Mary E. W. Collier
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.
Authors
Dr Camille Ettelaie C.Ettelaie@hull.ac.uk
Lecturer
Benjamin T. Goult
Anthony Maraveyas
Alison H. Goodall
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 |
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©2018 The authors
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