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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.


Mary E. W. Collier

Camille Ettelaie

Benjamin T. Goult

Alison H. Goodall


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.


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.

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 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
Keywords Tissue factor, Filamin-A, Microvesicles, Protease-activated receptor 2, Lipid rafts
Public URL
Publisher URL
Additional Information This is a description of an article published in Thrombosis and haemostasis, 2017.


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