1. Plasminogen-mediated matrix invasion and degradation by macrophages is dependent on surface expression of annexin II
by Domenick J. Falcone, Wolfgang Borth, K. M. Faisal Khan, and Katherine A. Hajjar
Blood
Volume 97(3):
February 1, 2001
©2001 by American Society of Hematology

2. Demonstration of annexin II surface expression on RAW264
Demonstration of annexin II surface expression on RAW264.7 macrophages by flow cytometric analysis.Control and EGTA-treated RAW264.7 macrophages were incubated with anti–annexin IgG (clone Z014, Zymed) in the absence or presence of EGTA followed by FITC-con...
Demonstration of annexin II surface expression on RAW264.7 macrophages by flow cytometric analysis.Control and EGTA-treated RAW264.7 macrophages were incubated with anti–annexin IgG (clone Z014, Zymed) in the absence or presence of EGTA followed by FITC-conjugated goat antimouse IgG. Relative fluorescence units are shown for each sample. To verify that annexin II was removed from the cell surface by treatment with EGTA, eluates were examined by Western blot as described in “Materials and methods” (inset).
Domenick J. Falcone et al. Blood 2001;97:
©2001 by American Society of Hematology

3. Identification of plasminogen binding proteins in RAW264
Identification of plasminogen binding proteins in RAW264.7 macrophage membrane preparation.For ligand blot, cell lysates and isolated membranes (Mem) were electrophoresed in gradient polyacrylamide gels and transferred to a PVDF membrane.
Identification of plasminogen binding proteins in RAW264.7 macrophage membrane preparation.For ligand blot, cell lysates and isolated membranes (Mem) were electrophoresed in gradient polyacrylamide gels and transferred to a PVDF membrane. PVDF membranes were blocked and subsequently incubated with buffer alone (Ctrl) or buffer containing 10 μg/mL Lys-plasminogen (+Lys-Plg). Bound plasminogen was visualized using monoclonal antiplasminogen IgG as described in “Materials and methods.” The presence of immunoreactive annexin II in macrophage lysates and isolated membranes was determined by Western blot as described in “Materials and methods.”
Domenick J. Falcone et al. Blood 2001;97:
©2001 by American Society of Hematology

4. Binding of 125I-Lys-plasminogen to RAW264
Binding of 125I-Lys-plasminogen to RAW264.7 macrophages is inhibited by anti–annexin II.(Left panel) Adherent cells (105/well) were washed with DPBS (3 ×).
Binding of 125I-Lys-plasminogen to RAW264.7 macrophages is inhibited by anti–annexin II.(Left panel) Adherent cells (105/well) were washed with DPBS (3 ×). The media were replaced with cold MSFM alone or MSFM containing 0.1 to 6 μg/mL 125I-Lys-plasminogen (125Lys-Plg). Following incubation for 1 hour at 4°C with125I-Lys-plasminogen, cells were washed (3 ×) with DPBS and dissolved in 0.5 NaOH. Data represent the mean ± SE of 3 separate samples. (Right panel) Adherent cells (105/well) were washed with DPBS (3 ×). The media were replaced with cold MSFM alone or MSFM containing monoclonal anti–annexin II IgG (10-25 μg/mL; clone Z014, Zymed), mouse IgG (25 μg/mL), or 25 mM ε-ACA. The cells were incubated with the antibodies for 2 hours at room temperature before the addition of 125I-Lys-plasminogen (1.0 μg/mL). Cells were then incubated 1 hour at 4°C. The data represent the mean ± SEM of 6 replicate samples. nIgG indicates normal IgG.
Domenick J. Falcone et al. Blood 2001;97:
©2001 by American Society of Hematology

5. Plasmin binding to RAW264.7 macrophages is reduced following incubation with EGTA.(Left panel) Adherent cells (105/well) were washed with DPBS (3 ×).
Plasmin binding to RAW264.7 macrophages is reduced following incubation with EGTA.(Left panel) Adherent cells (105/well) were washed with DPBS (3 ×). The media were replaced with cold MSFM alone or MSFM containing 0.2 to 8 μg/mL Lys-plasmin. Following incubation for 1 hour at 4°C, cells were washed (3 ×) with DPBS and membrane-bound plasmin activity determined as described in “Materials and methods.” Data represent the mean ± SE of 3 separate samples. (Right panel) Adherent cells (105/well) were washed with versine buffer (PBS/0.5 mM EDTA) at 4°C and then incubated with versine buffer containing 25 mM EGTA for 20 minutes. The EGTA was removed and cells were washed with versine buffer. The cells were incubated with versine buffer containing 25 mM EGTA, 0.1% bovine serum albumin, and Lys-plasmin (1.0 μg/mL) for 1 hour at 4°C. Membrane-bound plasmin (Pls) activity was determined as described in “Materials and methods.” The data represent the mean ± SEM of 3 replicate samples. The inset is a Western blot for annexin II of membranes isolated from Ctrl (control) and EGTA-treated cells.
Domenick J. Falcone et al. Blood 2001;97:
©2001 by American Society of Hematology

6. Anti–annexin II inhibits plasmin binding to human monocyte-derived macrophages.Cells were aliquoted into 96-well plates (105/well).
Anti–annexin II inhibits plasmin binding to human monocyte-derived macrophages.Cells were aliquoted into 96-well plates (105/well). The next day 2 groups of cells were washed (3 ×) with DPBS. The media were replaced with MSFM containing monoclonal anti–annexin II (clone Z014, Zymed) or normal mouse IgG (nIgG; 25 μg/mL). Following incubation for 30 minutes at 37°C, plasmin (1 μg/mL) was added and cells were incubated for 1 hour at 4°C. The remaining groups of cells were washed and media replaced with plasmin (Pls) alone, plasmin and ε-ACA (25 mM), or plasmin and purified annexin II. Following incubation for 1 hour at 4°C, membrane-bound plasmin was determined as described in “Materials and methods.” The data represent the mean ± SEM of 3 replicate samples. Control (Ctrl) indicates cells in the absence of exogenous plasmin.
Domenick J. Falcone et al. Blood 2001;97:
©2001 by American Society of Hematology

7. Extracellular matrix degradation by RAW264
Extracellular matrix degradation by RAW264.7 macrophages is inhibited by anti–annexin II IgG.Cells (5 × 105/well) were plated on insoluble [3H]-glucosamine–labeled human smooth muscle cell matrices in MSFM. Following adherence, media were replaced and 25 μg...
Extracellular matrix degradation by RAW264.7 macrophages is inhibited by anti–annexin II IgG.Cells (5 × 105/well) were plated on insoluble [3H]-glucosamine–labeled human smooth muscle cell matrices in MSFM. Following adherence, media were replaced and 25 μg/mL monoclonal anti–annexin II (clone Z014, Zymed) or mouse IgG added. Cells were incubated with the antibodies (30 minutes, 37°C) prior to the addition of plasminogen (Plg, 1 μg/mL). Incubation media were recovered the next day and solubilized [3H]-activity determined as described in “Materials and methods.” The media and media plus plasminogen (Plg) controls depict the levels of [3H]-glucosamine released from insoluble extracellular matrix in the presence of plasminogen but in the absence of macrophages. Data represent the mean ± SEM of 4 replicate samples. nIgG indicates normal IgG.
Domenick J. Falcone et al. Blood 2001;97:
©2001 by American Society of Hematology

8. Matrix invasion by THP-1 monocytes is inhibited by anti–annexin II
Matrix invasion by THP-1 monocytes is inhibited by anti–annexin II.Cells (4 × 104) were suspended in MSFM and preincubated with either 25 μg/mL monoclonal anti–annexin II IgG (clone Z014, Zymed), monoclonal anti-CD16 IgG, or anti-CD11b IgG.
Matrix invasion by THP-1 monocytes is inhibited by anti–annexin II.Cells (4 × 104) were suspended in MSFM and preincubated with either 25 μg/mL monoclonal anti–annexin II IgG (clone Z014, Zymed), monoclonal anti-CD16 IgG, or anti-CD11b IgG. Plasminogen (Plg, 10 μg/mL) was added to the cell suspension and cells were aliquoted into an insert containing a porous (8 μm) polycarbonate membrane previously coated with Matrigel. The insert was placed into a well containing MCP-1 (50 ng/mL) and incubated 24 hours at 37°C. Cells that migrated into the lower well were counted using phase-contrast microscopy. The results of 2 independent experiments containing 3 replicate samples for each condition per experiment were pooled. The 2 experiments were identical except for the control IgG used. For the purposes of presentation and analysis the 2 groups were combined. Data are presented as the mean ± SEM. nIgG indicates normal IgG.
Domenick J. Falcone et al. Blood 2001;97:
©2001 by American Society of Hematology