Functional Role of Protein Disulfide Isomerase Isoforms in Platelets

描述（由申请方提供）：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。该建议集中在PDI家族的两个成员-传统的PDI和最近发现的PDI家族成员ERp 57。我们发现，PDI介导血小板聚集，血管内PDI已被证明是血栓形成所需的。我们最近发现ERp 57介导血小板聚集、止血和血栓形成。ERp 57和PDI参与α II β 3向其高亲和力状态的转化;然而，它们调节α II β 3和血小板聚集的机制尚不清楚。此外，现在有多达20种不同的PDI家族成员，其中一些在血小板中发现。这些酶如何共同发挥作用仍然是一个谜。先前的方法通常使用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ERp 57和PDI中具有血小板特异性缺陷的靶向敲除小鼠，以及具有突变PDI的转基因小鼠。我们还产生了针对ERp 57的抗体，尽管ERp 57和PDI之间具有高度同源性，但该抗体不抑制PDI。我们目前的目标是描述血管内和血小板源性ERp 57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。我们假设血小板是止血和血栓形成所需酶的重要来源。具体目标是：1.表征血管内和血小板源性ERp 57在血小板聚集和纤维蛋白生成中的作用，以及血小板源性ERp 57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、血小板积聚和纤维蛋白生成中的作用;以及3.描述ERp 57和PDI激活α II β 3的机制。使用的主要技术将是血栓形成的激光诱导损伤模型。为了确定ERp 57和PDI工作的机制，我们将采用巯基标记策略，并对标记的巯基进行质谱鉴定。我们将确定血小板衍生的ERp 57和PDI在血小板功能和血栓形成中的作用，并开始解开这些酶的工作机制。确定活化α II β 3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，这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 αIIß3 to its high affinity state; however, the mechanisms by which they regulate αIIß3 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 αIIß3 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 αIIß3 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.