Evidence that traces of anionic phospholipids present in commercially obtained bovine PDI is responsible for increasing sTF activity. (A) Bovine PDI but not human rPDI enhances sTF activity. The reaction mixtures were in a buffer containing 10 mM Hepes, 0.15 M NaCl, 1 mg/mL BSA, and 5 mM CaCl2 and contained sTF (10 nM), FVIIa (10 nM), and various concentrations of bovine PDI, rhPDI (0 to 100 nM) or PC/PS (80%:20% wt/wt) vesicles (0 to 10 μM). TF-FVIIa activity was measured by adding the substrate, factor X (1 μM), and measuring the amount of factor Xa generated at the end of a 10-minute reaction period in a chromogenic assay (note: y-axis is in a log scale). (B). Bovine PDI enhances prothrombin activation. Factor Xa (0.1 nM) plus FVa (10 nM) were incubated for 5 minutes with a control buffer or various concentrations of bovine PDI, rhPDI, or PC/PS vesicles, and then prothrombin (1.4 μM) was added to initiate the reaction. At the end of a 2-minute reaction period, an aliquot was removed from the reaction mixture and the amount of thrombin formed was measured in a chromogenic assay. (C) Annexin V, a phospholipid binding protein, inhibits bovine PDI-mediated increased sTF activity. Bovine PDI (10 nM) or PC/PS vesicles (1 μM) was preincubated with various concentrations of annexin V for 30 minutes and then added to sTF (10 nM). TF activity was measured as described in panel A. (D). Treatment of bovine PDI with phospholipase C abolishes the enhancing effect of bovine PDI on sTF activity. Bovine PDI (10 nM) or PC/PS vesicles (1 μM) were treated with phospholipase C (0.1 U/mL) for 15 minutes before they were added to sTF. TF activity was measured as in panel A. The data shown in panels A to D represent means plus or minus SEM (n = 3-5). (E) PDI fails to bind to sTF. A CM5 sensor chip was coated with sTF and the chip was equilibrated overnight with the buffer at a flow rate of 5 μL/min. various concentrations of FVIIa or bovine PDI (10, 50, 100 nM) were passed over the sensor chip for 5 minutes (association time), followed by 10 minutes dissociation period at a flow rate of 30 μL//min. Regeneration was performed with a 3-minute pulse of 10 mM EDTA in Hepes buffer. Similar to the data shown in Figure 1E, no binding was observed between rhPDI and sTF.
