Figure 5.
Thrombin-PAR1/2 signaling amplifies cell surface-associated TF procoagulant activity in poly(I:C)-treated cells. (A) EA.hy926 cells were stimulated with poly(I:C) (12.5 μg/mL) and PAR1-selective (SFLLR) or PAR2-selective agonist peptides (150 μM) for 4 hours, and TF and THBD antigen were detected by immunofluorescence in permeabilized cells. Original magnification, ×40; bar represents 10 μm; nuclear counterstain with 4′6-diamidino-2-phenylindole (n = 3). (B) EA.hy926 cells were stimulated with poly(I:C) (12.5 μg/ml) and PAR1 and PAR2 activation peptides for 6 hours in triplicate, and the protein levels of TF and β-tubulin were determined by western blot analysis with the respective antibodies. Data shown in the bar graph represent the mean ± standard deviation of the ratio of TF to β-tubulin, determined by quantitative densitometry of western blots (n = 3) . (C) Cell lysates were prepared from EA.hy926 cells stimulated with poly(I:C) (12.5 μg/mL) and PAR1- and PAR2-activation peptides (150 μM) for 6 hours. TF procoagulant activity (PCA) was measured in the presence or absence of anti-TF antibody (HTF-1; 10 µg/mL) or IgG control (IgG2a; 10 μg/mL) by a 1-stage clotting assay and converted into picograms TF per milligrams protein from a standard curve generated with recombinant TF (Innovin; n = 3). (D) Cell-surface TF activity on intact cells was determined by an fXa generation assay (n = 5-6). (E) EA.hy926 cells were stimulated for 6 hours with poly(I:C) and/or thrombin, and TF procoagulant activity was measured by a 1-stage clotting assay and converted into PCA via a standard curve generated with recombinant TF (Innovin; n = 3). (F) Immunofluorescence detection of cell-surface TF and Thbd antigen on EA.hy926 cells stimulated for 4 hours with poly(I:C) and/or thrombin (original magnification, ×100; bar represents 10 μm; n = 3). (G) EA.hy926 cells were pretreated for 45 minutes with inhibitors of PAR1 (vorapaxar, 1 μM) and/or PAR2 (GB83, 25 μM) followed by stimulation with poly(I:C) and/or thrombin for 6 hours. Cell-surface TF activity was determined by fXa generation (n = 8). (H) EA.hy926 cells were pretreated for 45 minutes with PAR-specific inhibitors (vorapaxar, 1 μM; GB83, 25 μM) followed by stimulation with poly(I:C) and/or thrombin for 6 hours. Abundance of TF and β-tubulin in whole-cell lysates were determined by western blot analysis (n = 3). (I) EA.hy926 cells were pretreated (45 minutes) with cleavage-blocking antibodies for PAR1 (WEDE15, 10 μg/mL; ATAP2, 10 μg/mL) and/or PAR2 (SAM-11, 10 μg/mL), or isotype-matched nonimmune IgG controls followed by stimulation with poly(I:C) and/or thrombin for 6 hours. Cell-surface TF activity was determined via FXa generation assay (n = 4-8). Statistical significance was determined by ANOVA followed by a multiple-comparison test. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, not significant.

Thrombin-PAR1/2 signaling amplifies cell surface-associated TF procoagulant activity in poly(I:C)-treated cells. (A) EA.hy926 cells were stimulated with poly(I:C) (12.5 μg/mL) and PAR1-selective (SFLLR) or PAR2-selective agonist peptides (150 μM) for 4 hours, and TF and THBD antigen were detected by immunofluorescence in permeabilized cells. Original magnification, ×40; bar represents 10 μm; nuclear counterstain with 4′6-diamidino-2-phenylindole (n = 3). (B) EA.hy926 cells were stimulated with poly(I:C) (12.5 μg/ml) and PAR1 and PAR2 activation peptides for 6 hours in triplicate, and the protein levels of TF and β-tubulin were determined by western blot analysis with the respective antibodies. Data shown in the bar graph represent the mean ± standard deviation of the ratio of TF to β-tubulin, determined by quantitative densitometry of western blots (n = 3) . (C) Cell lysates were prepared from EA.hy926 cells stimulated with poly(I:C) (12.5 μg/mL) and PAR1- and PAR2-activation peptides (150 μM) for 6 hours. TF procoagulant activity (PCA) was measured in the presence or absence of anti-TF antibody (HTF-1; 10 µg/mL) or IgG control (IgG2a; 10 μg/mL) by a 1-stage clotting assay and converted into picograms TF per milligrams protein from a standard curve generated with recombinant TF (Innovin; n = 3). (D) Cell-surface TF activity on intact cells was determined by an fXa generation assay (n = 5-6). (E) EA.hy926 cells were stimulated for 6 hours with poly(I:C) and/or thrombin, and TF procoagulant activity was measured by a 1-stage clotting assay and converted into PCA via a standard curve generated with recombinant TF (Innovin; n = 3). (F) Immunofluorescence detection of cell-surface TF and Thbd antigen on EA.hy926 cells stimulated for 4 hours with poly(I:C) and/or thrombin (original magnification, ×100; bar represents 10 μm; n = 3). (G) EA.hy926 cells were pretreated for 45 minutes with inhibitors of PAR1 (vorapaxar, 1 μM) and/or PAR2 (GB83, 25 μM) followed by stimulation with poly(I:C) and/or thrombin for 6 hours. Cell-surface TF activity was determined by fXa generation (n = 8). (H) EA.hy926 cells were pretreated for 45 minutes with PAR-specific inhibitors (vorapaxar, 1 μM; GB83, 25 μM) followed by stimulation with poly(I:C) and/or thrombin for 6 hours. Abundance of TF and β-tubulin in whole-cell lysates were determined by western blot analysis (n = 3). (I) EA.hy926 cells were pretreated (45 minutes) with cleavage-blocking antibodies for PAR1 (WEDE15, 10 μg/mL; ATAP2, 10 μg/mL) and/or PAR2 (SAM-11, 10 μg/mL), or isotype-matched nonimmune IgG controls followed by stimulation with poly(I:C) and/or thrombin for 6 hours. Cell-surface TF activity was determined via FXa generation assay (n = 4-8). Statistical significance was determined by ANOVA followed by a multiple-comparison test. *P < .05; **P < .01; ***P < .001; ****P < .0001. ns, not significant.

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