PDI is a negative regulator of coagulation on ECs. (A) TF-ECs were treated with anti-PDI antibodies (10 μg/mL) for 30 minutes or ionomycin (Iono, 5μM) for 10 minutes before TF procoagulant activity was measured by a 2-stage Xa generation assay. TF activity is normalized against control samples and represented as fold increase over control IgG-treated ECs. *P < .05, ***P < .001 vs control. (B) PDI enzymatic activity was determined by an insulin turbidity assay in the presence of antibody-treated PDI (□, rabbit polyclonal; ▴, BD34 mAb) or PDI alone (■) and compared with no PDI controls (○). Not shown: IgG1k isotype or irrelevant rabbit IgG treated PDI was indistinguishable from PDI alone (■), whereas insulin reduction in the presence of antibody without PDI was indistinguishable from no PDI samples (○). (C) Antibody inhibition of surface PDI enhanced Xa generation in EA.hy926 cells stimulated with TNF-α (TNF⁺, **P < .01) but not in vehicle-treated cells (TNF⁻). (D) Procoagulant effects of surface PDI inhibition are sensitive to anti-TF antibodies (**P < .01) similar to ionomycin-induced decryption (***P < .001). (E) The procoagulant effects of extracellular PDI inhibition are observed on both EC surface and in the supernatant released from the cells (P < .01). (F) PDI inhibition increases TF procoagulant function, which can be further enhanced by ionomycin treatment. In contrast, preincubation with recombinant human PDI limits the amplitude of ionomycin-induced TF decryption. **P < .01, ***P < .001, 1-way ANOVA. (A-F) Data are mean ± SD of triplicates.