Investigation of the filamin A-Dependent mechanisms of tissue factor incorporation into microvesicles

Collier, Mary E. W.; Ettelaie, Camille; Goult, Benjamin T.; Maraveyas, Anthony; Goodall, Alison H.

Abstract

We have previously shown that phosphorylation of tissue factor (TF) at Ser253 increases the incorporation of TF into microvesicles (MVs) following protease-activated receptor 2 (PAR2) activation through a process involving filamin-A, whereas Ser258 phosphorylation suppresses this process. Here we examined the contribution of the individual phosphorylation of these serine residues to the interaction between filamin-A and TF, and further examined how filamin-A regulates the incorporation of TF into MVs. In vitro binding assays using recombinant filamin-A C-terminal repeats 22-24 with biotinylated phospho-TF cytoplasmic domain peptides as bait, showed that filamin-A had the highest binding affinities for phospho-Ser253 and double-phosphorylated TF peptides, whilst the phospho-Ser258 TF peptide had the lowest affinity. Analysis of MDA-MB-231 cells using an in situ proximity ligation assay revealed increased proximity between the C-terminus of filamin-A and TF following PAR2 activation, which was concurrent with Ser253 phosphorylation and TF-positive MV release from these cells. Knock-down of filamin-A expression suppressed PAR2-mediated increases in cell surface TF procoagulant activity without reducing cell surface TF antigen expression. Disrupting lipid rafts by pre-incubation with methyl-beta cyclodextrin (MβCD) prior to PAR2 activation reduced TF-positive MV release and cell surface TF procoagulant activity to the same extent as filamin-A knock-down. In conclusion, this study shows that the interaction between TF and filamin-A is dependent on the differential phosphorylation of Ser253 and Ser258. Furthermore the interaction of TF with filamin-A may translocate cell surface TF to cholesterol-rich lipid rafts, increasing cell surface TF activity as well as TF incorporation and release into MVs.

Citation

Collier, M. E. W., Ettelaie, C., Goult, B. T., Maraveyas, A., & Goodall, A. H. (2017). Investigation of the filamin A-Dependent mechanisms of tissue factor incorporation into microvesicles. Thrombosis and haemostasis, 117(11), 2034-2044. https://doi.org/10.1160/TH17-01-0009

• Journal Article Type: Article
• Acceptance Date: Jul 12, 2017
• Online Publication Date: Nov 30, 2017
• Publication Date: 2017-11
• Deposit Date: Oct 31, 2017
• Publicly Available Date: Oct 10, 2018
• Journal: Thrombosis and haemostasis
• Print ISSN: 0340-6245
• Publisher: Schattauer
• Peer Reviewed: Peer Reviewed
• Volume: 117
• Issue: 11
• Pages: 2034-2044
• DOI: https://doi.org/10.1160/TH17-01-0009
• Keywords: Tissue factor, Filamin-A, Microvesicles, Protease-activated receptor 2, Lipid rafts
• Public URL: https://hull-repository.worktribe.com/output/456141
• Publisher URL: https://th.schattauer.de/en/contents/archive/issue/special/manuscript/27993.html
• Additional Information: This is a description of an article published in Thrombosis and haemostasis, 2017.
• Contract Date: Oct 31, 2017