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
Camille Ettelaie
Benjamin T. Goult
Professor Anthony Maraveyas A.Maraveyas@hull.ac.uk
Professor in Cancer Medicine
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 |
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Acceptance Date | Jul 12, 2017 |
Online Publication Date | Nov 30, 2017 |
Publication Date | 2017-11 |
Deposit Date | Oct 31, 2017 |
Publicly Available Date | Oct 27, 2022 |
Journal | Thrombosis and haemostasis |
Print ISSN | 0340-6245 |
Electronic ISSN | 2567-689X |
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. |
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©2018 The authors
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