|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
PHAGOCYTES
From the Departments of Pathology, Cell Biology,
Pediatrics, and Medicine, Joan and Sanford I. Weill Medical College of
Cornell University, New York, NY, and the Institute of Immunology,
University of Vienna, Vienna, Austria.
Genetic evidence demonstrates the importance of plasminogen
activation in the migration of macrophages to sites of injury and
inflammation, their removal of necrotic debris, and their clearance of
fibrin. These studies identified the plasminogen binding protein
annexin II on the surface of macrophages and determined its role in
their ability to degrade and migrate through extracellular matrices.
Calcium-dependent binding of annexin II to RAW264.7 macrophages was
shown using flow cytometry and Western blot analysis of EGTA eluates.
Ligand blots demonstrated that annexin II comigrates with one of
several proteins in lysates and membranes derived from RAW264.7
macrophages that bind plasminogen. Preincubation of RAW264.7
macrophages with monoclonal anti-annexin II IgG inhibited (35%) their
binding of 125I-Lys-plasminogen. Likewise, plasmin
binding to human monocyte-derived macrophages and THP-1 monocytes was
inhibited (50% and 35%, respectively) when cells were preincubated
with anti-annexin II IgG. Inhibition of plasminogen binding to annexin
II on RAW264.7 macrophages significantly impaired their ability to
activate plasminogen and degrade
[3H]-glucosamine-labeled extracellular matrices. The
migration of THP-1 monocytes through a porous membrane, in response to
monocyte chemotactic protein-1, was blocked when the membranes were
coated with extracellular matrix. The addition of plasminogen to the monocytes restored their ability to migrate through the matrix-coated membrane. Preincubation of THP-1 monocytes with anti-annexin II IgG
inhibited (60%) their plasminogen-dependent chemotaxis through the
extracellular matrix. These studies identify annexin II as a
plasminogen binding site on macrophages and indicate an important role
for annexin II in their invasive and degradative phenotype.
(Blood. 2001;97:777-784) The recruitment of monocytes to an inflammatory
site is a complex process involving their adhesion to endothelial
cells, passage into the perivascular connective tissue, and migration
toward a chemotactic gradient.1,2 The synthesis and
activation of matrix degrading proteinases have been suggested to play
an essential role in the migration of monocytes and macrophages through
tissue. In this regard, the results of in vitro and in vivo studies
have suggested that macrophage expression of urokinase-type plasminogen activator (uPA) and its receptor (uPA-R) are important regulators of
migration,3,4 degradation of the fibrin-rich matrix that forms following tissue injury,5 fibrinolysis in the
atheroma,6 degradation of extracellular
matrices,7,8 and the release of matrix-bound growth
factors.9,10
The availability of animals genetically deficient in selected
components of the plasminogen/plasminogen activator system has refined
our understanding of the role of plasminogen activation in macrophage
migration and tissue remodeling following injury. For example,
thioglycollate-induced recruitment of monocytes into the peritoneal
cavities of mice deficient in plasminogen was diminished when compared
to wild-type mice.11 In an experimental model of
glomerulonephritis, macrophage infiltration of glomeruli was diminished
in mice deficient in uPA.12 Likewise, the recruitment of
monocytes to a vascular injury site appears to be critically dependent
on uPA-mediated plasminogen activation. In the setting of
full-thickness electrical injury of the femoral artery, plasminogen deficiency was associated with reduced infiltration of leukocytes, delayed removal of the necrotic tissue, and reduced formation of a
neointima.13 Similar results were observed in mice
deficient in uPA but not tissue-type plasminogen activator
(tPA).14 The analysis of atherosclerosis in apolipoprotein
E-deficient mice demonstrated that superimposed uPA deficiency
protects against aortic aneurysm formation, whereas tPA deficiency had
no protective effect.15 Lesion infiltration by macrophages
was essentially absent in uPA-deficient mice, but appeared normal in
tPA-deficient mice.15 In acute myocardial infarction,
macrophage infiltration into necrotic tissue was significantly reduced
in uPA-deficient mice and these animals were protected from ventricular
rupture.16 In contrast, deficiency in uPA-R, tPA,
stromelysin-1 (MMP-3), or metalloelastase (MMP-12) did not protect
against ventricular rupture. Thus, although neither uPA-R nor tPA seems
to be essential for macrophage recruitment to the healing myocardium,
uPA appears to play a central role.
The broad substrate specificity of plasmin requires that macrophages
localize its activation to the pericellular environment to limit
collateral tissue damage. An important mechanism by which macrophages
regulate plasminogen activation is the differential expression of uPA
and plasminogen activator inhibitor.4,10,17-21 In
addition, localized activation is achieved through the expression of
uPA-R and binding sites for plasminogen.22-24 Despite
direct evidence demonstrating the importance of plasminogen activation in the ability of macrophages to migrate to sites of injury and inflammation, as well as their ability to degrade necrotic debris and
fibrin, the identity of plasminogen binding sites remains unclear.
The binding of plasminogen to cells is mediated by plasminogen's
kringle domains and is inhibited by the lysine analogue
Human umbilical vein endothelial cells express a protein (36 kd) that
independently binds both plasminogen and tissue plasminogen activator.30,34 Sequence analysis of internal tryptic
peptides indicated that this protein is annexin II.30
Annexin II is a member of the annexin family of calcium-dependent
phospholipid binding proteins.35 Annexins lack a
hydrophobic signal sequence and were initially considered intracellular
proteins.35 Nonetheless, surface expression of annexin II
by a variety of cells has been demonstrated,30,34,36,37
and other annexins have been identified bound to components of the
extracellular matrix.37,38 In addition to serving
as a receptor for plasminogen and tPA, annexin II has been identified
as a high-affinity binding site for tenascin-C. In experiments reported
here, we demonstrated that annexin II is a binding site for plasminogen
on murine RAW264.7 macrophages, human monocyte-derived macrophages, and
THP-1 monocytes. Inhibition of plasminogen binding to annexin II
significantly inhibited plasminogen activation and extracellular matrix
degradation by RAW264.7 macrophages, as well as the chemotaxis of THP-1
monocytes through the extracellular matrix.
Cell culture
Isolation of a membrane-rich fraction from macrophage
lysates
Identification of membrane plasminogen binding proteins by ligand blot Samples and biotinylated molecular weight markers (Amersham Pharmacia Biotech, Piscataway, NJ) were electrophoresed in 4% to 15% polyacrylamide gradient gels. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA). The membrane was blocked in fresh 5.0% dry defatted milk in Tris-buffered saline/0.05% Tween-20 (TTBS) for 1 hour, washed once with TTBS for 15 minutes, and then incubated 1 hour at room temperature with 0.25% dry defatted milk in TTBS with or without Lys-plasminogen (10 µg/mL; American Diagnostica Inc, Greenwich, CT). Following 2 washes with TTBS, the membrane was incubated in 3% dry defatted milk in TTBS containing a monoclonal antibody directed against kringles 1 to 3 of human plasmin(ogen) (2.5 µg/mL; American Diagnostica) for 1 hour. The membrane was washed twice (TTBS), incubated for 1 hour in 3% dry defatted milk in TTBS containing biotinylated rabbit antimouse IgG (1:30 000; Pierce, Rockford, IL), washed twice (TTBS), and incubated 1 hour with preformed avidin-biotin-horseradish peroxidase (HRP) complexes (Pierce) in DPBS/0.1% Tween-20. Bound HRP was visualized using enhanced chemiluminescence (ECL; Amersham Life Science).Western blot for annexin II Purified annexin II and samples were electrophoresed in 4% to 15% polyacrylamide gradient gels. Proteins were transferred to a PVDF membrane, following which the membrane was blocked for 1 hour in TTBS containing 5% dry defatted milk. Following 2 washes (TTBS), the membrane was incubated 1 hour with 0.5 µg/mL monoclonal antihuman annexin II IgG (clone 5, IgG1, Transduction Laboratories, Lexington, KY) in TTBS containing 3% dry defatted milk. The membrane was washed twice (TTBS) and incubated 1 hour in 3% dry defatted milk in TTBS containing biotinylated rabbit antimouse IgG (1:30 000; Pierce). The membrane was then washed twice (TTBS) and incubated 1 hour with preformed avidin-biotin-HRP complexes (Pierce) in DPBS/0.1% Tween-20. Bound HRP was visualized using ECL.Flow cytometric analysis The RAW264.7 cells were propagated in suspension in Teflon beakers and their reactivity with a monoclonal antibody directed against annexin II (clone Z014, IgG1, Zymed Laboratories Inc, San Francisco, CA) was analyzed by flow cytometry. Cells were washed 3 times with Hepes-buffered saline (HBS; 137 mM NaCl, 4 mM KCl, 11 mM glucose, and 11 mM Hepes, pH 7.4) with or without 10 mM EGTA and resuspended in HBS or HBS/EGTA containing clone Z014 (100 µg/mL). Cell suspensions were incubated on a rocking platform (4°C, 45 minutes), washed in 20 volumes of HBS (or HBS/EGTA), resuspended in HBS (or HBS/EGTA) containing fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG (20 µg/mL; Cappel/ICN, Costa Mesa, CA) and incubated 45 minutes at 4°C. Following 3 additional washes, cells were resuspended in HBS and analyzed on a Coulter Epics XL flow cytograph (Beckman Coulter).Iodination of Lys-plasminogen Human Lys-plasminogen was iodinated according to the method of McFarlane.44 Iodinated proteins were separated from unincorporated 125I by gel filtration on a PD-10 column (Pharmacia) pre-equilibrated with HBS containing 0.5% human albumin. Following iodination, the ability of 125I-Lys-plasminogen to be activated by uPA was determined using the fluorogenic plasmin substrate as described below.Determination of membrane-bound plasmin activity Membrane-bound plasmin activity was quantitated as previously described.45 Cells were incubated in macrophage serum-free media (MSFM; Gibco) containing plasminogen for 1 hour at 4°C. Following incubation, media containing unbound plasmin(ogen) were removed and cells were washed (3 ×) with DPBS. MSFM containing the plasmin substrate D-Val-Leu-Lys-amino-4-methylcoumarin (Enzyme Systems Products, Dublin, CA) was added and allowed to incubate 2.5 hours. Cleavage of the plasmin substrate generated a fluorescent product, which was quantitated in a Fluoroscan microplate reader (American Diagnostica) and extrapolated to the fluorescence generated by 0 to 40 ng/mL plasmin prepared in MSFM.Preparation of [3H]-glucosamine-labeled extracellular matrices The preparation of [3H]-glucosamine-labeled extracellular matrices has been described previously.8 Human aortic smooth muscle cells (SMC) (Clonetics, San Diego, CA) were plated into 12-well plates (105/well) in SMC Growth Medium (Clonetics). On day 3, media were replaced with media containing 5 µCi/well [3H]-glucosamine (Dupont/New England Nuclear, Boston, MA). On day 6, the cell layer was removed by sequential exposure to 0.5% Triton X-100 in PBS (10 minutes) and 0.20 mM NH4OH in PBS (3 minutes). The remaining insoluble extracellular matrices were washed 3 times with sterile DPBS and stored at 4°C.Matrix invasion assay Tissue culture inserts (10 mm) with porous (8 µm pore size) polycarbonate membrane (Nunc, Naperville, IL) were coated with Matrigel (Collaborative Biomedical) as follows. Matrigel was thawed on ice and diluted to 0.5 mg/mL with cold sterile water. An aliquot (50 µL/insert) of diluted Matrigel was spread over the surface of the prechilled inserts and allowed to dry overnight. The next day matrices were rehydrated with 300 µL MSFM for 1 hour. THP-1 monocytes were harvested, washed 3 times in DPBS, and suspended in MSFM. Cells (4 × 104/insert) were added to the matrix-coated insert and the insert was placed in a 12-well plate containing MSFM. Cells that crossed the membrane into the lower well were counted by phase contrast microscopy.
Annexin II is expressed on the surface of RAW264.7 macrophages Surface expression of annexin II by RAW264.7 cells was determined by flow cytometry. Macrophages were incubated with monoclonal anti-annexin II IgG followed by FITC-conjugated secondary antibody. The binding of annexin II to cell membranes is calcium dependent.35,46 Therefore, as a control, surface annexin II expression was measured in the presence of the calcium chelator EGTA. As seen in Figure 1, the mean fluorescence intensity of cells treated with EGTA was 0.6. In the absence of the calcium chelator, mean fluorescence was increased 40-fold. The frequency distribution of annexin II-positive cells (ie, numbers of cells plotted as a function of fluorescence level) indicates that the majority of RAW264.7 macrophages express annexin II on their surface. To confirm that EGTA treatment removed annexin II from the cell surface, the eluate was concentrated and tested for the presence of annexin II by Western blot (Figure 1, insert). The EGTA eluate contained an approximate 36-kd protein, which was immunoreactive with monoclonal anti-annexin II IgG.
To quantitate the amount of annexin II on the cell surface, cells were
incubated (20 minutes) with 20 mM EGTA. The EGTA eluate was
concentrated by ultrafiltration and mixed with 6 × sodium dodecyl
sulfate (SDS) sample buffer without Annexin II comigrates with a protein that binds plasminogen in cell lysates and membranes derived from RAW264.7 macrophages To determine whether annexin II serves as plasminogen binding site on RAW264.7 macrophages, we performed ligand blots on total cell lysates and isolated membranes. Lys-plasminogen was used as a ligand in these studies. Lys-plasminogen, the plasmin-modified form of native Glu-plasminogen, is formed on the cell surface and is preferentially bound by cells.47,48 Cellular proteins to which plasminogen binds were identified using a monoclonal antiplasminogen IgG. Following incubation with Lys-plasminogen, several bands were observed in a blot of cell lysate and isolated membranes (+Lys-Plg, Figure 2). These bands were not visible in blots, which were not incubated with Lys-plasminogen (Ctrl, Figure 2). Notably, the relative abundance of plasminogen binding proteins in the membrane preparation was different compared to the cell lysate. As seen in Figure 2, immunoreactive annexin II in RAW264.7 lysate and membranes shares electrophoretic mobility with a plasminogen binding protein observed in the ligand blot. Therefore, annexin II could act as a plasminogen binding site on these cells.
Anti-annexin II IgG inhibits binding of 125I-Lys-plasminogen to RAW264.7 macrophages We next determined the ability of monoclonal anti-annexin II IgG to inhibit binding of 125I-Lys-plasminogen to RAW264.7 macrophages. The dose-dependent binding of 125I-Lys-plasminogen to RAW264.7 macrophages is shown in Figure 3. Binding of 125I-Lys-plasminogen to macrophages was inhibited more than 90% by the lysine analogue -ACA. Preincubation of cells with
anti-annexin II IgG (clone Z014; Zymed) inhibited their binding of
125I-Lys-plasminogen by 35%. Results of experiments in
which a greater concentration of anti-annexin II IgG (50 µg/mL) was
used to block binding of 125I-Lys-plasminogen to
macrophages demonstrated no further inhibition of binding (data not
shown). In contrast to anti-annexin II IgG, isotype-matched mouse IgG
(25 µg/mL) had no effect on 125I-Lys-plasminogen
binding.
Because inhibition of 125I-Lys-plasminogen binding to RAW264.7 cells required relatively high concentrations of anti-annexin II, we determined whether the presence of carboxyl terminal lysines in the anti-annexin II IgG might be responsible. First, we determined whether anti-annexin II IgG (clone Z014; Zymed) binds directly to immobilized plasminogen. For this purpose, plasminogen (1 µg) and annexin II (0.1 µg), as a positive control, were run in SDS polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions and transferred to PVDF membrane or blotted directly to nitrocellulose via a slot blot apparatus to rule out an effect of the denaturing conditions of SDS-PAGE. Following blocking, the membrane was incubated with anti-annexin II IgG (1 µg/mL) for 1 hour and probed with biotinylated rabbit antimouse IgG. As expected, anti-annexin II IgG bound strongly to annexin II, whereas we could not detect anti-annexin II IgG bound to the immobilized plasminogen (data not shown). Second, we determined whether the incubation of anti-annexin II IgG with Sepharose-conjugated carboxypeptidase B affected its ability to block plasmin binding to cells. The enzymatic activity of carboxypeptidase B following conjugation to CNBr-Sepharose 4B was 3.8 mU. Incubation of 0.2 nmoles (~25 µg) of anti-annexin II IgG with carboxypeptidase B-Sepharose (capable of cleaving ~4 nmol substrate/min) for 1 hour did not affect the ability of anti-annexin II to block plasmin binding (data not shown). Therefore, it is unlikely that anti-annexin II IgG inhibits plasminogen binding to cells due to the presence of carboxyl terminal lysine residues. EGTA-treated RAW264.7 macrophages bind less plasmin Annexin II binds to the surface of RAW264.7 macrophages in a calcium-dependent manner (Figure 1). Therefore, we incubated cells with EGTA to remove surface annexin II and then determined their ability to bind plasmin. The dose-dependent binding of plasmin to RAW264.7 macrophages is shown in Figure 4. As demonstrated for 125I-Lys-plasminogen, the binding of plasmin to macrophages was blocked more than 90% by-ACA. In the
absence of exogenous plasmin, control macrophages had little detectable
plasmin activity on their surfaces (< 1 ng plasmin/105
cells). Following incubation with exogenous plasmin, membrane-bound plasmin activity increased to 21 ng/105 cells. The binding
of plasmin to macrophages preincubated with EGTA was decreased by 30%.
Western blots verified that membranes isolated from EGTA-treated
macrophages contained markedly less annexin II. The
persistent presence of annexin II in these membrane preparations, in
contrast to the absence of surface expression following EGTA treatment
(Figure 1), may represent annexin II associated with the inner
membrane, microsomes, and the cytoskeleton. Importantly, the observed
decrease in plasmin bound to the surface of macrophages treated with
EGTA is consistent with the inhibition of
125I-Lys-plasminogen binding to cells preincubated with
anti-annexin II. These data, derived from 2 experimental approaches,
demonstrate that annexin II is a plasminogen binding site on
macrophages.
Anti-annexin II IgG inhibits activation of plasminogen by RAW264.7 macrophages The binding of plasminogen to monocyte/macrophages is required for its efficient activation by uPA bound to uPA-R.22,24,49 Therefore, we determined the effect of anti-annexin II IgG on plasminogen activation by uPA bound to uPA-R. RAW264.7 cells (105/well) were washed with DPBS (3 ×) and media replaced with MSFM. Cells were incubated 30 minutes at room temperature with 25 µg/mL monoclonal anti-annexin II IgG or mouse IgG prior to adding Lys-plasminogen (1 µg/mL) and the plasmin substrate. Cells were incubated at 37°C for 2.5 hours and the plasmin activity in their conditioned media determined. Media derived from cells incubated with plasminogen or plasminogen and normal mouse IgG contained 21.3 ± 1.3 and 19.3 ± 1.3 ng/mL plasmin (mean ± SE; n = 6), respectively. In contrast, media derived from cells incubated with anti-annexin II IgG and plasminogen contained 7.3 ± 0.8 ng/mL plasmin. The reduced ability of RAW264.7 cells to activate plasminogen in the presence of anti-annexin II IgG was not due to inhibition of uPA because the activation of plasminogen by fluid-phase uPA was unaffected by the same concentration of anti-annexin II IgG. These data indicate that membrane-bound annexin II supports both the binding of plasminogen and its subsequent activation by membrane-bound uPA.Anti-annexin II IgG inhibits plasmin binding to human monocyte-derived macrophages We next determined whether annexin II was a binding site for plasmin on the surface of human monocyte-derived macrophages. Blood monocytes were isolated and cultured in Teflon beakers for 7 days. Cells were aliquoted into 96-well plates and allowed to adhere overnight before the binding studies. In the absence of exogenous plasmin, monocyte-derived macrophages expressed trace amounts of membrane-bound plasmin (Figure 5). Following incubation with plasmin, membrane-bound plasmin activity was 13 ng/105 cells. Preincubation of cells with monoclonal anti-annexin II IgG (25 µg/mL) inhibited about 50% of the binding of plasmin to monocyte-derived macrophages. In other experiments, anti-annexin II IgG inhibited plasmin binding to human THP-1 monocytes by 25% (data not shown). Therefore, in a manner similar to murine RAW264.7 macrophages, cell surface annexin II serves as a binding site for plasmin(ogen) on human monocyte-derived macrophages and THP-1 monocytes.
Degradation of extracellular matrix by RAW264.7 macrophages is inhibited by anti-annexin II IgG Urokinase PA-dependent plasminogen activation plays an important role in tissue remodeling directly via plasmin cleavage of matrix components and indirectly via the activation of metalloproteinases.15,50 Because anti-annexin II IgG was demonstrated to partially inhibit the binding and activation of plasminogen by RAW264.7 macrophages, we determined whether antiannexin IgG would affect the degradation of insoluble [3H]-glucosamine-labeled SMC extracellular matrices. The extracellular matrix consists of 3 principal classes of molecules: proteoglycans, adhesive glycoproteins (ie, fibronectin and laminin), and fibrous proteins (ie, collagen and elastin). [3H]-glucosamine is useful as a tracer in these studies because it labels proteoglycans and adhesive glycoproteins, which are susceptible to plasmin cleavage directly.51,52 When matrices were incubated with media alone, small amounts of labeled matrix were solubilized (Figure 6). The addition of plasminogen to the media controls had no effect, demonstrating the absence of significant plasminogen activator activity in the matrices. When RAW264.7 cells were plated directly on the matrices, solubilization was also unchanged in the absence of plasminogen. However, in the presence of plasminogen, RAW264.7 solubilization of SMC matrices increased 6-fold over media controls. The addition of anti-annexin II IgG (25 µg/mL) inhibited matrix solubilization approximately 40%. Normal mouse IgG had no effect. These data indicate that binding of plasminogen to annexin II plays a significant role in macrophage-mediated matrix degradation.
Invasion of extracellular matrix by THP-1 monocytes is inhibited by anti-annexin II IgG In these studies, we determined the effect of anti-annexin II IgG on the ability of THP-1 monocytes to migrate through extracellular matrix in response to monocyte chemotactic protein-1 (MCP-1). Cell culture inserts containing THP-1 monocytes suspended on a porous (8 µm) polycarbonate membrane were placed into media alone or media containing MCP-1. Relatively few cells migrated into the lower well in the absence of MCP-1 (Figure 7). The addition of MCP-1 to the lower well resulted in the accumulation of large numbers of monocytes. The addition of MCP-1 to the cells in the upper well did not stimulate migration into the lower well (data not shown). THP-1 monocyte migration induced by MCP-1 was markedly diminished or completely blocked when the polycarbonate membranes were thinly coated with a basement membrane-derived extracellular matrix (Matrigel). However, when plasminogen was added to the cells in the upper well, the number of cells that migrated through the matrix-coated membrane increased 10-fold. If THP-1 monocytes were preincubated with monoclonal anti-annexin II IgG (clone Z014; Zymed), migration was inhibited 60%. Preincubation of cells with control antibodies had no effect. To rule out the possibility that anti-annexin II inhibits THP-1 monocyte migration independent of its effects on plasminogen binding, we determined whether the antibody affected the migration of THP-1 cells through uncoated inserts. Migration induced by MCP-1 was increased 20- ± 10-fold over controls. MCP-1-induced migration of THP-1 cells incubated with anti-annexin II was increased 32- ± 3-fold. Thus, preincubation of THP-1 cells with anti-annexin II did not inhibit MCP-1-induced migration through uncoated inserts. Because the binding of plasminogen to macrophages and its subsequent activation are inhibited by anti-annexin II IgG, we conclude that the migration of THP-1 monocytes through matrix is in part dependent on binding of plasminogen to annexin II.
Recent genetic evidence has demonstrated the importance of plasminogen activation in the ability of macrophages to migrate to sites of injury and inflammation, and their removal of necrotic debris and fibrin.11,13,15,53 Despite earlier observations that plasminogen binding to the cell surface imparts a kinetic advantage to activation22,24,27,49 and protection of generated plasmin from inhibition,22,47,49 the identities of cell surface binding sites for plasminogen have not been completely elucidated. In studies reported here, we demonstrate that annexin II, a plasminogen binding protein first described on endothelial cells,54 is present on the surface of RAW264.7 macrophages. The binding of annexin II to macrophages is calcium dependent as evidenced by the loss of surface expression following treatment with EGTA and the identification of annexin II in EGTA eluates. When cell membranes were examined for the presence of plasminogen binding proteins using a ligand blot procedure, plasminogen bound to a membrane protein that comigrates with purified annexin II. Moreover, preincubation of cells with anti-annexin II IgG partially inhibited 125I-Lys-plasminogen binding to RAW264.7 macrophages and the binding of plasmin to human monocyte-derived macrophages. Likewise, the removal of membrane-bound annexin II with EGTA inhibited plasmin binding to RAW264.7 macrophages. Based on these data, we conclude that annexin II is a plasminogen binding site on the surface of murine and human macrophages. Annexin II is a member of a highly conserved family of proteins, which exhibit calcium-dependent binding to membranes and cytoskeleton.35,55 It can exist in a monomeric form or in a heterotetrameric complex with p11, an S-100-like protein.46,56 Although annexin II is primarily an intracellular protein, surface expression has been reported for rat, murine, and human cells.30,36,37 Although the mechanism(s) by which cytoplasmic annexin II is secreted is not known, its binding to the cell surface requires a Ca++-dependent phospholipid binding site located in core repeat 2.57 Annexin II appears to be involved in the trafficking of membrane vesicles,58-60 and is a major protein component of murine J774 macrophage phagosomes61 and vesicles secreted by fibroblasts following cycles of stretch and relaxation.62 In addition, calcium-dependent secretion by permeabilized chromaffin cells is stimulated by annexin II.63 The RAW264.7 macrophages and human monocyte-derived macrophages express
large numbers (~106/cell) of binding sites for
plasminogen.45,64 The partial inhibition of
125I-Lys-plasminogen or plasmin binding to macrophages by
anti-annexin II IgG or treatment with EGTA is consistent with the
observation that macrophages express several membrane proteins that
serve as plasminogen binding sites. In this regard, The loss of cell viability is reported to increase cell surface plasminogen binding and activation by U937 monocytes.72 Using flow cytometric analysis, the binding of FITC-plasminogen to nonviable cells (5%-10% of the cell population) was 100-fold greater than viable cells. Moreover, the binding of FITC-plasminogen to viable cells was neither lysine dependent nor inhibited by excess plasminogen, whereas the binding of FITC-plasminogen to nonviable cells was lysine dependent and blocked by unlabeled plasminogen.72 Because the mechanism by which cytoplasmic annexin II is secreted is unknown, we determined whether surface expression was limited to apoptotic cells. When adherent cultures of RAW264.7 macrophages were examined for the presence of apoptotic cells via TUNEL staining and the binding of biotinylated annexin V, less than 1% of the macrophages were positive (data not shown). In contrast, when surface annexin II expression was assessed by flow cytometry (Figure 1), the majority of cells were positive ruling out the possibility that annexin II is expressed on the surface of apoptotic cells only. However, these data do not exclude the possibility that the release of intracellular annexin II from injured or dead cells contributes to surface expression by adjacent viable cells. The essential role of uPA activation of plasminogen in the migration of macrophages and clearance of necrotic debris has been clearly demonstrated using transgenic mice.11-15 However, the role of cell surface plasminogen binding sites in these processes has not been explored. In the present studies, the degradation of [3H]-glucosamine-labeled vascular SMC matrices by macrophages was increased 6-fold in the presence of exogenous plasminogen. Matrix degradation was inhibited about 40% when the binding of plasminogen to macrophages' annexin II was inhibited by antibody. These data likely underestimate the contribution of plasminogen binding to cell surface annexin II, because under the conditions of these experiments plasminogen activation occurs both on the cell surface and the fluid phase. In an assay that is likely to be more sensitive to the localization of plasminogen activation, chemotaxis of THP-1 monocytes through an extracellular matrix-coated membrane was inhibited about 60% by preincubation with anti-annexin II IgG. This is the first demonstration that inhibition of plasminogen binding to a specific protein on the macrophages' surface impairs matrix degradation and migration through the extracellular matrix. In conclusion, these studies identify surface annexin II as a plasminogen binding site on macrophages. Inhibition of plasminogen binding to annexin II impaired the ability of macrophages to activate plasminogen as well as invade and degrade extracellular matrix. These data indicate an important role for the surface expression of annexin II in the invasive and degradative phenotype of macrophages.
Submitted April 6, 2000; accepted October 4, 2000.
Supported by research grants R01-HL40819, R01-HL42493, and P01-HL46403 from the National Heart, Lung and Blood Institute, and grant P11482 from the Austrian Science Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Domenick J. Falcone, Department of Pathology, Rm A678, Cornell University Weill Medical College, 1300 York Ave, New York, NY 10021; e-mail: dfalcone{at}mail.med.cornell.edu.
1. Luscinskas FW, Gimbrone MA Jr. Endothelial-dependent mechanisms in chronic inflammatory leukocyte recruitment. Annu Rev Med. 1996;47:413-421[CrossRef][Medline] [Order article via Infotrieve].
2.
Etzioni A, Doerschuk CM, Harlan JM.
Of man and mouse: leukocyte and endothelial adhesion molecule deficiencies.
Blood.
1999;94:3281-3288
3.
Estreicher A, Muhllauser J, Carpentier JL, Orci L, Vassalli JD.
The receptor for urokinase type plasminogen activator polarizes expression of the protease to the leading edge of migrating monocytes and promotes degradation of enzyme inhibitor complexes.
J Cell Biol.
1990;111:783-792 4. Gyetko MR, Todd RF III, Wilkinson CC, Sitrin RG. The urokinase receptor is required for human monocyte chemotaxis in vitro. J Clin Invest. 1994;93:1380-1387. 5. Schafer BM, Maier K, Eickhoff U, Todd RF, Kramer MD. Plasminogen activation in healing human wounds. Am J Pathol. 1994;144:1269-1280[Abstract]. 6. Lassila R. Inflammation in atheroma: implications for plaque rupture and platelet-collagen interaction. Eur Heart J. 1993;14(suppl k):94-97.
7.
Werb Z, Bainton DF, Jones PA.
Degradation of connective tissue matrices by macrophages; III, morphological and biochemical studies on extracellar, pericellular, and intracellular events in matrix proteolysis by macrophages in culture.
J Exp Med.
1980;152:1537-1553 8. Falcone DJ, Ferenc MJ. Acetyl-LDL stimulates macrophage-dependent plasminogen activation and degradation of extracellular matrix. J Cell Physiol. 1988;135:387-396[CrossRef][Medline] [Order article via Infotrieve].
9.
Falcone DJ, McCaffrey TA, Haimovitz-Friedman A, Vergilio J-A, Nicholson AC.
Macrophage and foam cell release of matrix bound growth factors: role of plasminogen activation.
J Biol Chem.
1993;268:11951-11958
10.
Falcone DJ, McCaffrey TA, Haimovitz-Friedman A, Garcia M.
Transforming growth factor-
11.
Ploplis VA, French EL, Carmeliet P, Collen D, Plow EF.
Plasminogen deficiency differentially affects recruitment of inflammatory cell populations in mice.
Blood.
1998;91:2005-2009
12.
Kitching AR, Holdsworth SR, Ploplis VA, et al.
Plasminogen and plasminogen activators protect against renal injury in crescentic glomerulonephritis.
J Exp Med.
1997;185:963-968 13. Carmeliet P, Moons L, Ploplis V, Plow E, Collen D. Impaired arterial neointima formation in mice with disruption of the plasminogen gene. J Clin Invest. 1997;99:200-208[Medline] [Order article via Infotrieve].
14.
Carmeliet P, Moons L, Herbert JM, et al.
Urokinase but not tissue plasminogen activator mediates arterial neointima formation in mice.
Cir Res.
1997;81:829-839 15. Carmeliet P, Moons L, Lijnen HR, et al. Urokinase-generated plasmin activates matrix metalloproteinases during aneurysm formation. Nat Genet. 1997;17:439-444[CrossRef][Medline] [Order article via Infotrieve]. 16. Heymans S, Luttun A, Nuyens D, et al. Inhibition of plasminogen activators or matrix metalloproteinases prevents cardiac rupture but impairs therapeutic angiogenesis and causes cardiac failure. Nat Med. 1999;5:1135-1142[CrossRef][Medline] [Order article via Infotrieve]. 17. Saksela O, Hovi T, Vaheri AJ. Urokinase-type plasminogen activator and its inhibitor secreted by cultured human monocyte-macrophage. J Cell Physiol. 1985;122:125-132[CrossRef][Medline] [Order article via Infotrieve]. 18. Chapman HA, Stone OL. Characterization of a macrophage-derived plasminogen-activator inhibitor: similarities with placental urokinase inhibitor. Biochem J. 1985;230:109-116[Medline] [Order article via Infotrieve].
19.
Genton C, Kruithof EKO, Schleuning WD.
Phorbol ester induces the biosynthesis of glycosylated and nonglycosylated plasminogen activator inhibitor 2 in high excess over urokinase-type plasminogen activator in human U-937 lymphoma cells.
J Cell Biol.
1987;104:705-712 20. Hamilton JA, Whitty GA, Wojta J, Gallichio M, McGrath K, Ianches G. Regulation of plasminogen activator inhibitor-1 levels in human monocytes. Cell Immunol. 1993;152:7-17[CrossRef][Medline] [Order article via Infotrieve].
21.
Hamilton JA, Whitty GA, Stanton H, et al.
Macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor stimulate the synthesis of plasminogen-activator inhibitors by human monocytes.
Blood.
1993;82:3616-3621
22.
Plow EF, Freany DE, Plescia J, Miles LA.
The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type.
J Cell Biol.
1990;103:2411-2420 23. Kirchheimer JC, Remold HG. Endogenous receptor-bound urokinase mediates tissue invasion of human monocytes. J Immunol. 1989;143:2634-2639[Abstract].
24.
Ellis V, Behrendt N, Dano K.
Plasminogen activation by receptor-bound urokinase: a kinetic study with both cell-associated and isolated receptor.
J Biol Chem.
1991;266:12752-12758 25. Wu HL, Wu IS, Fang RY, et al. The binding of plasminogen fragments to cultured human umbilical vein endothelial cells. Biochem Biophys Res Commun. 1992;188:703-711[CrossRef][Medline] [Order article via Infotrieve]. 26. Burge SM, Marshall JM, Cederholm-Williams SA. Plasminogen binding sites in normal human skin. Br J Dermatol. 1992;126:35-41[CrossRef][Medline] [Order article via Infotrieve].
27.
Hajjar KA, Harpel PC, Jaffe EA, Nachman RL.
Binding of plasminogen to cultured human endothelial cells.
J Biol Chem.
1986;261:11656-11662 28. Dudani AK, Pluskota A, Ganz PR. Interaction of tissue plasminogen activator with a human endothelial cell 45-kilodalton plasminogen receptor. Biochem Cell Biol. 1994;72:126-131[Medline] [Order article via Infotrieve].
29.
Gonzalez-Gronow M, Gawdi G, Pizzo SV.
Characterizations of the plasminogen receptors of normal and rheumatoid arthritis human synovial fibroblasts.
J Biol Chem.
1994;269:4360-4366
30.
Hajjar KA, Jacovina AT, Chacko J.
An endothelial cell receptor for plasminogen/tissue activator, I: identity with annexin II.
J Biol Chem.
1994;269:21191-21197 31. Miles LA, Dahlberg CM, Plescia J, Felez J, Kato K, Plow EF. Role of cell-surface lysines in plasminogen binding to cells: identification of alpha-enolase as a candidate plasminogen receptor. Biochemistry. 1991;30:1682-1691[CrossRef][Medline] [Order article via Infotrieve]. 32. Redlitz A, Fowler BJ, Plow EF, Miles LA. The role of an enolase-related molecule in plasminogen binding to cells. Eur J Biochem. 1995;227:407-415[Medline] [Order article via Infotrieve]. 33. Bergman AC, Linder D, Sakaguchi K, et al. Increased expression of alpha-enolase in c-jun transformed rat fibroblasts without increased activation of plasminogen. FEBS Lett. 1997;417:17-20[CrossRef][Medline] [Order article via Infotrieve].
34.
Kassam G, Choi K-S, Ghuman J, et al.
The role of annexin II tetramer in the activation of plasminogen.
J Biol Chem.
1998;273:4790-4799
35.
Kaetzel MA, Dedman JR.
Annexins: novel Ca+2-dependent regulators of membrane function.
News in Physiol Sci.
1995;10:171-176 36. Yeatman TJ, Updyke TV, Kaetzel MA, Dedman JR, Nicolson GL. Expression of annexins on the surfaces of non-metastatic and metastatic human and rodent tumor cells. Clin Exp Metastasis. 1993;11:37-44[CrossRef][Medline] [Order article via Infotrieve].
37.
Chung CY, Erickson HP.
Cell surface annexin II is a high affinity receptor for the alternatively spliced segment of tenascin-C.
J Cell Biol.
1994;126:539-548 38. Lse TL, Lin YC, Mochitaka K, Grinnell F. Stress-relaxation of fibroblasts in collagen matrices triggers ectocytosis of plasma membrane vesicles containing actin, annexins II and VI, and beta 1 integrin receptors. J Cell Sci. 1998;105:167-177[Abstract]. 39. Raschke WC, Baird S, Ralph P, Nakoinz I. Functional macrophage cell lines transformed by Abelson leukemia virus. Cell. 1978;15:261-267[CrossRef][Medline] [Order article via Infotrieve]. 40. Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterization of a human acute leukemia cell line (THP-1). Int J Cancer. 1980;26:171-176[Medline] [Order article via Infotrieve]. 41. Tarsi-Tsuk D, Levy R. Stimulation of the respiratory burst in peripheral blood monocytes by lipoteichoic acid: the involvement of calcium ions and phospholipase A2. J Immunol. 1990;144:2665-2670[Abstract]. 42. Gown AM, Tsukada T, Ross R. Human atherosclerosis, II: immunocytochemical analysis of the cellular composition of human atherosclerotic lesions. Am J Pathol. 1986;125:191-207[Abstract].
43.
Pulford KA, Rigney EM, Micklem KJ, et al.
KP1: a new monoclonal antibody that detects a monocyte/macrophage associated antigen in routinely processed tissue sections.
J Clin Pathol.
1989;42:414-421 44. McFarlane AS. Efficient trace-labelling of proteins with iodine. Nature. 1958;182:53[Medline] [Order article via Infotrieve].
45.
Falcone DJ, Borth W, McCaffrey TA, Mathew J, McAdam K.
Regulation of macrophage receptor-bound plasmin by autoproteolysis.
J Biol Chem.
1994;269:32660-32666 46. Evans TC Jr, Nelsestuen GL. Biochemistry. 1994;33:13231-13238[CrossRef][Medline] [Order article via Infotrieve]. 47. Hajjar KA, Nachman RL. Endothelial cell mediated conversion of glu-plasminogen to lys-plasminogen: further evidence for assembly of the fibrinolytic system on the endothelial cell surface. J Clin Invest. 1988;82:1769-1778. 48. Silverstein RL, Friedlander RJ, Nicholas RL, Nachman RL. Binding of Lys-plasminogen to monocytes/macrophages. J Clin Invest. 1988;82:1948-1955.
49.
Kirchheimer JC, Remold HG.
Functional characteristics of receptor-bound urokinase on human monocytes: catalytic efficiency and susceptibility to inactivation by plasminogen activator inhibitors.
Blood.
1989;74:1396-1402 50. Mignatti P. Extracellular matrix remodeling by metalloproteinases and plasminogen activators. Kidney Int. 1995;47(suppl 49):S12-S14. 51. Richardson M, Hatton MW, Moore S. The plasma proteases, thrombin and plasmin, degrade the proteoglycan of rabbit aorta segments in vitro: an integrated ultrastructural and biochemical study. Clin Invest Med (Canada). 1988;11:139-150[Medline] [Order article via Infotrieve].
52.
Moser TL, Enghild JJ, Pizzo SV, Stack MS.
The extracellular matrix proteins laminin and fibronectin contain binding domains for human plasminogen and tissue plasminogen activator.
J Biol Chem.
1993;268:18917-18923 53. Romer J, Bugge TH, Pyke C, et al. Impaired wound healing in mice with a disrupted plasminogen gene. Nat Med. 1996;2:287-292[CrossRef][Medline] [Order article via Infotrieve]. 54. Hajjar KA, Jacovina AT, Chacko J. An endothelial cell receptor for plasminogen/tissue plasminogen activator, I: identity with annexin II. J Biol Chem. 1994;269:21191-21197.
55.
Lundgren CH, Hirofumi S, Sobel B, Fuji S.
Modulation of expression of monocyte/macrophage plasminogen activator activity and its implications for the attenuation of vasculopathy.
Circulation.
1994;90:1927-1934 56. Bianchi R, Garbuglia M, Verzini M, Giambanco I, Spreca A, Donato R. S-100 protein and annexin II2-p112 (calpactin I) act in concert to regulate the state of assembly of GFAP intermediate filaments in vitro. Biochem Biophys Res Commun. 1995;208:910-918[CrossRef][Medline] [Order article via Infotrieve].
57.
Hajjar KA, Guevara CA, Lev E, Dowling K, Chacko J.
Interaction of the fibrinolytic receptor, annexin II, with the endothelial cell surface
58.
Harder T, Gerke V.
The subcellular distribution of early endosomes is affected by the annexin II2p11(2) complex.
J Cell Biol.
1993;123:1119-1132
59.
Emans N, Gorvel JP, Walter C, et al.
Annexin II is a major component of fusogenic endosomal vesicles.
J Cell Biol.
1993;120:1357-1369 60. Sjolin C, Stendahl O, Dahlgren C. Calcium-induced translocation of annexins to subcellular organelles of human neutrophils. Biochem J. 1994;300:325-330.
61.
Desjardins M, Celis JE, Van Meer G, et al.
Molecular characterization of phagosomes.
J Biol Chem.
1994;269:32194-32200 62. Lee TL, Lin YC, Motchitaka K, Grinell F. Stress-relaxation of fibroblasts in collagen matrices triggers ectocytosis of plasma membrane vesicles containing actin, annexins II and VI and beta 1 integrin receptors. J Cell Sci. 1993;105:167-177. 63. Roth D, Morgan A, Burgoyne RD. Identification of a key domain in annexin and 14-3-3 proteins that stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells. FEBS Lett. 1993;320:207-210[CrossRef][Medline] [Order article via Infotrieve].
64.
Felez J, Miles LA, Plescia J, Plow EF.
Regulation of plasminogen receptor expression on human monocytes and monocytoid cell lines.
J Cell Biol.
1990;111:1673-1683 65. Redlitz A, Fowler BJ, Plow EF, Miles LA. The role of an enolase-related molecule in plasminogen binding to cells. Eur J Biochem. 1995;227:407-415. 66. Nakajima K, Nagata K, Hamanoue M, Takemoto N, Shimada A, Kohsaka S. Plasminogen-binding protein associated with the plasma membrane of cultured embryonic rat neocortical neurons. FEBS Lett. 1993;333:223-228[CrossRef][Medline] [Order article via Infotrieve]. 67. Nakajima K, Hamanoue M, Takemoto N, Hattori T, Kato K, Kohsaka S. Plasminogen binds specifically to alpha-enolase on rat neuronal plasma membrane. J Neurochem. 1994;63:2048-2057[Medline] [Order article via Infotrieve]. 68. Lopez-Alemany R, Correc P, Camoin C, Burtin P. Purification of the plasmin receptor from human carcinoma cells and comparison to alpha-enolase. Thromb Res. 1994;75:371-381[CrossRef][Medline] [Order article via Infotrieve].
69.
Kanalas JJ, Makker SP.
Analysis of a 45-kDa protein that binds to the Heymann nephritis autoantigen GP330.
J Biol Chem.
1993;268:8188-8192
70.
Hembrough TA, Li L, Gonias SL.
Cell-surface cytokeratin 8 is the major plasminogen receptor on breast cancer cells and is required for the acceleration of cell-associated plasminogen by tissue-type plasminogen activator.
J Biol Chem.
1996;271:25684-25691 71. Kralovich KR, Li L, Hembrough TA, Webb DJ, Karns LR, Gonias SL. Characterization of the binding sites for plasminogen and tissue-type plasminogen activator in cytokeratin 8 and cytokeratin 18. J Protein Chem. 1998;17:845-854[CrossRef][Medline] [Order article via Infotrieve]. 72. O'Mullane MJ, Baker MS. Loss of cell viability dramatically elevates cell surface plasminogen binding and activation. Exp Cell Res. 1998;242:153-164[CrossRef][Medline] [Order article via Infotrieve].
© 2001 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. Wygrecka, L. M. Marsh, R. E. Morty, I. Henneke, A. Guenther, J. Lohmeyer, P. Markart, and K. T. Preissner Enolase-1 promotes plasminogen-mediated recruitment of monocytes to the acutely inflamed lung Blood, May 28, 2009; 113(22): 5588 - 5598. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hastie, J. R. Masters, S. E. Moss, and S. Naaby-Hansen Interferon-{gamma} Reduces Cell Surface Expression of Annexin 2 and Suppresses the Invasive Capacity of Prostate Cancer Cells J. Biol. Chem., May 2, 2008; 283(18): 12595 - 12603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Das, T. Burke, and E. F. Plow Histone H2B as a functionally important plasminogen receptor on macrophages Blood, November 15, 2007; 110(10): 3763 - 3772. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. F. A. Swisher, U. Khatri, and G. M. Feldman Annexin A2 is a soluble mediator of macrophage activation J. Leukoc. Biol., November 1, 2007; 82(5): 1174 - 1184. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Du, H. Leung, K.M. F. Khan, C. G. Miller, K. Subbaramaiah, D. J. Falcone, and A. J. Dannenberg Tobacco Smoke Induces Urokinase-Type Plasminogen Activator and Cell Invasiveness: Evidence for an Epidermal Growth Factor Receptor Dependent Mechanism Cancer Res., September 15, 2007; 67(18): 8966 - 8972. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Zhang, Z.-H. Zhou, T. H. Bugge, and L. M. Wahl Urokinase-Type Plasminogen Activator Stimulation of Monocyte Matrix Metalloproteinase-1 Production Is Mediated by Plasmin-Dependent Signaling through Annexin A2 and Inhibited by Inactive Plasmin J. Immunol., September 1, 2007; 179(5): 3297 - 3304. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Jung, J. Wang, J. Song, Y. Shiozawa, J. Wang, A. Havens, Z. Wang, Y.-X. Sun, S. G. Emerson, P. H. Krebsbach, et al. Annexin II expressed by osteoblasts and endothelial cells regulates stem cell adhesion, homing, and engraftment following transplantation Blood, July 1, 2007; 110(1): 82 - 90. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Laumonnier, T. Syrovets, L. Burysek, and T. Simmet Identification of the annexin A2 heterotetramer as a receptor for the plasmin-induced signaling in human peripheral monocytes Blood, April 15, 2006; 107(8): 3342 - 3349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Gveric, B. M. Herrera, and M. L. Cuzner tPA Receptors and the Fibrinolytic Response in Multiple Sclerosis Lesions Am. J. Pathol., April 1, 2005; 166(4): 1143 - 1151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. B. Deora, G. Kreitzer, A. T. Jacovina, and K. A. Hajjar An Annexin 2 Phosphorylation Switch Mediates p11-dependent Translocation of Annexin 2 to the Cell Surface J. Biol. Chem., October 15, 2004; 279(42): 43411 - 43418. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V M Diaz, M Hurtado, T M Thomson, J Reventos, and R Paciucci Specific interaction of tissue-type plasminogen activator (t-PA) with annexin II on the membrane of pancreatic cancer cells activates plasminogen and promotes invasion in vitro Gut, July 1, 2004; 53(7): 993 - 1000. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. M. F. Khan, L. R. Howe, and D. J. Falcone Extracellular Matrix-induced Cyclooxygenase-2 Regulates Macrophage Proteinase Expression J. Biol. Chem., May 21, 2004; 279(21): 22039 - 22046. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Pluskota, D. A. Soloviev, K. Bdeir, D. B. Cines, and E. F. Plow Integrin {alpha}M{beta}2 Orchestrates and Accelerates Plasminogen Activation and Fibrinolysis by Neutrophils J. Biol. Chem., April 23, 2004; 279(17): 18063 - 18072. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Lei, G. Romeo, and A. Kazlauskas Heat Shock Protein 90{alpha}-Dependent Translocation of Annexin II to the Surface of Endothelial Cells Modulates Plasmin Activity in the Diabetic Rat Aorta Circ. Res., April 16, 2004; 94(7): 902 - 909. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zhang, D. K. Fogg, and D. M. Waisman RNA Interference-mediated Silencing of the S100A10 Gene Attenuates Plasmin Generation and Invasiveness of Colo 222 Colorectal Cancer Cells J. Biol. Chem., January 16, 2004; 279(3): 2053 - 2062. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Brownstein, A. B. Deora, A. T. Jacovina, R. Weintraub, M. Gertler, K. M. F. Khan, D. J. Falcone, and K. A. Hajjar Annexin II mediates plasminogen-dependent matrix invasion by human monocytes: enhanced expression by macrophages Blood, January 1, 2004; 103(1): 317 - 324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kirshner, D. Schumann, and J. E. Shively CEACAM1, a Cell-Cell Adhesion Molecule, Directly Associates with Annexin II in a Three-dimensional Model of Mammary Morphogenesis J. Biol. Chem., December 12, 2003; 278(50): 50338 - 50345. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Seth, M. A. Leo, P. H. McGuinness, C. S. Lieber, Y. Brennan, R. Williams, X. M. Wang, G. W. McCaughan, M. D. Gorrell, and P. S. Haber Gene Expression Profiling of Alcoholic Liver Disease in the Baboon (Papio hamadryas) and Human Liver Am. J. Pathol., December 1, 2003; 163(6): 2303 - 2317. [Abstract] [Full Text]
|
|
|
|
|
|
T. J. MacLeod, M. Kwon, N. R. Filipenko, and D. M. Waisman Phospholipid-associated Annexin A2-S100A10 Heterotetramer and Its Subunits: CHARACTERIZATION OF THE INTERACTION WITH TISSUE PLASMINOGEN ACTIVATOR, PLASMINOGEN, AND PLASMIN J. Biol. Chem., July 3, 2003; 278(28): 25577 - 25584. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2001 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
-enolase.
-mercaptoethanol.
essential role of endonexin repeat.
J Biol Chem.
1996;271:21652-21659