Endothelium-derived but not platelet-derived protein disulfide isomerase is required for thrombus formation in vivo

Abstract

Protein disulfide isomerase (PDI) catalyzes the oxidation reduction and isomerization of disulfide bonds. We have previously identified an important role for extracellular PDI during thrombus formation in vivo. Here, we show that endothelial cells are a critical cellular source of secreted PDI, important for fibrin generation and platelet accumulation in vivo. Functional PDI is rapidly secreted from human umbilical vein endothelial cells in culture upon activation with thrombin or after laser-induced stimulation. PDI is localized in different cellular compartments in activated and quiescent endothelial cells, and is redistributed to the plasma membrane after cell activation. In vivo studies using intravital microscopy show that PDI appears rapidly after laser-induced vessel wall injury, before the appearance of the platelet thrombus. If platelet thrombus formation is inhibited by the infusion of eptifibatide into the circulation, PDI is IntroductionA considerable body of evidence implicates the oxidation state of labile disulfide bonds in critical hemostatic proteins in regulating the process of thrombus formation. 1 The oxidation state of these bonds is regulated by an enzyme(s) of the thiol isomerase family. Thiol isomerases, including protein disulfide isomerase (PDI), while containing endoplasmic reticulum retention signals, are found extracellularly. Among the cells that secrete PDI and display the enzyme on their surface are platelets and endothelial cells. [2][3][4][5][6][7] The importance of thiol-disulfide balance for platelet function has long been recognized. For example, reduced glutathione and cysteine inhibit platelet aggregation induced by several agonists, while dithiothreitol and ␤-mercaptoethanol promote aggregation. 4 PDI likely plays an important role in maintaining this balance. The levels of both PDI and ERp5, another member of the PDI family, on the platelet surface increase significantly upon agonist stimulation. 4,6 PDI has been implicated in ␣ IIb ␤ 3 and ␣ 2 ␤ 1 activity, 8,9 and glycoprotein Ib ␣ expresses one or more free thiols on the activated platelet surface, but not on resting platelets. 4 Inhibitory anti-PDI antibodies or bacitracin, a nonspecific inhibitor of thiol isomerases, inhibit platelet activation in vitro, suggesting that ␣ IIb ␤ 3 -dependent platelet aggregation and secretion require thiol isomerases. 10 PDI may play a role in the de-encryption of tissue factor. [11][12][13] In contrast, there is less information to support potential roles of extracellular thiol isomerases in the function of endothelial cells. Endothelial cells in culture secrete PDI, which then is bound to the cell surface. 5 A novel thiol isomerase that appears to be endothelial cell specific, EndoPDI or ERp46, has been reported. 14 Recent evidence indicates that the protein disulfide isomerases, ERp46 and ERp57, are present in endothelial cell plasma membrane preparations. 15 Endothelial cells in culture secrete an activity that reduces the size of very large multimers of von Willebrand factor (VW...

Application of whole‐exome sequencing to direct the specific functional testing and diagnosis of rare inherited bleeding disorders in patients from the Öresund Region, Scandinavia

“…We also examined EDS and HT predisposing genes. As protein disulphide isomerase (PDI) was recently described to play a key role in clot formation (Jasuja et al , 2010), the P4HB gene was included to examine whether loss‐of‐function in P4HB could be a previously undiscovered cause of bleeding tendency. Finally, the search was expanded to genes associated with specific phenotypic (bleeding, thrombocytopenia, thrombocytopathy or symptomatic (hypermobility) search terms.…”

Protein Disulfide Isomerase Directly Interacts with β-Actin Cys374 and Regulates Cytoskeleton Reorganization

“…PDI has multiple biological functions and interacts with numerous proteins (12). Extracellular PDI is known to mediate platelet aggregation and thrombus formation (13,14). After activation of the platelets and endothelial cells, it can be found both inside and outside of the cells, in association with F-actin microfilaments or with cellular membranes, respectively (14 -18).…”

Differential proteomic analysis of platelets suggested target-related proteins in rabbit platelets treated with Rhizoma Corydalis

“…Protein disulphide isomerase-associated 3, a membrane of protein disulphide isomerase (PDI), was also downregulated by RC treatment. Evidence indicates that the oxidation state of labile disulphide bonds play critical roles in regulating the process of thrombus formation, and important for fibrin generation and platelet accumulation (Cho et al 2008;Jasuja et al 2010).…”