Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles

Collier, Mary E. W.; Ettelaie, Camille; Maraveyas, Anthony

Abstract

We previously reported that the incorporation of tissue factor (TF) into cell-derived microvesicles (MVs) is regulated by the phosphorylation of the cytoplasmic domain of TF. Since the cytoskeletal protein filamin-A is known to bind to the cytoplasmic domain of TF in a phosphorylation-dependent manner, the involvement of filamin-A in the incorporation of TF into MVs was examined. Endothelial cells were transfected to express TF, whereas MDA-MB-231 cells were used to examine endogenously expressed TF. MV release was induced by activating protease-activated receptor-2 (PAR2). Partial suppression of filamin-A expression using two different filamin-A siRNA sequences resulted in significant reductions in the incorporation of TF antigen into MVs as determined by TF-ELISA and western blot analysis, and was reflected in reduced thrombin-generation and FXa-generation capacities of these MVs. Deletion of the cytoplasmic domain of TF also resulted in reduced incorporation of TF into MVs, whereas the suppression of filamin-A expression had no additional effect on the incorporation of truncated TF into MVs. Partial suppression of filamin-A expression had no effect on the number and size distribution of the released MVs. However, >90 % suppression of filamin-A expression resulted in increased MV release, possibly as a result of increased instability of the plasma membrane and underlying cytoskeleton. In conclusion, the presence of filamin-A appears to be essential for the incorporation of TF into MVs following PAR2 activation, but is not required for the process of MV formation and release following PAR2 activation.

Citation

Collier, M. E. W., Ettelaie, C., & Maraveyas, A. (2014). Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles. Thrombosis and haemostasis, 111(4), 647-655. https://doi.org/10.1160/TH13-09-0769

• Acceptance Date: Oct 23, 2013
• Online Publication Date: Nov 21, 2013
• Publication Date: 2014-03
• Deposit Date: Jun 5, 2015
• Publicly Available Date: Jun 5, 2015
• Journal: Thrombosis and haemostasis
• Print ISSN: 0340-6245
• Publisher: Schattauer
• Peer Reviewed: Peer Reviewed
• Volume: 111
• Issue: 4
• Pages: 647-655
• DOI: https://doi.org/10.1160/TH13-09-0769
• Keywords: Tissue factor; Endothelial cells; Microparticles; Microvesicles; Filamin-A
• Public URL: https://hull-repository.worktribe.com/output/374780
• Publisher URL: https://www.thieme-connect.de/DOI/DOI?10.1160/TH13-09-0769
• Additional Information: This article is not an exact copy of the original published article in Thrombosis and haemostasis. The definitive publisher-authenticated version of: Collier, M. E. W., Maraveyas, A., Ettelaie, C. Filamin-A is required for the incorporation of tissue factor into cell-derived microvesiclesis. Thrombosis and haemostasis, 2014, v.111, issue 4 - is available online at: http://th.schattauer.de/en/contents/archive/issue/1852/manuscript/20528.html
• Contract Date: Jun 5, 2015