Summary
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.
Keywords
Tissue factor - filamin-A - microvesicles - microparticles - endothelial cells
