据表明，这些 蛋白质对于肌醇磷脂的生物合成和代谢是必不可少的。血小板有两个主要的PITP家族成员，PITP1和PITP2。这一建议的总体假设是，这些单独的PITP亚型具有不重叠的功能，每个功能对于血小板内离散的磷脂酰肌醇物种的产生和空间定位是必不可少的。第二个假设是，两种PITP亚型的酶活性对于正常的血小板黏附、聚集和颗粒分泌是必需的。为了了解这些单独的PITP亚型在血小板生物学中的独特和离散的作用，我们培育了在PITP1和PITP2基因内含有条件性零突变的小鼠。这份R01是为了申请资金，使我们能够表征PITP1fl/fl PF4Cre+、PITP2fl/fl PF4Cre+和PITP1fl/fl PITP2fl/fl PF4Cre+(双基因敲除)小鼠。这些小鼠的血小板和成熟的巨核细胞缺乏PITP1或PITP2，但它们在所有其他组织中都正常表达这些蛋白。任何一种亚型的缺失都会导致血小板减少。我们的初步数据表明，在最大剂量的凝血酶刺激下，缺乏PITP1的血小板第二信使INS(3，4，5)P3(也称为IP3)完全丧失。我计划对这些不同的PITP亚型在血小板中的功能进行全面和系统的研究，以了解它们在血小板中的独特生化作用和生物学意义。在目标1中，我们将确定PITP异构体和聚磷脂酰肌醇合成之间的联系，并分析PITP在血小板信号转导中的作用。在目标2中，我提出了旨在了解不同PITP亚型不同生化功能的实验。最终，我们将确定PITP亚型在体内和体外对血小板活化的作用。

DESCRIPTION (provided by applicant): Phosphorylated phosphatidylinositols (phosphoinositides) are a type of membrane bound phospholipid that contributes to multiple diverse processes required during platelet activation. PhosphatidylInositol Transfer Proteins (PITPs) are a small family of proteins that have been demonstrated in vitro to bind and transfer phosphoinositide monomers from one cellular compartment to another in an energy independent manner during vesicle trafficking and phospholipid signaling. Although there are no studies on the role of PITPs in hematopoietic cells, there is evidence in yeast cells that these proteins are essential for the biosynthesis and metabolism of phosphoinositides. Platelets have two dominant PITP family members, PITP1 and PITP2. The overall hypothesis of this proposal is that these individual PITP isoforms have non- overlapping functions that are each essential for the generation and spatial localization of discrete species of phosphoinositides within platelets. A secondary hypothesis is that the enzymatic activities of both PITP isoforms are necessary for normal platelet adhesion, aggregation, and granule secretion. To understand the unique and discrete roles of these individual PITP isoforms in platelet biology, we have generated mice containing conditional null mutations within the PITP1 and PITP2 genes. This R01 is to request funds that will allow us to characterize PITP1fl/fl PF4Cre+, PITP2fl/fl PF4Cre+, and PITP1fl/fl PITP2fl/fl PF4Cre+ (double knockout) mice. These mice have platelets and mature megakaryocytes lacking either PITP1 or PITP2, but they have normal expression of these proteins in all other tissues. Loss of either isoform results in thrombocytopenia. Our preliminary data indicates that platelets lacking PITP1 have a complete loss in the second messenger, Ins(3,4,5)P3 (also known as IP3) following stimulation by maximal doses of thrombin. I plan to perform a comprehensive and systematic study of the function of these individual PITP isoforms in platelets in order to understand their unique biochemical role and biologic importance within platelets. In Aim 1, we will determine the link between PITP isoforms and polyphosphoinositide synthesis, as well as analyze the contribution of PITPs to platelet signaling. In Aim 2, I propose experiments designed to understand the distinct biochemical functions of the individual PITP isoforms. In the final Aim, we will identify the role of PITP isoforms in platelet activation ex vivo and in vivo.