The stimulatory effect of phospholipids requires the heparin-binding site of PCI. (A) aPC was incubated with recombinant wild-type PCI (wtPCI) or mutated PCI, in which the H-helix was deleted (ΔH-PCI) in the absence and presence of heparin or PAPS for 20 minutes as indicated. Remaining aPC activity was determined using S-2366 as described in “Materials and methods.” (Inset) Western blots performed after SDS-PAGE of wtPCI and ΔH-PCI. (B) PAPS, OxPAPS, and OxPAPE (100 μg/mL each) were immobilized on microtiter plates and incubated with wild-type PCI or ΔH-PCI (4 nM each). Bound PCI was detected as described in “Materials and methods.” Means of triplicates ± SEM are shown. (C) aPC was incubated with PCI (10 nM) in the presence of heparin (0.5 μg/mL) or PAPS (50 μg/mL) and in the presence of different concentrations of peptide AA264-283 for 20 minutes at 37°C. Remaining enzymatic activity was determined as described in “Materials and methods.” (D) Thrombin (1 nM) was incubated for 20 minutes with different concentrations of PCI (i) or ATIII (ii) in the absence or presence of heparin (2.5 μg/mL), PAPE (50 μg/mL), or OxPAPE (50 μg/mL). Remaining thrombin activity was determined as described in “Materials and methods.” Data shown represent means of quadruplicates (i) or duplicates (ii). *P ≤ .02; **P ≤ .004. Similar results were obtained in 3 independent experiments.