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.

Porphyromonas gingivalis triggers the shedding of inflammatory endothelial microvesicles that act as autocrine effectors of endothelial dysfunction

“…Future studies should also determine the precise composition of EMV Pg as their content is clearly influenced by the quantity and type of cellular stress trigger. Indeed, a specific insight should be made on mRNA, miRNA, proteins and membrane receptors 64,74,75 .…”

Apixaban Suppresses the Release of TF-Positive Microvesicles and Restrains Cancer Cell Proliferation through Directly Inhibiting TF-fVIIa Activity

“…40,41 Moreover, the examination of MDA-MB-231 cell line with a monoclonal antibody, indicated the low level expression of the zymogen form of fVII. 42 However, our previous 43 and current examination of cell lines including MDA-MB-231 cells demonstrates the presence of heavy and light chains of the activated form of fVIIa. The expression of fVIIa in MDA-MB-231 cell line was also corroborated by analysing the expression of fVII mRNA in the cells by RT-PCR 43 but was lower in AsPC-1 cell line.…”

“…42 However, our previous 43 and current examination of cell lines including MDA-MB-231 cells demonstrates the presence of heavy and light chains of the activated form of fVIIa. The expression of fVIIa in MDA-MB-231 cell line was also corroborated by analysing the expression of fVII mRNA in the cells by RT-PCR 43 but was lower in AsPC-1 cell line. Moreover, no detectable endogenous fXa antigen was present.…”

Accumulation of tissue factor in endothelial cells promotes cellular apoptosis through over-activation of Src1 and involves β1-integrin signalling

“…Cell numbers were determined by staining with crystal violet as previously described [50,51] and calculated from a standard curve. In addition, cellular apoptosis was quantified using the TiterTACS™ Colorimetric Apoptosis Detection Kit (AMS Biotechnology, Abingdon, UK) according to the manufacturer's instructions and as described before [18,19]. The criteria for endothelial cell apoptosis were evaluated according to the published consensus guidelines [52] and the apoptosis established as described previously [53].…”

“…Furthermore, TF is capable of inducing cellular apoptosis through intracellular signalling mechanisms [15][16][17]. Recently, we reported that the accumulation of TF within endothelial cells, either through increased retention, or by acquisition of TF can promote cellular apoptosis [18,19] through mechanisms mediated by p38 mitogen-activated protein kinases (MAPKs) [18]. TF-VIIa complex is capable of activating protease activated receptor (PAR)-2 which results in cellular signalling directly [11][12][13][14].…”