Functional impact of the EGFRvIII oncogene on the ability of the TF pathway to regulate procoagulant and angiogenic phenotype of glioblastoma cells. (A) Differential, but low-level FXa-generating activity exhibited by U373 and U373vIII cells in the absence of exogenous FVIIa (compare with panel D). In this setting, U373vIII cells exhibit greater ability to activate FX, possibly in relation to their production of endogenous FVII. (B) Simultaneous up-regulation of TF, PAR1, and PAR2 proteins, as a function of EGFRvIII-dependent cellular transformation of U373 glioblastoma cells (Western blot; β-actin is used as loading control). These changes correspond to those observed at the mRNA level, as shown in Figure 1C. (C) Fluorescence-activated cell sorter analysis documenting the results of the enforced expression of the exogenous human TF in parental U373 cells (human TF sequence was introduced using pcDNA3.1hygro vector). Viable TF U373D11 and TF U373G11 cells and cells transfected with empty vector (EV1 U373 and EV2 U373) were stained for surface TF antigen. Two of several similar clones in each category are shown. (D) Manifestation of a robust TF-dependent procoagulant activity (TF-PCA; generation of FXa) by EGFRvIII-transformed U373vIII cells expressing endogenous TF. Their EGFRvIII nonexpressing U373 counterparts transfected with TF (TF U373D11 and TF U373G11) exhibit comparable levels of TF PCA. In contrast, parental U373 cell line and several control transfectants (EV1 U373 and EV2 U373) display a negligible procoagulant activity. Unlike in the case of data presented in panel A, this assay was conducted in the presence of the exogenous recombinant FVIIa. **P < .005. N = 4. (E) VEGF release on addition of FVIIa or PAR agonistic peptides (PAR1-AP and PAR2-AP) to EGFRvIII, TF, and control U373-derived cells. VEGF in conditioned medium was detected by enzyme-linked immunosorbent assay. Dashed line represents the limit of the assay sensitivity, as defined by the supplier; dark bars, cells treated as indicated by “+”. Only in EGFRvIII-transformed U373vIII cells, but not in their parental (U373),TF transfected (TF U373D11 and TF U373G11), or mock-transfected (EV1 U373 and EV2 U373) counterparts, an appreciable increase in VEGF secretion was detected (24-hour stimulation with FVIIa 10nM or PAR1/2APs 100μM each). *P < .05. **P < .005. N = 2. (F) IL-8 up-regulation in glioma cell lines on activation of the TF/PAR pathway (test carried out using human IL-8 enzyme-linked immunosorbent assay; designations as in panel E). Appreciable increase in IL-8 production was observed on exposure to FVIIa, PAR1-AP, or PAR2-AP only in the case of EGFRvIII-transformed cells, but not in cells expressing TF in the absence of EGFRvIII (TF U373D11 and TF U373G11) or in cells with low levels of TF and PARs (U373, EV1 U373, and EV2 U373). All experimental conditions are as in panel E. Responses to treatments were compared with the corresponding untreated cells. *P < .05. **P < .005. N = 6 (“Experimental procedures” and supplemental data).