PB1365 - The Ig-like repeat 24 of filamin A is essential for the induction of migration by tissue factor containing extracellular vesicles in human coronary artery smooth muscle cells

Background: The interaction of the cytoplasmic domain of tissue factor (TF) with immunoglobulin (Ig)-like repeats 22-24 within filamin A (FlnA) has been implicated in TF induced cell migration and also, the incorporation and release of TF within extracellular vesicles (EV). However, the specific Ig-like repeat responsible for interaction with TF has not been identified.

Aims: To examine the contribution of the interaction of TF to Ig-like repeats 22-24 of FlnA in HCASMC migration and the incorporation of TF within EV.

Methods: HCASMC and MDA-MB-231 cells were transfected with the pcDNA 3 c-myc plasmid containing the wild type or mutant FlnA, lacking Ig-like repeats 22-24, 23-24 or 24, and the cells were permitted to express the proteins for 48h. Cell migration was assessed using Transwell chambers (8 µm pore). HCASMC (3 × 104) migration was stimulated using EV derived from endothelial cells expressing TFWt-tGFP, tGFP, or with PDGF-BB (10 ng/ml) placed in the lower chamber. After 18 h incubation, migrating cells were visualised by microscopy and cell numbers determined by crystal violet assay. MDA-MB-231 cells (3 × 105) were incubated in serum free media for 4h. TF+EV were isolated from the media by ultracentrifugation and qualified by TF-ELISA.

Results: Induction of migration by TF+EV in HCASMC was significantly reduced in cells expressing mutant FlnA lacking Ig-like repeats (22-24, 23-23 or 24). In contrast, migration induced by PDGF-BB remained unaltered by expression of FlnA mutants. Additionally, release of TF+EV from MDA-MB-231 cells was not significantly impacted by the expression of FlnA mutants.

Conclusion(s): The binding of TF to FlnA is required for induction of migration by TF+EV in HCASMC, however does not influence to incorporation of TF into EV. Furthermore, Ig-like repeat 24 of FlnA was specifically shown to be required for TF-mediated migration, but not in response to other stimuli such as that by growth-factor receptors.