Inhibitory effect of SM on TF coagulant activity. (A) Full-length recombinant human TF was reconstituted in liposomes with PC alone or PC with varying % molar concentrations of SM. TF coagulant activity was measured by its ability to support FVIIa activation of FX using 10 pM of relipidated TF, 1 nM FVIIa, and 175 nM of FX. FXa generation was measured in a chromogenic assay. (B) TF (1.3 nM) relipidated in PC, PC:SM (60:40 mol %), or PC:ceramide (60:40 mol %) was incubated with FVIIa (10 pM) and FX (175 nM), and the activation of FX was measured in a chromogenic assay. (C) TF (10 pM) relipidated in PC (○) or PC: SM (60:40 mol %) (●) was incubated with varying concentrations of FVIIa (1 to 100 pM FVIIa), and TF-FVIIa activation of FX was measured. (D) TF (10 nM) reconstituted in PC or PC with varying % molar concentrations of SM was incubated with FVIIa (100 nM), and TF-FVIIa amidolytic activity was measured using tissue plasminogen activator chromogenic substrate (Chromozym-tPA). (E) TF (10 nM) relipidated in PC (○) or PC: SM (60:40 mol %) (●) was incubated with varying concentrations of FVIIa (1 to 100 nM), and TF-FVIIa amidolytic activity was measured in a chromogenic assay. FVIIa amidolytic activity in the absence of TF was also measured (□). (F) TF (0.5 nM) relipidated in PC alone, PC:SM (60:40 mol %), PC:PS (94:6 mol %), or PC:PS:SM (54:6:40 mol %) was incubated with FVIIa (10 pM) and FX (175 nM), and TF-FVIIa activation of FX was measured. *P < .05; ** P < .01; ns, no statistical significance.