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.
Highlights
Material and MethodsTissue factor (TF) initiates the coagulation mechanism through the formation of a complex with factor VIIa which activates factors X and IX.[1,2] TF is expressed on the surface of cells and may be released as cell-derived microvesicles following cellular activation.[3,4,5,6,7,8,9] TF is capable of initiating cellular signals in cells expressing this protein, and on exposure of recipient cells to exogenous TF-containing microvesicles
Examination of the factor VIIa (fVIIa) by western blot indicated the presence of active fVIIa in HepG2, MDA-MB-231, and MCF-7 cell lines, frequently exhibiting multiple bands (►Fig. 1C) which have previously been attributed to the presence of glycosylation variants.[46,47]
In this study, by measuring the ratios of fVII/fVIIa and TF, we examined a possible mechanism by which TF-containing microvesicles may confer different outcomes in cultured primary endothelial cells
Summary
Material and MethodsTissue factor (TF) initiates the coagulation mechanism through the formation of a complex with factor VIIa (fVIIa) which activates factors X and IX.[1,2] TF is expressed on the surface of cells and may be released as cell-derived microvesicles following cellular activation.[3,4,5,6,7,8,9] TF is capable of initiating cellular signals in cells expressing this protein, and on exposure of recipient cells to exogenous TF-containing microvesicles. 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.
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