Extracellular protein disulfide isomerase regulates coagulation on endothelial cells through modulation of phosphatidylserine exposure

Abstract

Tissue factor (TF) is the cellular receptor for plasma protease factor VIIa (FVIIa), and the TF-FVIIa complex initiates coagulation in both hemostasis and thrombosis. Cell surface-exposed TF is mainly cryptic and requires activation to fully exhibit the procoagulant potential. Recently, the protein disulfide isomerase (PDI) has been hypothesized to regulate TF decryption through the redox switch of an exposed disulfide in TF extracellular domain. In this study, we analyzed PDI contribution to coagulation using an in vitro endothelial cell model. In this model, extracellular PDI is detected by imaging and flow cytometry. Inhibition of cell surface PDI induces a marked increase in TF procoagulant function, whereas exogenous addition of PDI inhibits TF decryption. The coagulant effects of PDI inhibition were sensitive to annexin V treatment, suggesting exposure of phosphatidylserine (PS), which was confirmed by prothrombinase assays and direct labeling.In contrast, exogenous PDI addition enhanced PS internalization. Analysis of fluorescent PS revealed that PDI affects both the apparent flippase and floppase activities on endothelial cells. In conclusion, we identified a new mechanism for PDI contribution to coagulation on endothelial cells, namely, the regulation of PS exposure, where PDI acts as a negative regulator of coagulation. (Blood. 2010; 116(6):993-1001) IntroductionTissue factor (TF) is a transmembrane glycoprotein that binds with high affinity to the plasma protease factor VII in either zymogen (FVII) or activated form (FVIIa). The formation of the TF-FVIIa complex is crucial for initiation of coagulation, leading to thrombin generation and fibrin formation. Although the primary role of TF-FVIIa is to maintain hemostasis after vascular injury, aberrant activation of coagulation underlies thrombosis, the major cause of mortality in most industrialized countries. 1 In physiologic conditions, initiation of coagulation is maintained silent by restricting the exposure of TF to the plasma factors. 2,3 In pathologic conditions, however, TF may become exposed on the endothelium 4,5 and on circulating monocytes. 6 At these sites, TF can initiate thrombotic events associated with sepsis, 4,7 cancer, 8,9 or atherosclerosis. 10,11 Multiple in vitro studies have indicated that most of the cell-exposed TF is "cryptic," thus not fully active toward coagulation, 12,13 and TF "decryption" has been proposed as the initial step in the activation of coagulation. 14 Although the molecular mechanisms of TF decryption are not completely understood, many of the stimuli that decrypt TF also increase the exposure of phosphatidylserine (PS), [15][16][17][18] which is known to enhance coagulation. PS-independent mechanisms of TF decryption have also been postulated, such as TF self-association, 19 association with lipid rafts, [20][21][22] and the redox switch of an exposed disulfide in the membrane proximal domain of TF. 23 Protein disulfide isomerase (PDI) is an oxidoreductase 24 localized mainly in the endoplasmic reticulum (ER...

Inhibition of thiol isomerase activity diminishes endothelial activation of plasminogen, but not of protein C

“…Thiol isomerases may alter allosteric disulphides in a component of the AIIt heterotetramer to which tPA binds on the endothelial surface. Alternatively, PDI-mediated alteration of PS exposure [13] may destabilise the anchoring of AIIt by annexin A2, and thus diminish endothelial surface plasminogen activation.…”

“…PDI associates with tissue factor and targets the allosteric cys186-cys209 disulphide bond [12]. An alternative mechanism for the action of PDI on tissue factor is via modulated exposure of phosphatidylserine (PS) on the cell surface, since PDI has been shown to increase PS internalisation by affecting both flippase and floppase enzymes [13].…”

Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase

“…Furthermore, pretreatment of THP1 cells with RL90 had no effect on ATG-induced PS externalization ( Figure 5G), indicating that inhibition of cellular PCA was not caused by decreased complement activation or modulation of PDI-dependent regulation of PS asymmetry. 34 Despite similar binding to various cell lines (supplemental Figure 5B), ATG only activated TF on monocytic U937 and myelomonocytic HL60 cells, but not on colorectal cancer HT29, myeloma IM9, glioma G44, or breast cancer MDA-MB231 cells (supplemental Figure 5C), pointing to a cell type-specific effect of ATG. PDI antigen was detected on HL60 cells ( Figure 5H), and both surface-associated PDI activity and ATG-mediated TF activation in this cell line were inhibited by anti-PDI RL90, rutin, and bacitracin ( Figure 5I), demonstrating that functional PDI was required for the TF activation process.…”

The lipid peroxidation product 4-hydroxy-2-nonenal induces tissue factor decryption via ROS generation and the thioredoxin system

“…Overall, our data are in agreement with earlier reports that show thiol pathways play a major role in the regulation of PS dynamics in the plasma membrane. [39][40][41] In summary, our present study delineates potential mechanisms by which oxidative stress transforms cryptic TF into procoagulantactive TF on the cell surface of monocytes and macrophages. Our data show that oxidative stress can activate TF via at least 2 different nonoverlapping pathways: a ROS-dependent pathway and a TrxR/Trx-dependent pathway.…”

“…Earlier data suggested that sulfhydryl-modifying agents modulate cellsurface PS dynamics by regulating the aminophospholipid translocases. [37][38][39] Therefore, it is possible that a direct modification of aminophospholipid translocases by PAO may be responsible for blocking HNE-induced PS externalization and TF decryption. Overall, our data are in agreement with earlier reports that show thiol pathways play a major role in the regulation of PS dynamics in the plasma membrane.…”