Functional Role of Protein Disulfide Isomerase Isoforms in Platelets

蛋白质二硫键异构酶(PDI)催化二硫键的可逆形成和异构化 蛋白质，并支持血栓形成。最近的报告表明，PDI家族的其他成员ERP57， ERp5和ERp72也与血栓形成有关。PDI、ERp57、ERp5和ERp72在细胞周期调控中的作用 血栓形成明显或多余，不清楚。我们发现ERp72、PDI或 ERp57缺失的小鼠只能通过缺失的特定PDI恢复。这意味着这些酶 在激活支持血小板聚集的αIIbβ3血小板整合素方面有单独的靶点。这些 PDI家族成员含有催化蛋白质构象变化的CGHC活性中心基序 参与血栓形成。本提案重点介绍了PDI家族的两个新成员，它们的主题是 发现于血小板中；ERp46，以及PDI家族的跨膜成员TMX3。我们现在知道PDI， ERp57、ERp5和ERp72介导了血小板聚集和血栓形成，并参与了 αIIbβ3到其高亲和力状态。然而，这些PDI调节αIIbβ3和 目前尚不清楚血小板聚集情况。每种酶的单独靶标以及它们如何共同发挥作用 仍然是个谜。为了确定每个PDI在血小板中的特定功能，我们使用了有针对性的 基因敲除老鼠靠近了。具体目的是：1.研究ERp46在血栓形成中的作用 和血小板功能；2.表征TMX3在血栓形成和血小板功能中的作用；以及3. 鉴定半胱氨酸/二硫键靶标及其激活αIIbβ3的机制 ERP46和TMX3。使用的主要技术将是激光诱导血栓形成的损伤模型。至 确定PDI系列的多个成员共同工作的实际机制，我们将采用 硫醇标记策略与标记物的质谱鉴定。这将开始揭开 这些酶单独工作的机制，以及它们如何作为一个网络一起工作。澄清 在激活αIIb的最后步骤中所需的细胞外氧化还原网络β3代表了一个重要的 在血小板功能和血栓形成方面，可以作为激活其他整合素的模型。 确定具体的机制也可能导致新类型的抑制剂，双重调节血小板和 凝结。由于血小板与多种疾病状态有关，我们的发现可能会有更广泛的意义 对其他疾病状况的基本了解的影响，以及可能的治疗方法 这些条件。

Protein disulfide isomerase (PDI) catalyzes the reversible formation and isomerization of disulfide bonds in proteins, and supports thrombosis. Recent reports indicate that other members of the PDI family, ERp57, ERp5, and ERp72 also contribute to thrombosis. Whether the roles of PDI, ERp57, ERp5 and ERp72 in thrombosis were distinct or redundant was unclear. We showed the aggregation defect in ERp72, PDI or ERp57-null mice was only recovered by the specific PDI that was missing. This implies that these enzymes have individual targets in the activation of the αIIbβ3 platelet integrin that supports platelet aggregation. These PDI family members contain the CGHC active-site motif that catalyzes conformational changes in proteins involved in thrombosis. This proposal focuses on two novel members of the PDI family with this motif that are found in platelets; ERp46, and a transmembrane member of the PDI family, TMX3. We now know that PDI, ERp57, ERp5 and ERp72 mediate platelet aggregation and thrombosis, and are involved in conversion of αIIbβ3 to its high affinity state. However, the actual mechanisms by which these PDIs regulate αIIbβ3 and platelet aggregation are unknown. The individual targets of each enzyme and how they function together remains an enigma. To determine the specific function of each PDI in platelets we have used a targeted knockout mice approach. The specific aims are to: 1. Characterize the role of ERp46 in thrombus formation and platelet function; 2. Characterize the role of TMX3 in thrombus formation and platelet function; and 3. Characterize the cysteine/disulfide targets and mechanism of activation of αIIbβ3 by PDI, ERp57, ERp72, ERp46 and TMX3. A principal technique used will be the laser-induced injury model of thrombosis. To determine the actual mechanisms by which multiple members of the PDI family work together we will employ a thiol labeling strategy with mass spectrometry identification of the labeled thiols. This will begin to unravel the mechanisms by which these enzymes work individually, and how they work together as a network. Elucidation of the extracellular redox network required for the final steps in the activation of αIIbβ3 represents a significant aspect of platelet function and thrombus formation, and can be a model for activation of other integrins. Defining the specific mechanisms could also lead to novel types of inhibitors that dually regulate platelets and coagulation. Since platelets are involved in a variety of disease states, our findings will likely have broader implications for basic understanding of other disease conditions, and possible therapeutic approaches for these conditions.