Effect of phagocytosis on plasmin formation and annexin II expression. (A) Plasmin generation of 1 × 106 target APL cells, 5 × 105 phagocytes (THP-1–derived MΦs or HUVECs), or incubation of 1 × 106 target APL cells with 5 × 105 phagocytes was evaluated at the given times. Plasmin production of the coincubated cells was time dependently reduced. *P < .05 compared with 0 hour time point of each group. (B) Plasmin formation of 1 × 106 target APL cells, with or without 2nM annexin V or lactadherin, and with or without incubation with 5 × 105 phagocytes (THP-1–derived MΦs or HUVECs) after 2 hours was measured. Plasmin formation of 1 × 106 viable APL cells is also shown. *P < .05 compared with the mixture of target APL cells and phagocytes (MΦs and ECs separately). (C) NB4 cells were first labeled with goat anti–human annexin II IgG, and then with an Alexa Fluor 488–conjugated secondary Ab. Annexin II expression on permeabilized untreated NB4 cells (left) and 1μM DNR-treated NB4 cells (right) was viewed with confocal microscopy. The cell nuclei were counterlabeled with PI (red), and scale bar represents 10 μm. (D) Nonpermeabilized or permeabilized APL cells with and without 1μM DNR treatment were stained as in panel C and analyzed by flow cytometry. Annexin II expression of DNR-treated cells decreased compared with untreated viable APL cells. (E) Flow cytometry was used to quantitate annexin II expression on cells that were treated as in panel C. Cells stained with goat anti–human IgG and Alexa Fluor 488–conjugated secondary Ab were used as control (black). The percentage of annexin II–positive viable APL cells (green) and target APL cells (pink) from one patient with permeabilization were 98.3% and 35.1%, respectively (left). Middle panel showed that, compared with controls, annexin II was expressed on the surface of nonpermeabilized THP-1–derived MΦs (pink) and more so on permeabilized cells (green). Permeabilized HUVECs showed an increase in annexin II (green), but, compared with controls, nonpermeabilized HUVECs (pink) showed no increase in annexin II (right).
