Figure 6.

Loss of annexin A2 and overexpression of SLPI alter plasminogen activation. A: Annexin II siRNA (si-A) and an unrelated control siRNA (si-C) were transfected into monocytes using the Amaxa Human Dendritic Cell Nucleofector kit.²³ The cells were incubated 6 days before analysis of annexin II and α-tubulin (control) protein using Western blot (inset) and plasmin proteolytic activity in parallel with control (ctrl) cells. B: Adherent macrophages were treated with adenovirus vector (Ad), adenovirus-SLPI (Ad-SLPI), or no treatment. After 24 hours incubation, medium was removed and replaced with 10% DMEM. Day 3 to 7 supernatants were tested for SLPI by ELISA (inset, n = 2). Cells were detached, washed in PBS, and tested for plasminogen (Plg) activation (relative fluorescent units × 10³) in parallel with control cells. *P < 0.05. C: Fibrin gels were prepared in 12-well plates and the transfected (Ad-only, Ad-SLPI) and control macrophages were resuspended in gelatin barbital buffer and 50 μL (2 × 10⁶ cells) added to fibrin plates for 1 to 3 days at 37°C. The 3-day wells were then stained with Coomassie Blue (n = 2) and areas of degradation visualized.