Functional Role of Protein Disulfide Isomerase Isoforms in Platelets

描述(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。本提案关注PDI家族的两个成员--传统的PDI和最近发现的PDI家族成员ERp57。我们发现PDI介导了血小板聚集，而血管内PDI被证明是血栓形成所必需的。我们最近发现ERp57介导了血小板聚集、止血和血栓形成。ERp57和PDI参与了IIb 3向高亲和力状态的转化；然而，它们调节IIb 3和血小板聚集的机制尚不清楚。此外，PDI家族现在有多达20种不同的成员，其中许多成员存在于血小板中。这些酶是如何共同发挥作用的仍是个谜。以前的方法通常使用PDI的非特异性抑制剂来证明PDI在血小板功能和血栓形成中的作用。因此，需要更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。我们已经产生了具有ERp57和PDI中血小板特异性缺陷的靶向基因敲除小鼠，以及具有突变PDI的转基因小鼠。我们还产生了一种针对ERp57的抗体，尽管ERp57与PDI之间有很高的同源性，但它并不抑制PDI。我们目前的目标是确定血管内和血小板来源的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。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。其具体目的是：1.鉴定血管内和血小板衍生的ERp57在血小板聚集和纤维蛋白生成中的作用，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、血小板聚集和纤维蛋白生成中的作用；以及3.表征ERp57和PDI激活IIb的机制。使用的主要技术将是激光诱导血栓形成的损伤模型。为了确定ERp57和PDI的作用机制，我们将采用硫醇标记策略，并对标记的硫醇进行质谱鉴定。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，并开始揭示这些酶的作用机制。确定激活IIb的最后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，可能导致新型抑制剂或调节血小板聚集的方法。

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.