Differential Effects of Protein Disulfide Isomerase (PDI) Inhibitors On the Expression of Tissue Factor Procoagulant Activity (TF PCA) by AML Blasts
Authors: Fischer Cornelia , Spath Brigitte , Amirkhosravi Ali , Fiedler Walter , Bokemeyer Carsten , Langer Florian 
Published: October 2019

Abstract
Abstract 1112
Acute myelogenous leukemia (AML) may be complicated by DIC. TF plays a critical role in AML-associated coagulopathy, and induction of apoptosis significantly increases TF PCA on leukemic blasts, mainly via phosphatidylserine (PS) membrane exposure. However, PDI, a thiol isomerase with oxidoreductase and chaperone activity, has also been implicated in cellular TF regulation. Particularly, PDI inhibitors have been shown to exert antithrombotic activity in animal models. Besides its predominant localization in the endoplasmic reticulum, PDI is present on cell surfaces, where it may represent a promising therapeutic target. We investigated the effect of PDI inhibitors on the expression of TF PCA by leukemic HL60 and THP1 cells to explore their potential as anticoagulant drugs for the prevention and/or treatment of AML-associated DIC. Using a fluorescence-based insulin reduction assay, we confirmed inhibition of recombinant human PDI by bacitracin and quercetin-3-rutinoside (also known as rutin and recently shown to be a specific PDI inhibitor) with IC50 values of 0.6 mM and 14 μM, respectively, showing >95% inhibition at 1 mM (bacitracin) and 50 μM (rutin). Significant insulin reductase activity was observed on HL60 cells, and this activity was inhibited by 75% and 49% using 1 mM bacitracin and 100 μM rutin, respectively, suggesting the presence of additional, PDI-independent thiol isomerase activity. Short-term treatment with 100 μM rutin for 15 min also inhibited TF PCA on HL60 cells by 37%. Importantly, the inhibitory effect of rutin on cell-associated PDI and TF activity was completely abolished by cell washing, confirming previous evidence that rutin is a reversible PDI inhibitor. When HL60 cells were exposed to rutin (100 μM) for 24 hrs, cell-associated TF PCA was increased 2.3-fold (P<0.01), an effect that was accompanied by enhanced PS exposure, as assessed by annexin V-FITC binding (positive cells, 32±11 vs. 10±4%; P<0.01), and increased PCA of cellular microparticles (MPs) isolated from culture supernatants, as evidenced by the thrombin generation parameters lag phase (LP, 14±1 vs. 19±4 min), peak thrombin (PT, 55±17 vs. 22±14 nM), and area under the curve (AUC, 1193±329 vs. 476±347 nM*min; P<0.01). Interestingly, treatment with 100 μM rutin also resulted in a 1.7-fold increase in total cellular TF antigen (P=0.07). The effects of long-term incubation with bacitracin (1 mM) were even more pronounced, involving an 8.3-fold and 4.6-fold increase in cell-associated TF PCA and total cellular TF antigen, respectively. PS exposure (45±9%) and shedding of procoagulant MPs (LP, 7±1 min; PT, 175±49 nM; AUC, 2756±402 nM*min) were also significantly increased. While neither short-term nor long-term exposure to rutin affected TF PCA on THP1 cells, co-incubation with rutin dose-dependently (10–100 μM) inhibited daunorubicin-induced TF PCA in this cell model, an effect that could not be explained by decreased PS exposure. Importantly, both the reaction pattern of HL60 and that of THP1 cells were reproduced ex vivo using myeloblasts from AML patients. In summary, our findings suggest a highly complex and context-dependent role of PDI in leukemic-cell TF PCA expression. While short-term exposure to rutin can reversibly inhibit both PDI and TF activity, long-term exposure may result in significantly increased cellular TF PCA and MP shedding, pointing to a possible role of PDI in PS homeostasis, cytoskeleton rearrangement, and/or TF recycling. In addition, induction of leukemic-cell apoptosis and necrosis by cytotoxic drugs, which is associated with an early loss in membrane integrity and enhanced accessibility of cytoplasmic enzymes, may involve an additional role of (intracellular) PDI in the efficient presentation of TF PCA by AML blasts.

Disclosures:
No relevant conflicts of interest to declare.