TF expression in stimulated endothelial cells and PBMC treated with tunicamycin
To determine whether glycosylation of TF modulates its cell surface expression or procoagulant activity, HUVEC were stimulated with TNFα + IL1β for 6 h and simultaneously treated with varying concentrations of tunicamycin, an inhibitor of the N-linked glycosyl reaction. As shown in Fig. 1(A), treatment of HUVEC with increasing concentrations of tunicamycin progressively reduced the amount of TF protein that migrates at 47 kDa, representing the fully glycosylated form of TF. Non-glycosylated TF protein in tunicamycin-treated cells migrated near 35 kDa. However, the intensity of this band was very low and faintly detectable. As a control, the same samples were analyzed for ICAM-1, a cell surface glycoprotein that is absent on unperturbed endothelial cells but induced by cytokines in a similar fashion to that of TF. Immunoblot analysis of ICAM-1 showed clearly that tunicamycin treatment, in a dose-dependent manner, inhibited glycosylation of ICAM-1, with 1 μg mL−1 tunicamycin completely inhibiting glycosylation of ICAM-1. In contrast to TF, a decrease in intensity of the glycosylated ICAM-1 band in tunicamycin-treated cells correlated well with an increase in non-glycosylated ICAM-1. These data raise the possibility that tunicamycin treatment either selectively inhibited the synthesis and/or surface expression of TF protein, or TF antibodies fail to effectively recognize the non-glycosylated TF protein in Western blot analysis. Next, we investigated the effect of tunicamycin (1 μg mL−1) on the expression of TF antigen and its activity at the cell surface. As shown in Fig. 1(B), cell surface TF activity was reduced by about 80% in HUVEC treated with tunicamycin. Similarly, tunicamycin treatment also reduced the expression of TF protein at the cell surface by about 80% or more (Fig. 1C). These data indicate that reduced TF activity observed in cells treated with tunicamycin is the result of reduced TF protein expression at the cell surface rather than reduced TF procoagulant activity of non-glycosylated TF. Measurement of total TF antigen levels and activity in cell lysates revealed that tunicamycin treatment also inhibited total TF protein levels and subsequently the activity by about 60% and 80%, respectively (Fig. 1D,E). These data suggest that the impaired TF protein expression at the cell surface in tunicamycin-treated cells primarily stems from reduction in total TF protein synthesis rather than impaired transport of TF protein from intracellular sources to the cell surface membrane. In additional studies, we investigated whether tunicamycin inhibited TF protein synthesis via a pre- or post-transcriptional mechanism. Analysis of TF mRNA levels with quantitative RT-PCR showed that TNFα + IL1β induction increased TF mRNA levels by about 150-fold over the control unstimulated HUVEC and tunicamycin (1 μg mL−1) treatment had no effect on cytokine-induced TF mRNA (TF mRNA levels as fold increase over the uninduced HUVEC: TNFα + IL1β, 158 ± 27; tunicamycin + TNFα + IL1β, 169 ± 35, n = 3, mean ± SEM). These data indicate that tunicamycin inhibited TF protein expression via a post-transcriptional mechanism.
Figure 1. Tunicamycin treatment inhibits tissue factor (TF) protein expression and activity in stimulated endothelial cells. (A) Monolayers of human umbilical vein endothelial cells (HUVEC) were stimulated with TNF-α + IL1-β (20 ng mL−1 each) in the presence or absence of varying concentrations of tunicamycin (A) or 1 μg mL−1 (B–D) for 6 h. At the end of stimulation, (A) cells were lysed in non-reducing SDS-PAGE buffer and the samples were subjected to immunoblot analysis using polyclonal antibodies against TF or ICAM-1; (B) cell surface TF activity was determined by adding factor (F) VIIa (10 nm) and FX (175 nm) and measuring FXa generation; (C) cell surface TF antigen levels were determined in a binding assay using 125I-TF9 9C3 mAb (10 nm); (D) total TF antigen in cell lysates was determined in ELISA with TF polyclonal antibody; (E) TF activity in cell lysates was determined by adding FVIIa (10 nm) and FX (175 nm) and measuring FXa generation. *Denotes significant difference from TNF-α + IL1-β alone treated cells (P ≤ 0.02).
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Next, we examined the effect of tunicamycin on TF expression in PBMC. As shown in Fig. 2(A), as expected, no detectable TF protein was expressed in unperturbed PBMC and LPS stimulation markedly increased TF protein expression in PBMC. LPS-stimulated PBMC were more resistant to tunicamycin treatment as compared with HUVEC and complete deglycosylation of TF required a five times higher concentration of tunicamycin; that is, 5 μg mL−1. Analysis of intact cells showed LPS markedly increased TF activity while pretreatment of PBMC with tunicamycin had no statistically significant effect on TF activity induced by LPS (Fig. 2B). A similar trend was noted in TF activity measured in cell lysates (data not shown). Although TF protein levels appeared to decrease in LPS-stimulated PBMC upon treatment with tunicamycin on western blots (Fig. 2A), measurement of TF antigen levels quantitatively by ELISA revealed no significant differences in TF protein levels in PBMC treated with tunicamycin or control vehicle and stimulated with LPS (Fig. 2C). These data suggest that TF lacking carbohydrate is not recognized well by western blot analysis (see the following section for more on this). Because inhibition of N-glycosylation by tunicamycin treatment affected the cell surface expression of TF activity and protein differently in HUVEC and PBMC and seeing that it is difficult to draw a firm conclusion on whether TF glycosylation plays a role in regulating TF procoagulant activity from these data alone, we examined next the role of glycosylation on TF activity by site-specific mutagenesis in which one or more of the Asn residues in potential glycosylation sites were mutated to Ala.
Figure 2. Effect of tunicamycin on tissue factor (TF) activity and antigen in peripheral blood mononuclear cells (PBMC). PBMC (2 × 106 cells mL−1) in serum-rich RPMI 1640 medium were stimulated with 100 ng mL−1 LPS for 6 h in the presence or absence of varying concentrations of tunicamycin. Where tunicamycin was added, cells were preincubated with it for 1 h. (A) Immunoblot analysis using TF polyclonal antibodies; (B) TF activity on intact cells as determined in factor X activation assay; (C) TF antigen levels determined in ELISA. Data are mean ± SEM (n = 4). ns, not statistically significant.
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Tissue factor mutants lacking the potential glycosylation sites are expressed normally and exhibit no defect in tissue factor procoagulant activity
To determine the functional importance of N-glycosylation, Asn residues at one, or various combinations, of the four potential glycosylation sites were mutated to Ala, and plasmid constructs encoding wild-type TF-GFP or one of these TF-GFP mutants were used for transfection in CHO cells. Only potential glycosylation sites in the extracellular domain of TF were used in double- or triple-mutation combinations. Analysis of total TF antigen in the transfected cells by immunoblot analysis with TF polyclonal antibodies demonstrated an apparent increase in mobility of the glycosylation-deficient mutants as compared with the wild-type TF-GFP protein (Fig. 3A). In view of the fact that these constructs had GFP tagged to the cytoplasmic tail of TF, wild-type TF fusion protein migrated at approximately 72 kDa, whereas glycosylation-deficient mutants migrated at 69 (single mutant, TFN11A, TFN124A, TFN137A), 65 (double mutant, TFN11A/N124A, TFN11A/N137A and TFN124A/N137A mutant) or 62 kDa (triple mutant, TFN11A/N124A/N137A). The intensity of the TF band recognized by TF antibodies on immunoblot analysis was significantly lower in cell lysates harvested from cells transfected with double mutants, and markedly lower in cells transfected with the triple mutant, thereby giving the impression that lack of carbohydrates at more than one N-linked glycosylation site impairs TF protein expression. However, when cells transfected with wild-type TF-GFP or TF-GFP mutants were examined for GFP fluorescence by fluorescence microscopy, all transfectants showed similar levels of TF expression (data not shown). Similarity in levels of TF expression among cells transfected to express wild-type TF-GFP and TF-GFP mutants was confirmed by immunoblot analysis using GFP antibodies that recognize the TF fusion protein (Fig. 3B). Both wild-type TF-GFP and mutant TF-GFP were in monomeric form as we observed a single band corresponding to the molecular weight of monomeric fusion protein.
Figure 3. Glycosylation does not play a role in tissue factor (TF) cell surface expression or procoagulant activity. CHO cells were transfected with wild-type TF-GFP, single, double or triple glycosylation defective TF-GFP mutants. After 48 h of transfection, cells were lysed in non-reducing SDS-PAGE and an equal amount of protein was subjected to SDS-PAGE followed by immunoblot analysis with TF polyclonal antibodies (A), GFP peptide antibodies (B), or different TF monoclonal antibodies (C). Cell surface expression of TF was determined in radioligand binding assays using 125I-FVIIa (10 nm) (D) or 125I-TF9 9C3 mAb (10 nm) (E). (F) TF procoagulant activity at the cell surface was measured in a factor (F) Xa generation assay after adding FVIIa (10 nm) and FX (175 nm). Data are mean ± SEM (n = 4–6).
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Carbohydrate moieties on a protein are known to be highly antigenic and thus it may be possible that our TF polyclonal antibodies raised using native (presumably fully glycosylated) TF purified from human brain extract  may have reduced reactivity towards non-glycosylated TF protein. This could be a possible explanation for the observed differences in band intensities of wild-type and mutant TF fusion protein when probed with anti-TF antibodies and GFP antibodies. In additional studies, we evaluated TF protein expression levels using multiple TF mAb (TF9 10H10, TF8 5G9, TF9 6B4, TF9 9C3 and TF811 D12) that may bind to TF at different epitopes . Interestingly, all of the antibodies barely detected TF from cells transfected with the triple mutant (Fig. 3C). The mutation at Asn11 resulted in a marked reduction in detection of TF protein by immunoblotting when this mutation was present alone or in combination with other Asn mutants (TFN11A/N124A and TFN11A/N137A). The inability of TF antibodies to detect the non-glycosylated TF mutant appears to be limited to immunoblot analysis as we were able to measure similar trends in TF antigen levels in cell lysates of cells transfected with wild-type TF-GFP or glycosylation-deficient mutant TF-GFP in ELISA using TF polyclonal antibodies (data not shown). Next, we evaluated the expression of TF antigen at the cell surface in cells transfected with wild-type TF-GFP or glycosylation-deficient TF-GFP mutants in TF-specific binding assays using 125I-FVIIa or 125I-TF mAb (TF9 9C3). As shown in Fig. 3(D,E), there were no significant differences in 125I-FVIIa or 125I-TF mAb binding to cells expressing wild-type-GFP or mutant TF-GFP. Measurement of TF procoagulant activity in a FX activation assay showed no significant differences between wild-type and TF mutants, including the triple mutant that is completely devoid of glycosylation (Fig. 3F). Overall, these data indicate that glycosylation does not affect TF synthesis, transport to the cell surface, or its coagulant activity.
As the above studies showed that mutation at different potential glycosylation sites, at one, two or all three of the extracellular sites, resulted in similar behavior of TF, further studies were employed to examine the role of TF glycosylation in TF procoagulant activity and signaling function when expressed in endothelial cells. For these studies, we used the triple mutant (TFN11A/N124A/N137A) as this TF mutant would be completely devoid of glycosylation. In order to have a better control of expression levels and be able to perform these studies in HUVEC, we generated an adenovirus expression system encoding wild-type TF-GFP or TFN11A/N124A/N137A-GFP (both contain GFP at the C-terminus). To determine if N-linked glycosylation plays a role in subcellular localization and distribution of TF, HUVEC transduced with adenovirus encoding wild-type TF-GFP or TFN11A/N124A/N137A-GFP were analyzed for TF distribution by confocal microscopy. Analysis of TF expression by examining the fluorescence of tagged-GFP revealed that both wild-type and the triple mutant exhibited a similar pattern of TF distribution, that is, predominantly localized at the cell surface with a substantial amount present in the perinuclear compartment, and a small fraction in endosome-like structures. Immunostaining of non-permeabilized cells with TF polyclonal antibodies also showed comparable expression of wild-type TF and the triple mutant TF at the cell surface. TF antibody staining completely overlapped with the GFP fluorescence. These data further support the observation that N-linked glycosylation of TF is not necessary for transport of TF to the cell surface (Fig. 4).
Figure 4. No impairment in cellular expression of glycosylation-deficient tissue factor (TF). Human umbilical vein endothelial cells (HUVEC) monolayers cultured on glass coverslips were transduced with 20 MOI per cell of wild-type or TFN11A/N124A/N137A adenovirus tagged with GFP. After 48 h of transduction, the monolayers were fixed, and one set permeabilised with 0.025% Triton-X-100 and stained with TF polyclonal antibodies (5 μg mL−1) and DAPI (nuclear staining). Fluorescence was analyzed by confocal microscopy. Upper two rows, non-permeabilised cells; bottom two rows, permeabilised cells. Left panel, TF expression analyzed by immunofluorescence staining; middle panel, TF expression by GFP fluorescence; right panel, merged image of GFP and TF immunofluorescence.
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Next, we evaluated the procoagulant function of the triple mutant in comparison with wild-type TF-GFP in a FX activation assay in HUVEC. As observed with CHO cells transfected with wild-type and TFN11A/N124A/N137A plasmid constructs, mutation of all three putative glycosylation sites in the extracellular domain did not alter the procoagulant activity of TF in endothelial cells. Given that the glycosylation sites are located in the region of FVIIa interaction, it is conceivable that carbohydrates attached to TF may contribute to FVIIa interaction with TF. However, if this effect is not substantial, measuring FX activation at 10 nm FVIIa may mask the subtle effect of TF glycosylation on its interaction with FVIIa. Nevertheless, analysis of FVIIa interaction with TF using varying concentrations of FVIIa in a FX activation assay showed similar Kd values for FVIIa binding to coagulant-active wild-type TF-GFP and the triple mutant (Fig. 5A; Kd values as follows – wild-type TF-GFP, 0.18 ± 0.028 nm and TFN11A/N124A/N137A-GFP, 0.17 ± 0.039 nm). Recent studies have shown that deglycosylation of natural placental TF decreases the affinity of FX for both the TF-FVIIa complex (albeit minimally) and Kcat (significantly), thus contributing to a 6-fold difference in TF activity between glycosylated and deglycosylated TF . Our comparative analysis of wild-type TF-GFP and the glycosylation-deficient mutant expressed at the endothelial cell surface for their ability to activate varying concentrations of FX showed no significant differences in the Vmax or the Km of FX activation (Fig. 5B: values for Vmax for wild-type TF-GFP, 7.70 ± 0.61 nm min−1, and TFN11A/N124A/N137A-GFP, 7.21 ± 0.81 nm min−1; values for Km for wild-type TF-GFP, 0.65 ± 0.16 μm, and TFN11A/N124A/N137A-GFP, 1.0 ± 0.30 μm; a small difference in Km values between them is not statistically significant). In order to minimize error as the result of differences in the expression levels between wild-type and mutant TF, cell surface 125I-TF9 9C3 mAB binding was performed and data were normalized with protein amounts. Finally, to evaluate FVIIa binding to cryptic TF, which generally has lower affinity for FVIIa compared with coagulant-active TF, we analyzed 125I-FVIIa binding to HUVEC expressing wild-type TF-GFP or the triple mutant (because most of the TF on cell surfaces is cryptic [22,23], the binding parameters observed with 125I-FVIIa reflect its binding to cryptic TF). The binding data showed no significant differences between the mutant and wild-type TF-GFP in their affinity and binding capacity (Fig. 5C; Kd values as follows – wild-type TF-GFP, 15.5 ± 3.57 nm, and TFN11A/N124A/N137A-GFP, 13.4 ± 4.00 nm; Bmax values as follows – wild-type TF-GFP, 63 ± 4.7 fmol 10−5 cells, and TFN11A/N124A/N137A-GFP, 52 ± 4.8 fmol 10−5 cells).
Figure 5. Non-glycosylated tissue factor (TF) interacts with factor (F) VIIa and FX with a similar efficiency to that of wild-type TF. Human umbilical vein endothelial cells (HUVEC) monolayers were transduced with 20 MOI per cell of wild-type TF-GFP or TFN11A/N124A/N137A-GFP adenovirus. After 48 h of transduction, TF activity at the cell surface was measured in a FX activation assay using varying concentrations of FVIIa (0.01–10 nm) and a fixed concentration of FX (175 nm) (A), or varying concentrations of FX (0.025–3 μm) and a fixed concentration of FVIIa (10 nm) (B). Specific binding of 125I-FVIIa to the surface TF was measured in a radioligand binding assay (C). (•) Wild-type TF-GFP; (○) TFN11A/N124A/N137A-GFP. Data are mean ± SEM (n = 3).
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Tissue factor glycosylation does not affect TF-FVIIa-mediated PAR2-dependent cell signaling
To investigate whether TF glycosylation plays a role in modulating TF-FVIIa-mediated signaling, we examined TF-FVIIa cleavage of PAR2 and PAR2-dependent p44/42 MAPK activation. CHO cells were co-transduced with wild-type TF-GFP or TFN11A/N124A/N137A-GFP and AP-PAR2. The transduced cells were exposed to FVIIa and TF-FVIIa cleavage of PAR2 was determined by measuring the alkaline phosphatase activity released into the medium. As shown in Fig. 7(A), both wild-type TF-GFP and TFN11A/N124A/N137A-GFP activated PAR2 to a similar extent; that is, approximately 15–20% of that observed with trypsin. Consistent with the observation that FVIIa bound to wild-type TF-GFP or the triple mutant cleaves PAR2 with a similar efficiency, there was no observable difference in FVIIa-induced, time-dependent, activation of p44/42 MAPK in cells expressing wild-type TF-GFP or TFN11A/N124A/N137A-GFP (Fig. 7B). Consistent with the data obtained in heterologus CHO cells, HUVEC transduced with either wild-type TF-GFP or TFN11A/N124A/N137A-GFP supported FVIIa cleavage of PAR2 to a similar extent (Fig. 7C). We also analyzed FVIIa-induced p44/42 MAPK activation in HUVEC transduced with wild-type TF-GFP or TFN11A/N124A/N137A-GFP in the presence of EPCR blocking mAb (to block EPCR-dependent FVIIa-induced p44/42 MAPK activation ). The data showed that endothelial cells expressing either wild-type TF-GFP or TFN11A/N124A/N137A-GFP supported FVIIa activation of p44/42 MAPK to a similar extent (approximately 2.5-fold increase over the control; data not shown). In additional studies, we examined TF-FVIIa-PAR2 signaling in endothelial cells by measuring FVIIa induction of TR3 in HUVEC transduced to express PAR2 and either wild-type TF-GFP or TFN11A/N124A/N137A-GFP. As shown in Fig. 7(D), FVIIa induction of TR3 is strictly dependent on TF and no significant differences were found in TR3 levels between the cells expressing wild-type TF or the glycosylation-deficient TF mutant (Fig. 7D).
Figure 7. Factor (F) VIIa cleavage of PAR2 and activation of p44/42 MAPK in cells expressing wild-type or non-glycosylated tissue factor (TF). (A and B) CHO cells were co-transduced with 100 MOI per cell of wild-type TF-GFP and TFN11A/N124A/N137A-GFP adenovirus with 25 MOI per cell of AP-PAR2 adenovirus or with AP-PAR2 alone for 48 h. (A) The transduced cells were incubated at 37 °C with a control vehicle, FVIIa (10 nm) or trypsin (5 nm), and at the end of 1 h incubation, soluble alkaline phosphatase activity released in the medium was measured. The values obtained in control vehicle treatment were subtracted from FVIIa and trypsin treatments and the value obtained in trypsin treatment was designated 100% of PAR2 cleavage. (B) Cells deprived of serum overnight were treated with FVIIa (10 nm) for varying times (0–30 min) or trypsin (5 nm) for 5 min and the cell lysates were subjected to SDS-PAGE and immunoblotted with phospho p44/42 or total MAPK antibodies. (C) Human umbilical vein endothelial cells (HUVEC) were infected with 20 MOI per cell of wild-type TF-GFP or TFN11A/N124A/N137A-GFP adenovirus and 10 MOI per cell of AP-PAR2 adenovirus. Cells were treated with FVIIa and trypsin and cleavage was determined as described for panel A. (D) HUVEC were transduced with PAR2 (10 MOI per cell) and either wild-type TF-GFP or TFN11A/N124A/N137A-GFP (20 MOI per cell) adenovirus for 48 h. The cells were serum starved for 2 h in EBM-2 SFM and then treated with FVIIa (10 nm) for 90 min. Total RNA was isolated and subjected to real-time qPCR analysis in triplicates. Fold-increase in TR3 mRNA levels was measured relative to TR3 mRNA levels in cells expressing PAR2 (no TF) and treated with control vehicle. ns, no statistically significant difference.
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In additional studies, we also examined whether TF glycosylation plays a role in cell adhesion by plating CHO cells expressing wild-type TF-GFP or TFN11A/N124A/N137A-GFP on different extracellular matrix proteins (collagen, laminin and fibronectin) or BSA-coated wells. The results showed no differences between wild-type TF-GFP and TFN11A/N124A/N137A-GFP in their attachment to extracellular matrix proteins, their morphology or spreading pattern (data not shown). We also attempted to investigate the role of carbohydrate attachment in TF in its interaction with β1-integrin. Co-immunoprecipitation studies revealed that although TF constitutively expressed in cancer cells (MDA-MB-231) could associate with the β1-integrin as reported earlier , this interaction is not detectable in HUVEC transduced with wild-type TF-GFP or TFN11A/N124A/N137A-GFP either in the presence or absence of FVIIa.