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

• 批准号: 8666045
• 负责人: DAVID W ESSEX
• 金额: $ 38.44万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 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/8666045
• 关键词: Active Sites; Affinity; Agonist; Antibodies; Blood Cells; 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

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（由适用提供）：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。该提案的重点是PDI家族的两个成员 - 传统的PDI，以及最近发现的PDI家族成员ERP57。我们发现PDI介导了血小板聚集，并且已经证明血管内血栓形成需要PDI。我们最近表明，ERP57介导了血小板聚集，止血和血栓形成。 ERP57和PDI参与将„ IIB€3转换为高亲和力状态；但是，它们调节IIB课和血小板聚集的机制尚不清楚。此外，现在有多达20个不同的PDI家族成员，其中许多是在血小板中发现的。这些酶如何一起起作用仍然是一个谜。以前的方法通常使用PDI的非特异性抑制剂来记录PDI在血小板功能和血栓形成中的作用。因此，需要较新的方法来定义每种酶的分子作用，以及我们已经在ERP57和PDI中产生的具有血小板特异性缺乏的靶向敲除小鼠以及具有突变体PDI的转基因小鼠。我们还产生了一种对ERP57的抗体，该抗体希望ERP57和PDI之间的高同源性不抑制PDI。我们目前的目标是表征血管内和血小板衍生的ERP57在血小板功能和血栓形成中的作用。我们还将表征血小板衍生的PDI在血小板功能和血栓形成中的作用。我们假设血小板为止血和血栓形成提供了这些酶的重要来源。具体目的是：1。表征血管内和血小板衍生的ERP57在血小板积累和纤维蛋白产生中的作用，以及血小板衍生的ERP57在血小板功能中的作用； 2。表征血小板衍生的PDI在血小板功能，血栓形成，降低了纤维蛋白的产生中的作用；和3。表征ERP57和PDI激活„IIB¿3的机理。使用的主要技术将是激光诱导的血栓形成模型。为了确定ERP57和PDI工作的机制，我们将采用硫醇标记策略，并鉴定标记的硫醇。我们将确定血小板衍生的ERP57和PDI在血小板功能和血栓形成中的作用，并开始揭示这些酶起作用的机制。确定激活»iib课的最终步骤所需的细胞外氧化还原机制是血小板功能和血栓形成的一个非常重要的方面，它可能导致新型的抑制剂或调节血小板聚集的方法。