Functional Role of Protein Disulfide Isomerase Isoforms in Platelets

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。 蛋白质，并支持血栓形成。最近的报道表明PDI家族的其他成员，ERp 57， ERp 5和ERp 72也有助于血栓形成。是否PDI、ERp 57、ERp 5和ERp 72在 血栓形成是明显的还是多余的尚不清楚。我们发现ERp 72、PDI或 ERp 57缺失小鼠仅通过缺失的特异性PDI恢复。这意味着这些酶 在支持血小板聚集的αIIbβ3血小板整合素的活化中具有单独的靶点。这些 PDI家族成员含有CGHC活性位点基序，催化蛋白质的构象变化 与血栓形成有关该建议集中于具有该基序的PDI家族的两个新成员， 在血小板中发现; ERp 46和PDI家族的跨膜成员TMX 3。我们现在知道PDI， ERp 57、ERp 5和ERp 72介导血小板聚集和血栓形成，并参与血小板聚集和血栓形成的转化。 αIIbβ3的高亲和力状态。然而，这些PDIs调节αIIbβ3和α IIbβ3的实际机制还不清楚。 血小板聚集是未知的。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血小板中每个PDI的具体功能，我们使用了靶向的 敲除小鼠接近。具体目标是：1.表征ERp 46在血栓形成中的作用 和血小板功能; 2.表征TMX 3在血栓形成和血小板功能中的作用;以及3. 表征半胱氨酸/二硫键靶点以及PDI、ERp 57、ERp 72激活αIIbβ3的机制， ERp 46和TMX 3。使用的主要技术将是血栓形成的激光诱导损伤模型。到 确定PDI家族的多个成员共同工作的实际机制，我们将采用 巯基标记策略与标记的巯基的质谱鉴定。这将开始解开 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。阐发 αIIbβ3激活的最后步骤所需的细胞外氧化还原网络代表了一个显著的 在血小板功能和血栓形成方面，可以作为其他整合素活化的模型。 明确具体的机制也可能导致新型抑制剂的出现，这种抑制剂可以双重调节血小板， 凝血由于血小板参与了多种疾病状态，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理解的影响，以及可能的治疗方法 了以下条件

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.