@article { , title = {Investigation of the filamin A-Dependent mechanisms of tissue factor incorporation into microvesicles}, 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.}, doi = {10.1160/TH17-01-0009}, eissn = {2567-689X}, issn = {0340-6245}, issue = {11}, journal = {Thrombosis and haemostasis}, pages = {2034-2044}, publicationstatus = {Published}, publisher = {Schattauer}, url = {https://hull-repository.worktribe.com/output/456141}, volume = {117}, keyword = {Health and Health Inequalities, Tissue factor, Filamin-A, Microvesicles, Protease-activated receptor 2, Lipid rafts}, year = {2017}, author = {Collier, Mary E. W. and Ettelaie, Camille and Goult, Benjamin T. and Maraveyas, Anthony and Goodall, Alison H.} }