De-Palmitoylation of Tissue Factor Regulates Its Activity, Phosphorylation and Cellular Functions

Abstract

In this study, the role of de-palmitoylation of tissue factor (TF) in the decryption of its activity was explored. TF-tGFP constructs were prepared by mutagenesis-substitution at Cys245 to prevent or mimic palmitolyation. Additionally, to reduce TF de-palmitoylation, the expression of palmitoyl-protein thioesterases (PPT) was suppressed. Other TF mutants were prepared with altered flexibility, hydrophobicity or length of the transmembrane domain. The outcome of these alterations on fXa-generation, fVIIa binding, Ser253 phosphorylation and TF-microvesicle release were assessed in endothelial cells, and the influence on endothelial and MCF-7 cell proliferation and apoptosis was analysed. Preventing TF palmitoylation (TFSer245-tGFP), increasing the hydrophobicity (TFPhe241-tGFP) or lengthening (TFLongTM-tGFP) of the transmembrane domain enhanced fXa-generation in resting cells compared to cells expressing TFWt-tGFP, but fXa-generation was not further increased following PAR2 activation. Extending the available length of the transmembrane domain enhanced the TF-tGFP release within microvesicles and Ser253 phosphorylation and increased cell proliferation. Moreover, prevention of PKCα-mediated Ser253 phosphorylation with Gö6976 did not preclude fXa-generation. Conversely, reducing the hydrophobicity (TFSer242-tGFP), shortening (TFShortTM-tGFP) or reducing the flexibility (TFVal225-tGFP) of the transmembrane domain suppressed fXa-generation, fVIIa-HRP binding and Ser253 phosphorylation following PAR2 activation. PPT knock-down or mimicking palmitoylation (TFPhe245-tGFP) reduced fXa-generation without affecting fVIIa binding. This study has for the first time shown that TF procoagulant activity is regulated through de-palmitoylation, which alters the orientation of its transmembrane domain and is independent of TF phosphorylation. However, Ser253 phosphorylation is facilitated by changes in the orientation of the transmembrane domain and can induce TF-cellular signalling that influences cellular proliferation/apoptosis.