Ettelaie Camille, Featherby Sophie, Rondon Araci M R, Greenman John, Versteeg Henri H, Maraveyas Anthony
Biomedical Section, University of Hull, Cottingham Road, Hull HU6 7RX, UK.
Einthoven Laboratory for Vascular and Regenerative Medicine, Division of Thrombosis and Hemostasis, Department of Internal Medicine, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands.
Cancers (Basel). 2021 Jul 30;13(15):3837. doi: 10.3390/cancers13153837.
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 (TF-tGFP), increasing the hydrophobicity (TF-tGFP) or lengthening (TF-tGFP) of the transmembrane domain enhanced fXa-generation in resting cells compared to cells expressing TF-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 (TF-tGFP), shortening (TF-tGFP) or reducing the flexibility (TF-tGFP) of the transmembrane domain suppressed fXa-generation, fVIIa-HRP binding and Ser253 phosphorylation following PAR2 activation. PPT knock-down or mimicking palmitoylation (TF-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.
在本研究中，探讨了组织因子（TF）去棕榈酰化在其活性解密中的作用。通过将半胱氨酸245进行诱变取代制备TF-tGFP构建体，以防止或模拟棕榈酰化。此外，为减少TF去棕榈酰化，抑制了棕榈酰蛋白硫酯酶（PPT）的表达。还制备了其他跨膜结构域柔韧性、疏水性或长度改变的TF突变体。在内皮细胞中评估了这些改变对凝血因子Xa（fXa）生成、凝血因子VIIa（fVIIa）结合、丝氨酸253磷酸化和TF微泡释放的影响，并分析了对内皮细胞和MCF-7细胞增殖及凋亡的影响。与表达TF-tGFP的细胞相比，阻止TF棕榈酰化（TF-tGFP）、增加跨膜结构域的疏水性（TF-tGFP）或延长其长度（TF-tGFP）可增强静息细胞中的fXa生成，但在蛋白酶激活受体2（PAR2）激活后fXa生成并未进一步增加。延长跨膜结构域的可用长度可增强TF-tGFP在微泡中的释放以及丝氨酸253磷酸化，并增加细胞增殖。此外，用Gö6976阻止蛋白激酶Cα（PKCα）介导的丝氨酸253磷酸化并不妨碍fXa生成。相反，降低跨膜结构域的疏水性（TF-tGFP）、缩短其长度（TF-tGFP）或降低其柔韧性（TF-tGFP）可抑制PAR2激活后的fXa生成、fVIIa-辣根过氧化物酶（HRP）结合和丝氨酸253磷酸化。PPT敲低或模拟棕榈酰化（TF-tGFP）可减少fXa生成，而不影响fVIIa结合。本研究首次表明，TF促凝活性通过去棕榈酰化来调节，这会改变其跨膜结构域的方向，且独立于TF磷酸化。然而，跨膜结构域方向的改变会促进丝氨酸253磷酸化，并可诱导影响细胞增殖/凋亡的TF细胞信号传导。
