The Ratio of Factor VIIa:Tissue Factor Content within Microvesicles Determines the Differential Influence on Endothelial Cells

Abstract

Tissue factor (TF)-positive microvesicles from various sources can promote cellular proliferation or alternatively induce apoptosis, but the determining factors are unknown. In this study the hypothesis that the ratio of fVIIa:TF within microvesicles determines this outcome was examined. Microvesicles were isolated from HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7 cell lines and microvesicle-associated fVIIa and TF antigen and activity levels were measured. Human coronary artery endothelial cells (HCAECs) were incubated with these purified microvesicles, or with combinations of fVIIa-recombinant TF, and cell proliferation/apoptosis was measured. Additionally, by expressing mCherry-PAR2 on HCAEC surface, PAR2 activation was quantified. Finally, the activation of PAR2 on HCAEC or the activities of TF and fVIIa in microvesicles were blocked prior to addition of microvesicles to cells. The purified microvesicles exhibited a range of fVIIa:TF ratios with HepG2 and 786-O cells having the highest (54:1) and lowest (10:1) ratios, respectively. The reversal from proapoptotic to proliferative was estimated to occur at a fVIIa:TF molar ratio of 15:1, but HCAEC could not be rescued at higher TF concentrations. The purified microvesicles induced HCAEC proliferation or apoptosis according to this ruling. Blocking PAR2 activation on HCAEC, or inhibiting fVIIa or TF-procoagulant function on microvesicles prevented the influence on HCAEC. Finally, incubation of HCAEC with recombinant TF resulted in increased surface exposure of fVII. The induction of cell proliferation or apoptosis by TF-positive microvesicles is dependent on the ratio of fVIIa:TF and involves the activation of PAR2. At lower TF concentrations, fVIIa can counteract the proapoptotic stimulus and induce proliferation.

Keywords: apoptosis; cell proliferation; factor VIIa; microvesicles; protease-activated receptor-2; tissue factor.

Fig. 1

Analysis of the TF and fVIIa content of microvesicles. Five cell lines (HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7) were propagated in 25 cm ² flasks, washed with phosphate-buffered saline (PBS) pH 7.4 and adapted to respective serum-free medium, for 1 hour. Microvesicles were prepared from the conditioned media by ultracentrifugation and the functional density determined using the Zymuphen MP-assay kit. ( A ) The TF antigen concentration of the microvesicles was measured using the Quantikine TF-ELISA kit and ( B ) the associated TF activities were measured using the fXa-generation assay ( n  = 5). ( C ) The presence of fVII/fVIIa antigen in the cells was detected by western blot analysis. The samples were separated by 12% (w/v) SDS-PAGE, transferred onto nitrocellulose membranes and then blocked with TBST (10 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20). The membranes were then probed with a rabbit polyclonal anti-fVII antibody diluted 1:2000 (v/v) in TBST and developed with an alkaline phosphatase-conjugated goat antirabbit antibody, diluted 1:4000 (v/v). The bands were visualized using the Western Blue stabilized alkaline phosphatase-substrate and ( D ) quantified by ImageJ program. (The micrographs are representative of three separate experiments). ( E ) Microvesicle-associated factor VII/VIIa antigen levels were quantified using the Assaymax FVII-ELISA kit ( n  = 5). ( F ) Microvesicle-associated fVIIa activity was also measured by fXa-generation assay but in the absence of exogenous fVIIa and presence of TF (1 U/mL) ( n  = 5). ( G ) The molar ratio of fVIIa:TF in microvesicles derived from the cells lines was calculated using the data generated above. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TF, tissue factor.

Fig. 2

Examination of the influence of cell line-derived microvesicles on HCAEC cell numbers. Microvesicles were prepared from cell lines (HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7), supplemented (50 nM) to HCAEC (2 × 10 ⁴ ) and incubated in for up to 48 hours. ( A ) Cell numbers were determined using the crystal violet staining assay and ( B ) the change in cell numbers was calculated as a percentage of the untreated cells ( n  = 5; * =  p  < 0.05 vs. the respective untreated sample at each time point). ( C ) Total RNA was extracted from samples of the treated HCAEC and the relative expression of cyclin D1 mRNA was analyzed by RT-PCR against β-actin as housekeeping ( n  = 3; * =  p  < 0.05 vs. the untreated sample). Cellular apoptosis was also measured (as absorption at 450 nm) in sets of HCAEC using the TiterTACS chromogenic TUNEL assay and ( D ) the change in level of apoptosis was calculated as a percentage of the untreated cells ( n  = 5; * =  p  < 0.05 vs. the untreated sample). ( E ) Total RNA was extracted from samples of the treated HCAEC and the relative expression of bax mRNA was analyzed by RT-PCR against β-actin as housekeeping ( n  = 3; * =  p  < 0.05 vs. the untreated sample). HCAEC, human coronary artery endothelial cell; RT-PCR, reverse transcription polymerase chain reaction.

Fig. 3

Examination of the effect of different ratios of fVIIa:TF on HCAEC cell numbers. ( A ) HCAECs (2 × 10 ⁴ ) were incubated for 24 hours with a range of recombinant human TF (Innovin thromboplastin reagent) at a range of 0–10 U/mL (1 U/mL = 1.3 ng/mL), in the presence or absence of purified fVIIa (0–10 nM). Cell numbers were determined by crystal violet staining assay and percentage change in cell numbers calculated ( n  = 5; * =  p  < 0.05 vs. the untreated sample). ( B ) HCAECs (2 × 10 ⁴ ) were also incubated for 24 hours with a range of concentrations of purified fVIIa (0.1–10 nM) in the presence of recombinant human TF (0–4 U/mL). Cell numbers were determined by crystal violet staining assay and percentage change in cell numbers calculated ( n  = 5; * =  p  < 0.05 vs. the untreated sample). Total RNA was extracted from samples of the treated HCAEC and the relative expression of ( C ) cyclin D1 mRNA and ( D ) bax mRNA was analyzed by RT-PCR against β-actin as housekeeping ( n  = 3; * =  p  < 0.05 vs. the untreated sample). HCAEC, human coronary artery endothelial cells; RT-PCR, reverse transcription polymerase chain reaction; TF, tissue factor.

Fig. 4

Examination of the involvement of PAR2 in microvesicle-induced HCAEC apoptosis. Sets of HCAEC (2 × 10 ⁴ ) were preincubated with a blocking anti-PAR2 antibody (SAM11; 20 µ/mL) or a control isotype. Sets of cells were then incubated with microvesicles derived from ( A ) HepG2 cell line (50 nM) for 24 hours and cell numbers were determined by crystal violet staining assay ( n  = 5; * =  p  < 0.05 vs. the untreated sample, # =  p  < 0.05 vs. the sample devoid of antibody). ( B ) A similar set of cells was incubated for 24 hours with microvesicles derived from 786-O cell line (50 nM) and the rate of apoptosis determined using the TiterTACS chromogenic TUNEL assay ( n  = 5; * =  p  < 0.05 vs. the untreated sample, # =  p  < 0.05 vs. the sample devoid of antibody). Total RNA was extracted from samples of the treated HCAEC and ( C ) the relative expression of cyclin D1 mRNA, and ( D ) bax mRNA was analyzed by RT-PCR against β-actin as housekeeping ( n  = 3; * =  p  < 0.05 vs. the untreated sample, # =  p  < 0.05 vs. the sample devoid of antibody). ( E ) HCAECs (2 × 10 ⁴ ) were incubated for 24 hours with microvesicles derived from 786-O cell line (50 nM) in the presence or absence of PAR2-activating peptide (20 µM) and the rate of apoptosis determined ( n  = 5; * =  p  < 0.05 vs. the untreated sample, # =  p  < 0.05 vs. microvesicles only). ( F ) HCAECs (2 × 10 ⁴ ) were incubated for 30 minutes with microvesicles derived from 786-O cell line (0–130 nM), or PAR2-activting peptide (20 µM). The cells were then incubated with a mouse antihuman PAR2 antibody (SAM11; 20 µg/mL) and probed with a HRP-conjugated goat antimouse antibody diluted 1:1000 (v/v). The relative amount of cell-surface PAR2 was then measured using the TMB substrate ( n  = 5; * =  p  < 0.05 vs. the untreated sample). HCAEC, human coronary artery endothelial cells; PAR2, protease-activated receptor-2; RT-PCR, reverse transcription polymerase chain reaction.

Fig. 5

Examination of the activation of PAR2 in response to fVIIa:TF complex and microvesicles. Sets of HCAEC (5 × 10 ³ ) were transfected to express mCherry-PAR2 hybrid protein. ( A ) Sets of transfected and untransfected cells were then fixed and stained phalloidin-iFlour 488 (diluted 1:1000 v/v) and DAPI (2 µg/mL) and examined by fluorescence microscopy for mCherry-PAR2 (Red), phalloidin-iFlour 488 (Green), and DAPI (Blue). ( B ) As a comparison, untransfected cells which were labeled with FITC-conjugated anti-PAR2 (SAM11; 20 µg/mL) or unlabeled cells. The cells were examined by fluorescence microscopy for PAR2 (Green) and DAPI (Blue). ( C ) HCAECs expressing the mCherry-PAR2 protein were incubated for 1 hour with combinations of TF (0–4 U/mL) together with fVIIa (0–10 nM) and the release of mCherry in the media was measured by determining the fluorescence at Em. 630 nm (Ext. 580 nm) ( n  = 5; * =  p  < 0.05 vs. the untreated sample). ( D ) Sets of transfected cells were also incubated with microvesicles derived from the five cell lines (HepG2, BxPC-3, 786-O, MDA-MB-231, and MCF-7) at 50 nM and the release of mCherry measured by determining the fluorescence at Em. 630 nm (Ext. 580 nm) ( n  = 5; * =  p  < 0.05 vs. the untreated sample). HCAEC, human coronary artery endothelial cells; PAR2, protease-activated receptor-2; TF, tissue factor.

Fig. 6

Active fVIIa both attenuates and is required for the induction of HCAEC apoptosis by microvesicle-associated TF. ( A ) Sets of HCAEC (2 × 10 ⁴ ) were incubated with microvesicles derived from 786-O cells alone, or supplemented with fVIIa (5 nM) and cellular apoptosis was measured after 24 hours ( n  = 5; * =  p  < 0.05 vs. the untreated sample, # =  p  < 0.05 vs. the respective sample without fVIIa). ( B ) Microvesicles derived from 786-O cells were preincubated with HTF-1 anti-TF antibody (20 µg/mL) to block fVIIa binding, 10H10 anti-TF antibody (20 µg/mL) to block TF signaling, or an isotype mouse antibody (20 µg/mL), and cellular apoptosis was measured after 24 hours ( n  = 5; * =  p  < 0.05 vs. the untreated sample). ( C ) Microvesicles derived from 786-O cells were preincubated with an inhibitory anti-fVIIa antibody (20 µg/mL) prior to addition to HCAEC (2 × 10 ⁴ ) and cellular apoptosis was measured after 24 hours ( n  = 5; * =  p  < 0.05 vs. the untreated sample, # =  p  < 0.05 vs. sample with microvesicle alone). ( D ) Microvesicles derived from HepG2 cells were preincubated with an inhibitory anti-fVIIa antibody (20 µg/mL) prior to addition to HCAEC (2 × 10 ⁴ ). HCAEC numbers were determined after 24 hours using the crystal violet staining assay and the cell numbers calculated ( n  = 5; * =  p  < 0.05 vs. the untreated sample, # =  p  < 0.05 vs. sample with microvesicle alone). HCAEC, human coronary artery endothelial cells; TF, tissue factor.

Fig. 7

HCAECs express fVIIa on the cell surface in response to TF and PAR2 activation. Two separate sets of HCAEC (2 × 10 ⁴ ) were incubated with ( A ) TF (2 U/mL) or ( B ) PAR2-activating peptide (20 µM) for the durations shown and the cells were then fixed using 4% (v/v) formaldehyde. The cells were washed and one set was permeabilized while the other kept intact. Total and surface expression of fVII were measured in situ, by incubating all samples with a mouse anti-fVIIa antibody (20 µg/mL). The samples were then probed with a HRP-conjugated goat antimouse antibody (dilute 1:1000 v/v) and developed using the TMB substrate. The percentage ratio of surface:total fVII was then calculated ( n  = 5; * =  p  < 0.05 vs. the observed ratio at time zero). HCAECs (2 × 10 ⁴ ) were also subjected to repeated treatment with recombinant TF (2 U/mL) at 60 minute intervals. After each treatment, the relative amount of ( C ) cell-surface fVII antigen and ( D ) the released microvesicle-associated fVII antigen were measured and the percentages were calculated against the amount of cell-surface fVII in the untreated cells. HCAEC, human coronary artery endothelial cells; PAR2, protease-activated receptor-2; TF, tissue factor.