Fig. 1. Effects of bPDI and PC/PS on TF procoagulant reactions.

A bPDI or PC/PS were subjected to 80% acetone precipitation at −20°C for 30 minutes, precipitates were recovered by centrifugation for 10 min and redissolved in HBS (10 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 1.5 mM CaCl₂). The factor Xa generation assay was carried out in HBS, 1 mg/ml BSA with 10 nM VIIa, 500 nM substrate X, and 10 nM soluble TF_1–219 with or without 100 nM bPDI or 10 μM PC/PS (70% phosphatidyl choline/30% phosphatidylserine; w/w). Samples were quenched after 45 minutes for chromogenic assay. The efficiency of precipitation was confirmed by Western blotting of non-reduced samples of the starting material and the resuspended acetone precipitate. Note that the dimer content was not changed by the precipitation step and that no other bands were visible on this blot. B RL90 or 1D3 monoclonal anti-PDI antibody (Stressgen, Ann Arbor, MI) or irrelevant IgG control (0.4 μg) were incubated with PDI or PC/PS for 15 minutes followed by immunoprecipitation with Protein G Dynabeads. Controls and depleted supernatants at volumes equivalent to the starting material were added to the soluble TF assay described in A. C TF⁺ micro particles were recovered from 2 day cultures of adherent HaCaT keratinocytes by a brief wash in 15% isopropanol in HBS which yielded micro particles with properties indistinguishable from those released into serum-free culture supernatant. Cell-free supernatants were subjected to ultracentrifugation and assayed for TF activity under the conditions described above. Rates of Xa generation were calculated from multiple samples over a 20 minute time period. The rate enhancing effects of 100 nM bPDI or 10 μM PC/PS were calculated for the indicated range of mastoparan concentrations or 100 μM inactive control peptide mastoparan 17 (Sigma). Data are mean ± SD (n = 4–5), * p < 0.05. D TF cell surface activity of HaCaT monolayers grown for two days was measured in the absence or presence of 10 μM PC/PS, 100 nM bPDI, 100 nM NEM-blocked bPDI, or 100 nM bPDI subjected to antibody depletion as indicated. Data are mean ± SD (n = 3–5).