Functional Role of Protein Disulfide Isomerase Isoforms in Platelets

血小板中蛋白质二硫键异构酶亚型的功能作用

基本信息

批准号：
8483001
负责人：
DAVID W ESSEX
金额：
$ 38.36万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-06-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8483001
关键词：
Active Sites Affinity Agonist Antibodies Blood Cells Blood Clot Blood Platelets Blood coagulation Cell surface Cells Cellular biology Coagulation Process DNA Sequence Rearrangement Deposition Development Disease Disulfides ERp57 Enzymes Event Family Fibrin Generations Goals Hemorrhage Hemostatic function Injury Integrins Knockout Mice Label Laboratories Lasers Lead Location Mass Spectrum Analysis Mediating Membrane Proteins Modeling Modification Molecular Monoclonal Antibodies Morbidity - disease rate Mus Myocardial Infarction Oxidation-Reduction Pattern Physiological Platelet Activation Platelet Inhibitors Platelet aggregation Prevention Protein Disulfide Isomerase Protein Isoforms Proteins Reaction Reagent Relative (related person)Reporting Role Signal Transduction Source Stroke Sulfhydryl Compounds System Techniques Testing Thrombosis Thrombus Time Transgenic Mice Transgenic Organisms United States Work disulfide bond extracellular in vivo inhibitor/antagonist insight member mortality mutant novel novel strategies prevent public health relevance response

项目摘要

DESCRIPTION (provided by applicant): Protein disulfide isomerase (PDI) catalyzes the reversible formation and isomerization of disulfide bonds in proteins. This proposal focuses on two members of the PDI family-the traditional PDI, and a more recently discovered member of the PDI family, ERp57. We found that PDI mediates platelet aggregation, and intravascular PDI has been shown to be required for thrombus formation. We recently showed that ERp57 mediates platelet aggregation, hemostasis and thrombosis. ERp57 and PDI are involved in conversion of ¿IIb¿3 to its high affinity state; however, the mechanisms by which they regulate ¿IIb¿3 and platelet aggregation are unknown. Furthermore, there are now up to 20 different members of the PDI family, and a number of these are found in platelets. How these enzymes function together remains a mystery. Previous approaches have generally used non-specific inhibitors of PDI to document a role for PDI in platelet function and thrombosis. Newer approaches are therefore required to define the molecular roles of each enzyme, as well as the intravascular sources of these enzymes. We have generated targeted knockout mice with platelets specific deficiencies in ERp57 and in PDI, and transgenic mice with a mutant PDI. We have also generated an antibody to ERp57 that despite the high homology between ERp57 and PDI does not inhibit PDI. Our current goal is to characterize the role of intravascular and platelet-derived ERp57 in platelet function and thrombus formation. We will also characterize the role of platelet-derived PDI in platelet function and thrombosis. We hypothesize that platelets provide an essential source of these enzymes for hemostasis and thrombosis. The specific aims are to: 1. Characterize the role of intravascular and platelet-derived ERp57 in platelet accumulation and fibrin generation, and the role of platelet-derived ERp57 in platelet function; 2. Characterize the role of platelet-derived PDI in platelet function, thrombosis, platelt accumulation, and fibrin generation; and 3. Characterize the mechanism of activation of ¿IIb¿3 by ERp57 and PDI. A principal technique used will be the laser-induced injury model of thrombosis. To determine the mechanisms by which ERp57 and PDI work, we will employ a thiol labeling strategy with mass spectrometry identification of the labeled thiols. We will determine the role of platelet-derived ERp57 and PDI in platelet function and thrombosis, and begin to unravel the mechanisms by which these enzymes work. Determining the extracellular redox mechanisms required for the final steps in the activation of ¿IIb¿3 is a highly significant aspect of platelet function and thrombus formation that could lead to novel types of inhibitors or ways to regulate platelet aggregation.

描述（由申请人提供）：蛋白质二硫键异构酶（PDI）催化蛋白质中二硫键的可逆形成和异构化。该提案重点关注PDI家族的两个成员——传统的PDI和最近发现的PDI家族成员。，我们发现 PDI 介导血小板聚集，并且血管内 PDI 已被证明是血栓形成所必需的。 ERp57 介导血小板聚集、止血和血栓形成，ERp57 和 PDI 参与 ¿ IIb?? 3 其高亲和力状态；然而，它们的调节机制 ¿ IIb??此外，PDI 家族目前有多达 20 种不同的成员，其中许多酶在血小板中如何共同发挥作用仍然是一个谜。因此，需要新的方法来确定每种酶的分子作用以及这些酶的血管内来源，以记录 PDI 在血小板功能和血栓形成中的作用。我们还产生了一种针对 ERp57 的抗体，尽管 ERp57 和 PDI 之间具有高度同源性，但它不会抑制 PDI 的作用。血小板衍生的 ERp57 在血小板功能和血栓形成中的作用我们还将描述血小板衍生的 PDI 在血小板功能和血栓形成中的作用。这些酶在止血和血栓形成中的作用的具体目的是： 1. 表征血管内和血小板源性 ERp57 在血小板聚集和纤维蛋白生成中的作用，以及血小板源性 ERp57 在血小板功能中的作用；血小板衍生的 PDI 在血小板功能、血栓形成、血小板积累和纤维蛋白生成中的作用；以及 3. 表征 ¿ IIb?? ERp57 和 PDI 的主要技术是激光诱导的血栓形成模型。为了确定 ERp57 和 PDI 的作用机制，我们将采用硫醇标记策略，并通过质谱法鉴定标记的硫醇。确定血小板衍生的 ERp57 和 PDI 在血小板功能和血栓形成中的作用，并开始揭示这些酶的细胞外作用机制。激活最终步骤所需的氧化还原机制IIb?? 3是血小板功能和血栓形成的一个非常重要的方面，可能会导致新型抑制剂或调节血小板聚集的方法的出现。