Endothelial Cell Thiol Isomerases: Potential Role in Thrombus Formation.
Authors: Jasuja Reema , Furie Bruce , Furie Barbara C. 
Published: April 2020

Abstract
Thiol isomerases are multifunctional enzymes that contain a variable number of thioredoxin-like domains and catalyze the formation and isomerization of disulfide bonds. Members of the thiol isomerase family, including PDI, ERp5, ERp46, ERp57 and ERp72, are found in the endoplasmic reticulum (ER) where they play important roles during protein synthesis. Despite having an ER retention sequence some members of this family have been identified at cellular locations outside the ER with the potential to regulate the activity of proteins with labile disulfide bonds. Some of these proteins are involved in hemostasis, and include tissue factor and platelet surface receptors. Here, we describe the cellular localization and secretion of PDI and the first identification of two additional thiol isomerases in human umbilical vein endothelial cells (HUVEC). Our studies show a rapid secretion of PDI from HUVECs upon activation with thrombin or the calcium ionophore A23187 as detected by immunoblotting of proteins in the culture medium. We have estimated approximately 48000 ± 3600 molecules of PDI per cell. Approximately 8% of HUVEC PDI is secreted over 5 minutes upon cell activation. This secreted PDI is functionally active as measured by the reduction of disulfide bonds in the insulin transhydrogenase assay. Subcellular fractionation studies demonstrated PDI localized in different cellular compartments in activated and quiescent HUVECs. PDI was detected in the ER colocalized with sarcoplasmic and endoplasmic reticulum calcium ATPase (SERCA2) in both resting and activated cells. However PDI was detected in plasma membrane-containing fractions colocalized with alkaline phosphatase in thrombin-activated HUVECs while it was predominantly microsomal in buffer-treated control cells. PDI does not appear colocalized with the Weibel-Palade body marker von Willebrand factor (vWF) in subcellular fractions of activated or resting HUVECs nor is it detected in preparations of isolated Weibel-Palade bodies. Immunocytochemistry confirms localization of PDI in the ER but also shows punctate localization in the cytoplasm. Immunostaining for PDI in human aortic endothelial cells (HAEC) showed a similar staining pattern as HUVEC. Since PDI is secreted following cell activation, it is likely that the punctate PDI staining observed in the cytoplasm indicates PDI localization in secretory granules distinct from Weibel Palade bodies. We have also demonstrated the presence of two thiol isomerases, ERp57 and Erp72, in endothelial cells. Both ERp57 and ERp72 are constitutively secreted from HUVECs with no difference in secretory pattern pre and post activation. In contrast to PDI, ERp57 and ERp72 are detected in the microsomal as well as the plasma membrane fractions regardless of the activation state of the HUVECs. ERp57 was also detected in nuclear fractions consistent with previous immunocytochemical observations for ERp57. The regulated secretion of active PDI from endothelial cells and its rapid kinetics of release along with its subcellular localization suggest a potential role for endothelial cell PDI in modulation of protein structure and function in proteins regulated by endothelial cell injury, such as inflammation and thrombosis.