Agonist-induced PDI secretion from HUVECs. PDI secretion from HUVECs into serum-free cell culture medium after overnight culture of resting or activated cells was detected by sodium dodecyl sulfate–electrophoresis of the medium followed by immunoblotting with anti-PDI antibodies. (A) PDI in culture medium (50 μL from confluent monolayer ∼ 1 × 10⁶ cells) detected with the monoclonal anti-PDI antibody, RL90, at 1 μg/mL. Lane 1, conditioned media from resting HUVECs; lane 2, conditioned medium from HUVECs activated with 1 U/mL thrombin; lane 3, conditioned medium from HUVECs activated with 10μM A23187 calcium ionophore. (B) Immunoblot showing PDI secretion after thrombin stimulation from 5 × 10⁶ HUVECs over a period of 30 minutes. Top panel, medium from thrombin-activated HUVECs. Bottom panel, cell lysates from the corresponding activated cells. (C) Time course study of PDI release from 5 × 10⁶ HUVECs after stimulation with (■) 100 ng/mL PMA, (♦) 1 U/mL thrombin, or (▴) 0.1mM histamine. (D) PDI activity was measured by the insulin transhydrogenase assay in the conditioned media from 5 × 10⁶ thrombin-activated HUVECs (mean of 3 experiments) in the presence (unfilled) or absence (filled) of 5 μg/mL monoclonal inhibitory antibody RL90. Circles, PDI activity secreted from cultured HUVECs; triangles, activity of recombinant human PDI (1 μg/mL).