Journal: Thrombosis and Haemostasis

Article Title: Investigation of the Filamin A–Dependent Mechanisms of Tissue Factor Incorporation into Microvesicles

doi: 10.1160/th17-01-0009

Figure Lengend Snippet: Fig. 2 Tissue factor–microvesicle (TF-MV) release and TF phosphorylation in MDA-MB-231 cells following protease-activated receptor 2 (PAR2) activation. MDA-MB-231 cells were activated with PAR2 agonist peptide (PAR2-AP; 20 μM) and MVs were isolated at intervals over 60 minutes. (A) The concentrations of the isolated MVs were measured using the calibrated automated thrombogram (CAT) assay with the PRP-Reagent (n ¼ 3). (B) The TF content of the isolated MVs was determined using the TF-ELISA (n ¼ 3). (C–E) MDA-MB-231 cells were activated with PAR2-AP (20 μM) for 0 to 60 minutes and TF was immunoprecipitated using the anti-TF antibody HTF1. The phosphorylation of (C) Ser253 and (D) Ser258 was examined by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and western blot analysis (n ¼ 5, p ¼ 0.035 vs. 0 minutes). (E) Western blot images for total TF (FL-295), phospho-Ser253 (phospho-serine protein kinase C site antibody) and phospho-Ser258.

Article Snippet: Cells were then treated with PAR2 agonist peptide (PAR2-AP; SLIGKV-NH2; 20 μM; Tocris Bioscience, Bristol, United Kingdom) or human factor VIIa (FVIIa; 5 nM; Enzyme Research Laboratories Ltd., Swansea, United Kingdom).

Techniques: Phospho-proteomics, Activation Assay, Isolation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, SDS Page, Polyacrylamide Gel Electrophoresis, Western Blot

Journal: Thrombosis and Haemostasis

Article Title: Investigation of the Filamin A–Dependent Mechanisms of Tissue Factor Incorporation into Microvesicles

doi: 10.1160/th17-01-0009

Figure Lengend Snippet: Fig. 3 Increased proximity of filamin A and TF in MDA-MB-231 cells and primary endothelial cells following protease-activated receptor 2 (PAR2) activation. (A) MDA-MB-231 cells were activated with PAR2 agonist peptide (PAR2-AP; SLIGKV, 20 μM) for 0 to 60 minutes, fixed and then incubated with anti-TF antibody (HTF1) together with the anti-filamin A antibody EP2405Y. In control reactions, HTF1 and EP2405Y were replaced with mouse and rabbit IgG, respectively. The proximity ligation assays were performed using the DuoLink assay. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) and the actin cytoskeleton was stained using Acti-stain phalloidin-488 (n ¼ 4). (B) HDBEC (human dermal blood endothelial cells) were transfected to express wild-type TF (WT), or TF with alanine (Ala253) or aspartate (Asp253) substitutions of serine 253. Cells were activated with PAR2-AP (20 µM) for 45 minutes and the proximity of TF and filamin A was examined using the PLA (proximity ligation assay) procedure. IgG ¼ TF antibody replaced with mouse control IgG (n ¼ 4,p < 0.05 vs. WT without PAR2 activation. p < 0.01 vs. WT with PAR2-AP, #p < 0.0001 vs. Asp253 with PAR2 activation).

Article Snippet: Cells were then treated with PAR2 agonist peptide (PAR2-AP; SLIGKV-NH2; 20 μM; Tocris Bioscience, Bristol, United Kingdom) or human factor VIIa (FVIIa; 5 nM; Enzyme Research Laboratories Ltd., Swansea, United Kingdom).

Techniques: Activation Assay, Incubation, Control, Ligation, Staining, Transfection, Proximity Ligation Assay

Journal: Thrombosis and Haemostasis

Article Title: Investigation of the Filamin A–Dependent Mechanisms of Tissue Factor Incorporation into Microvesicles

doi: 10.1160/th17-01-0009

Figure Lengend Snippet: Fig. 4 Filamin A knock-down suppresses protease-activated receptor 2 (PAR2) mediated increase in cell surface tissue factor (TF) activity. (A–C) MDA-MB- 231 cells were transfected with filamin A siRNA or control siRNA (2 nM). Following 48-hour incubation, the cells were activated with PAR2 agonist peptide (PAR2-AP; SLIGKV)for30minutes. (A) Cellswerethen incubated witheither theTFinhibitory antibodyHTF1 orcontrolIgG (40 μg/mL) for15minutesandcell surfaceTF-specific thrombingenerationwasdeterminedusingthecalibratedautomatedthrombogram(CAT)assayintheabsenceofanyexogenousreagent (n ¼ 5, p < 0.05 vs. untreated cells with control siRNA, p < 0.002 vs. SLIGKV-treated cells with control siRNA). (B,C) Cell surface TF on MDA-MB-231 cells with knock-down of filamin A expression following PAR2 activation was examined by flow cytometry and (B) the mean fluorescence intensity and (C) percentage of positive cells were measured (n ¼ 3, p ¼ 0.0001 vs. respective siRNA control).

Article Snippet: Cells were then treated with PAR2 agonist peptide (PAR2-AP; SLIGKV-NH2; 20 μM; Tocris Bioscience, Bristol, United Kingdom) or human factor VIIa (FVIIa; 5 nM; Enzyme Research Laboratories Ltd., Swansea, United Kingdom).

Techniques: Knockdown, Activity Assay, Transfection, Control, Incubation, Expressing, Activation Assay, Cytometry

Journal: Thrombosis and Haemostasis

Article Title: Investigation of the Filamin A–Dependent Mechanisms of Tissue Factor Incorporation into Microvesicles

doi: 10.1160/th17-01-0009

Figure Lengend Snippet: Fig. 5 Disrupting cholesterol-rich microdomains prevents protease-activated receptor 2 (PAR2) mediated increases in cell surface tissue factor (TF) activity. MDA-MB-231 cells were reverse transfected with filamin A siRNA or control siRNA (2 nM). Following 48-hour incubation, the cells were incubated with MβCD (7.5 mM) and PAR2 agonist peptide (PAR2-AP; SLIGKV, 20 µM) for 30 minutes. TF-MV (microvesicle) release was examined by measuring (A) phosphatidylserine-positive MV release using the calibrated automated thrombogram (CAT) assay with the PRP- Reagent (n ¼ 5, p ¼ 0.009 vs. SLIGKV, p ¼ 0.029 vs. untreated control) and (B) TF activity of the MVs using the CAT assay with the MP-Reagent (n ¼ 5, p 0.04 vs. untreated control). (C) Cells were incubated with either the anti-TF inhibitory antibody HTF1 or control IgG (40 μg/mL) for 15 minutes and cell surface TF-specific thrombin generation on cells with knock-down of filamin A expression was determined using the CAT assay in the absence of any exogenous reagent (n ¼ 5, p < 0.05 vs. untreated cells with control siRNA, p < 0.002 SLIGKV vs. SLIGKV-treated cells with control siRNA, p < 0.0001 MβCD vs. SLIGKV-treated cells with control siRNA).

Article Snippet: Cells were then treated with PAR2 agonist peptide (PAR2-AP; SLIGKV-NH2; 20 μM; Tocris Bioscience, Bristol, United Kingdom) or human factor VIIa (FVIIa; 5 nM; Enzyme Research Laboratories Ltd., Swansea, United Kingdom).

Techniques: Activity Assay, Transfection, Control, Incubation, Knockdown, Expressing

Journal: Thrombosis and Haemostasis

Article Title: Investigation of the Filamin A–Dependent Mechanisms of Tissue Factor Incorporation into Microvesicles

doi: 10.1160/th17-01-0009

Figure Lengend Snippet: Fig. 6 Filamin A knock-down does not reduce cell surface phospha- tidylserine. MDA-MB-231 cells were reverse transfected with filamin A or control siRNA (2 nM). Following 48-hour incubation, the cells were adapted to serum-free medium and activated with protease-activated receptor 2 agonist peptide (PAR2-AP; SLIGKV, 20 µM) for 30 minutes. Cells were detached using TrypLE Select, washed and then re-sus- pended in HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffered saline (HBS), pH 7.4 containing calcium chloride (2 mM). Negative controls were incubated with HBS lacking calcium chloride. Cells were incubated with 5 µL of annexin V-FITC (BD Biosciences) for 20 minutes on ice. The cells were then examined by flow cytometry using a Gallios flow cytometer. A gate was set at 2% of cells incubated with annexin V-FITC in the absence of calcium chloride (n ¼ 4).

Article Snippet: Cells were then treated with PAR2 agonist peptide (PAR2-AP; SLIGKV-NH2; 20 μM; Tocris Bioscience, Bristol, United Kingdom) or human factor VIIa (FVIIa; 5 nM; Enzyme Research Laboratories Ltd., Swansea, United Kingdom).

Techniques: Knockdown, Transfection, Control, Incubation, Saline, Cytometry