Detection of plasminogen bound to cells under pathologic conditions. (A) Peripheral blood was incubated for 6 hours with 10 μg/mL LPS (yellow tracing) or untreated (red tracing; left panel) or 1nM PMA (green tracing) or untreated (red tracing; right panel, as indicated), and FACS analysis was performed with antiplasminogen mAb49 as described in “Methods.” Purple tracings indicate FACS analyses of untreated blood with isotype control. Neutrophils were gated with anti-CD15 mAb. (B) FACS analyses with mAb49 of cell lines of different lineages incubated with 10μM plasminogen (black tracings) or without plasminogen (blue tracings). In other controls, the cell lines were treated with carboxypeptidase B (200 U/mL) before adding plasminogen (teal tracings). The cell events are different for each cell type because the calibration of the flow cytometer changes in each experiment. (C) Molt4 cells (2.5 × 10⁶ cells/mL) were incubated with 10μM plasminogen for 1 hour at 37°C in either the absence (black bar) or presence of 0.2M EACA (violet bar) or without plasminogen or EACA (blue bar). Radiolabeled mAb49 (20nM) was then added and incubated for 30 minutes, and cell-bound radioactivity was separated from free radioactivity as described in “Methods.” In controls, direct binding of mAb49 to immobilized plasminogen (absorbance OD₄₀₅ = 0.18 ± 0.003) was not decreased in the presence of EACA (absorbance OD₄₀₅ = 0.22 ± 0.002). Thus, these results suggest that mAb49 binding to the cells reports the full picture of cell bound plasminogen. (D) NB4 cells were incubated with 1μM all-trans-retinoic acid (ATRA) for 48 hours, washed and preincubated with plasminogen (10μM), followed by FACS analyses with mAb49 (orange tracing). FACS analyses with mAb49 of untreated NB4 cells preincubated with either plasminogen (black tracing) or buffer (blue tracing) detected by antiplasminogen mAb49 is also shown. (E) Top panel: FACS analysis using mAb49 of peripheral blood from a patient containing promyelocytic blast cells (CD33⁺; HLDR⁻) at day 0 of ATRA treatment (red tracing) and after ATRA treatment for either 4 days (orange tracing) or 5 days (pink tracing). As a negative control, FACS analysis with an isotype control antibody was performed on blood containing promyelocytic blast cells (CD33⁺; HLDR⁻) at day 0 (turquoise tracing). Bottom panel: FACS analysis using mAb49 (red tracing) or isotype control (turquoise tracing) of blood from a patient with an M1 leukemia.