Lung cancer chemotherapy agents increase procoagulant activity via protein disulfide isomerase-dependent tissue factor decryption

Abstract

Lung cancer patients undergoing chemotherapy have an elevated risk for thrombosis. However, the mechanisms by which chemotherapy agents increase the risk for thrombosis remains unclear. The aim of this study was to determine the mechanism(s) by which lung cancer chemotherapy agents cisplatin, carboplatin, gemcitabine, and paclitaxel elicit increased tissue factor activity on endothelial cells, A549 cells, and monocytes. Tissue factor activity, tissue factor antigen, and phosphatidylserine exposure were measured on chemotherapy-treated human umbilical vein endothelial cells (HUVEC), A549 cells, and monocytes. Cell surface protein disulfide isomerase (PDI) and cell surface free thiol levels were measured on HUVEC and A549 non-small cell lung carcinoma cells. Treatment of HUVECs, A549 cells, and monocytes with lung cancer chemotherapy significantly increased cell surface tissue factor activity. However, elevated tissue factor antigen levels were observed only on cisplatin-treated and gemcitabine-treated monocytes. Cell surface levels of phosphatidylserine were increased on HUVEC and monocytes treated with cisplatin/gemcitabine combination therapy. Chemotherapy also resulted in increased cell surface levels of PDI and reduced cell surface free thiol levels. Glutathione treatment and PDI inhibition, but not phosphatidylserine inhibition, attenuated tissue factor activity. Furthermore, increased tissue factor activity was reversed by reducing cysteines with dithiothreitol. These studies are the first to demonstrate that lung cancer chemotherapy agents increase procoagulant activity on endothelial cells and A549 cells by tissue factor decryption through a disulfide bond formation in a PDI-dependent mechanism.

Regulation of tissue factor in NT2 germ cell tumor cells by cisplatin chemotherapy

“…mechanism, in addition to pleiotropic effects on normal vascular cells such as monocytes and endothelial cells [6][7][8][9], could thus contribute to the clinically well described cisplatin-induced hypercoagulability in GCT patients. In this regard, it is important to note that the presence of liver metastases was associated with an almost 5-fold increased risk for major thromboembolic events in GCT patients undergoing cisplatin-based combination chemotherapy [2], suggesting that direct effects of administered cytotoxic agents on cancer cells in gross tumor manifestations are likely to play a role in vivo.…”

“…Most recently, protein disulfide isomerase (PDI) has been implicated in the activation of TF on cisplatin-treated NSCLC cells, an effect that was not associated with an increase in cellular TF antigen [8]. To investigate the role of PDI in our in vitro system, NT2 cells were co-incubated with cisplatin and either of two PDI inhibitors, bacitracin or rutin, for 48 hrs.…”

“…Consistently, systemic markers of endothelial perturbation are increased in patients receiving cisplatin chemotherapy [7]. In contrast, cisplatin may increase TFspecific procoagulant activity (PCA) on peripheral blood monocytes and non-small cell lung cancer (NSCLC) cells through a mechanism that, at least to some extent, involves activation of preexisting TF, rather than de novo TF synthesis [8,9]. These findings suggest that cisplatin exerts pleiotropic effects in a cell type-specific manner.…”

Procoagulant effects of lung cancer chemotherapy

“…Isolated monocytes were re-suspended in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin at a density of 1 Â 10 6 cells/ml and treated with chemotherapy agents cisplatin, gemcitabine, carboplatin, and paclitaxel diluted in RPMI-1640 to 20, 60, 40, and 4 mmol/l, respectively, based on combinations and maximal plasma concentrations observed in cancer patients as previously reported [10].…”

“…The procoagulant activity of cancer cells (pancreatic, breast, and colorectal) has been shown to be linked with the cell surface expression of tissue factor (TF) [8,9]. We have recently demonstrated that chemotherapy agents can induce procoagulant TF activity on the cell surface of lung cancer cells via TF decryption [10]. However, other potential mechanisms by which cancer and/or chemotherapy induce a hypercoagulable state include platelet activation [11], increasing levels of circulating TF and/or phosphatidylserine-bearing microparticles [12][13][14], and increasing levels of procoagulant cell-free DNA (CFDNA) [15].…”