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

Collier, Mary EW and Ettelaie, Camille and Goult, Benjamin T and Maraveyas, Anthony and Goodall, Alison H (2017) Investigation of the filamin-A dependent mechanisms of tissue factor incorporation into microvesicles. Thrombosis and Haemostasis, (11). pp. 2009-2211. ISSN 0340-6245. (doi:https://doi.org/10.1160/TH17-01-0009) (Access to this publication is currently restricted. You may be able to access a copy if URLs are provided)

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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.

Item Type: Article
Subjects: Q Science > QH Natural history > QH581.2 Cell Biology
Divisions: Faculties > Sciences > School of Biosciences
Depositing User: Ben Goult
Date Deposited: 12 Sep 2017 11:48 UTC
Last Modified: 10 Jan 2018 14:02 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/62507 (The current URI for this page, for reference purposes)
Goult, Benjamin T: https://orcid.org/0000-0002-3438-2807
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