title: Filamin-A is required for the incorporation of tissue factor into cell-derived microvesicles.
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.
authors.affiliation: Dr. Mary Collier, Biomedical Section, Department of Biological Sciences, University of Hull, Hull, HU6 7RX, UK, Tel.: +44 1482 465528; Fax: +44 1482 465458, E-mail: mary_collier8@hotmail.com.
authors.forename: Mary E W
authors.initials: ME
authors.lastname: Collier
authors.forename: Anthony
authors.initials: A
authors.lastname: Maraveyas
authors.forename: Camille
authors.initials: C
authors.lastname: Ettelaie
mesh_terms: D055252:Cell-Derived Microparticles / Q000737:chemistry / Q000378:metabolism*; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000502:physiology*; D015951:Factor Xa / Q000096:biosynthesis* / Q000235:genetics; D064448:Filamins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D010316:Particle Size; D015202:Protein Engineering; D017434:Protein Structure, Tertiary / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D044464:Receptor, PAR-2 / Q000378:metabolism*; D055571:Secretory Pathway; D017384:Sequence Deletion / Q000235:genetics; D013917:Thrombin / Q000096:biosynthesis* / Q000235:genetics; D013925:Thromboplastin / Q000235:genetics / Q000378:metabolism*
chemical_list: D064448:Filamins; D034741:RNA, Small Interfering; D044464:Receptor, PAR-2; D013925:Thromboplastin; D013917:Thrombin; D015951:Factor Xa
keywords: Tissue factor; endothelial cells; filamin-A; microparticles; microvesicles