Thiol Isomerases During Thrombus Formation

描述（由申请人提供）：在内质网内蛋白质合成过程中，硫醇异构酶对二硫键的形成至关重要，但位于内质网外的蛋白质硫醇异构酶可能对蛋白质中氧化还原敏感的变构二硫键的调节至关重要。血小板和内皮细胞中的硫醇异构酶可能在血栓形成过程中的蛋白质功能调节中发挥重要作用。我们的假设是，每一种被认为影响体内血栓形成的硫醇异构酶在特定的底物上具有特定的功能。蛋白二硫异构酶和PDI家族成员将通过流式细胞术、免疫荧光显微镜和电子显微镜在静息血小板和未受刺激的内皮细胞中鉴定，并定位于细胞表面或内部细胞器，或两者。将探讨巯基异构酶在血小板活化和内皮细胞刺激过程中的再分配，特别关注酶的释放及其与细胞表面的结合。为了鉴定潜在的硫醇异构酶底物，将标记血小板和内皮细胞表面表达游离硫醇的蛋白质，纯化膜，并使用16-BAC/SDS进行2D凝胶电泳分离这些蛋白质。这些标记的膜蛋白将通过质谱鉴定，并将代表可能的硫醇异构酶底物。为了确定巯基异构酶在血栓形成过程中的作用，我们将通过活体多通道广角共聚焦显微镜研究包括PDI、ERp5、ERp54、ERp72、ERp46、ERdj5、TMX和TMX3在内的巯基异构酶在活体小鼠中的表达动力学。利用巯基异构酶特异性抗体抑制各巯基异构酶对血小板活化的影响：通过钙动员监测血小板活化，通过p -选择素表达检测血小板α颗粒释放，纤维蛋白形成，凝血酶活性，血小板积聚，血小板开/关率，血小板血栓栓塞，微粒积聚，vWF积聚，TF表达，并在活体小鼠激光血管壁损伤模型和氯化铁模型中对胶原暴露进行评价。这些研究旨在确定血小板和内皮细胞释放的硫醇异构酶的作用，以及它们对血栓和止血相关蛋白功能调节的贡献，包括但不限于GPIb、GPIIbIIIa和组织因子。公共卫生相关性：越来越多的证据表明，血栓形成过程中的重要步骤是由关键止血蛋白中不稳定二硫键的氧化状态调节的。这些键的氧化状态是由巯基异构酶家族中的一种酶调节的。提出的研究的中心假设是特定的硫醇异构酶作用于对血栓形成至关重要的特定底物，从而调节血栓平衡。本提案的研究设计和方法部分中描述的实验旨在验证这一假设。建立巯基异构酶在血栓形成调控中的作用，可以为干预血栓性疾病、动脉粥样硬化血栓形成、中风和血栓栓塞性疾病提供新的靶点，这些都是美国发病率和死亡率的主要原因。

DESCRIPTION (provided by applicant): Thiol isomerases are critical for disulfide bond formation during protein synthesis within the endoplasmic reticulum, but protein thiol isomerases located outside of the endoplasmic reticulum may be critical for regulation of redox-sensitive allosteric disulfide bonds in proteins. Thiol isomerases in platelets and endothelial cells may play an important role in the regulation of protein function during thrombus formation. Our hypothesis is that each thiol isomerase that putatively influences thrombus formation in vivo has a specific function on a specific substrate(s). Protein disulfide isomerase and members of the PDI family will be identified in resting platelets and unstimulated endothelial cells, and localized to either the cell surface or internal organelles, or both, by flow cytometry, immunofluorescence microscopy and electron microscopy. The redistribution of thiol isomerases during platelet activation and endothelial cell stimulation, with special focus on the release of the enzymes and their binding to the cell surface, will be explored. To identify potential thiol isomerase substrates, proteins expressing free thiols on the surface of platelets and endothelial cells will be labeled, the membranes purified, and these proteins separated by 2D gel electrophoresis using 16-BAC/SDS. These labeled membrane proteins will be identified by mass spectrometry and will represent possible thiol isomerase substrates. To determine the role of thiol isomerases during thrombus formation, the kinetics of expression of thiol isomerases, including PDI, ERp5, ERp54, ERp72, ERp46, ERdj5, TMX and TMX3 will be studied in a live mouse by intravital multichannel widefield and confocal microscopy. The effect on inhibition of each thiol isomerase using antibodies specific for thiol isomerases on platelet activation monitored by calcium mobilization, platelet alpha granule release detected by P-selectin expression, fibrin formation, thrombin activity, platelet accumulation, platelet on/off rates, platelet thrombus embolization, microparticle accumulation, vWF accumulation, TF expression, and collagen exposure will be evaluated in the laser-induced vessel wall injury model and the ferric chloride model in a living mouse. These studies are designed to determine the role of thiol isomerases released from platelets and endothelial cells, and their contribution to the modulation of function of proteins involved in thrombosis and hemostasis, including but not limited to GPIb, GPIIbIIIa and tissue factor. PUBLIC HEALTH RELEVANCE: Evidence is mounting that important steps in the process of thrombus formation are regulated by the oxidation states of labile disulfide bonds in critical hemostatic proteins. The oxidation state of these bonds is regulated by an enzyme(s) of the thiol isomerase family. The central hypothesis of the proposed research is that specific thiol isomerases act on specific substrates critical to thrombus formation and thus regulate the thrombotic balance. The experiments described in the Research Design and Methods section of this proposal are designed to test this hypothesis. Establishment of a role for thiol isomerases in regulation of thrombus formation could provide novel targets for intervention in thrombotic disorders, atherothrombosis, stroke and thromboembolic disease, major causes of morbidity and mortality in the United States.