Regulation of Urokinase/Urokinase Receptor Interaction by
Heparin-like Glycosaminoglycans*
Marco
Pucci
,
Gabriella
Fibbi,
Lucia
Magnelli, and
Mario
Del
Rosso
From the Department of Experimental Pathology and Oncology of
Florence University, Viale G. B. Morgagni 50, 50134 Florence, Italy
Received for publication, July 7, 2000, and in revised form, November 17, 2000
 |
ABSTRACT |
We show here that the interaction between the
urokinase-type plasminogen activator and its receptor, which plays a
critical role in cell invasion, is regulated by heparan sulfate present on the cell surface and in the extracellular matrix. Heparan sulfate oligomers showing a composition close to the dimeric repeats of heparin
(glucosamine-NSO3(6-OSO3)-iduronic
acid(2-OSO3)) n = 5 and n > 5, where iduronic acid may alternate with glucuronic acid, exhibit
affinity for urokinase plasminogen activator and confer specificity on
urokinase/urokinase receptor interaction. Cell surface clearance of
heparan sulfate reduces the affinity of such interaction with a
parallel decrease of specific urokinase binding in the presence of an
unaltered expression of receptor. Transfection of human urokinase
plasminogen activator receptor in normal Chinese hamster ovary
fibroblasts and in Chinese hamster ovary cells defective for the
synthesis of sulfated glycosaminoglycans results in specific urokinase/receptor interaction only in nondefective cells. Heparan sulfate/urokinase and receptor/urokinase interactions exhibit similar
Kd values. We concluded that heparan sulfate functions as an adaptor molecule that confers specificity on
urokinase/receptor binding.
 |
INTRODUCTION |
The urokinase receptor
(u-PAR)1 binds urokinase-type
plasminogen activator (u-PA) by specific interactions with u-PAR domain 1 and vitronectin (VN) by interactions with a site within u-PAR domains
2 and 3, the affinity of these bindings is increased when u-PAR is
occupied by (pro)-u-PA (1). u-PAR domains 2 and 3 may also reversibly
bind an enlarging series of integrins (including
1,
2, and
3
integrins). The interaction of integrins with u-PAR modulates the
affinity of the integrin for its corresponding ligand. Some experiments
indicate that interaction with u-PAR reduces the affinity of the
associated integrin for certain ligands, whereas in other experimental
setups the affinity of the interaction between integrin and ligand
increases after binding of u-PAR to the integrin (2). Both interactions
of u-PAR-associated integrins with extracellular matrix (ECM) and the
activation of a cell surface-associated proteolytic cascade by
u-PAR-bound u-PA (1, 3) are required for invasion of cells within ECM
in processes such as cancer and angiogenesis (1, 4). Therefore, the
regulation of u-PAR expression and u-PAR affinity for u-PA on the cell
surface is critical for the regulation of cell invasion. Activation of
certain cellular oncogenes leads to a higher expression of some of the
components of the u-PA/u-PAR system (5). Many hormones, growth factors,
cytokines, and tumor promoters regulate the expression of the system at
the transcriptional level (6, 7). A transcriptional activation of the
u-PAR gene has been observed in invasive colon cancer cells (8), and a
post-transcriptional regulation of u-PAR mRNA by integrin
engagement was reported in leukocytes (9).
Few studies deal with the post-translational regulation of the
u-PA/u-PAR system. The internalization and rapid degradation of
u-PAR-bound u-PA after complex formation with plasminogen activator inhibitors (10) was the first described mechanism regulating u-PAR
exposure and u-PAR-bound u-PA. On the cell surface u-PAR is present in
two forms as follows: a three-domain form (domain 1 + 2 + 3), which exhibits u-PA-binding properties, and a two-domain
form (domain 2 + 3), lacking domain 1 and therefore u-PA binding
activity. The two-domain form is the product of u-PA or plasmin
cleavage of the three-domain form. Such cleavage may represent a
post-translational mechanism exploited by proteinases to control their
own activity on cell surface (11). Moreover, the level of glycosylation
modulates u-PAR affinity for its ligand (12). u-PA has a significant
affinity for heparin (HP)-like glycosaminoglycans (GAGs), namely
heparan-sulfate (HS) (13), which is one of the most ubiquitous
molecules present on cell surface, ECM and basement membrane (14) in
the form of proteoglycans (PG). u-PA does not contain the typical
HP-binding consensus sequence reported by Cardin and Weintraub (15),
although it seems to bind to HP through the kringle domain of its
amino-terminal fragment (ATF), where several basic amino acid residues
are found (16-19). There is also evidence that the u-PA B-chain and
the u-PA catalytic site participate in u-PA/HP interactions (19, 20).
Moreover, injection of HP in mice increases the fibrinolytic activity
in the plasma euglobulin fraction by an increase of u-PA protein levels, probably following u-PA elution from endothelial cells (21). A
recent paper by Brunner et al. (22) has reported that sulfated GAGs enhance tumor cell invasion in vitro by
stimulating plasminogen activation both in the fluid phase and on the
cell surface.
We report here that HS promotes the interaction of u-PA with u-PAR,
thereby providing an efficient post-translational mechanism by which
the pericellular environment may regulate u-PA activity on the cell
surface and the subsequent degradation of anatomical barriers, which is
required for cell invasion. We show that a form of HS, characterized by
a hexosamine and uronic acid composition very close to that of HP,
exhibits affinity for u-PA and that in the absence of HS a specific
u-PA/u-PAR interaction does not occur. Such activity is expressed by a
sequence of more than 5 repeats of uronic acid-glucosamine. These data
suggest that HS functions as an adaptor molecule allowing the
interaction between u-PA and u-PAR.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture, Cell Transfections with the Human u-PAR Construct,
and Incorporation of Radioactive Precursors--
Human skin
fibroblasts (HSF) from skin biopsies and Chinese hamster ovary
fibroblasts (CHO K1) were grown as monolayers at 37 °C, in
175-cm2 dishes in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 2 mM
L-glutamine, and penicillin/streptomycin. The pgsA 745 clone of CHO fibroblasts, which lacks the xylosyltransferase that
initiates GAGs synthesis, was kindly provided by Dr. J. D. Esko,
of the University of Alabama at Birmingham. Both parental CHO and the
pgsA clone were stably cotransfected with Okayama-Berg vector (23)
containing u-PAR cDNA (24) and pGDJSV2 plasmid, conferring neomycin
resistance (kindly provided by Dr. Y. Tsujimoto, University of Osaka,
Japan), using the calcium phosphate technique. Incorporation of
radioactive precursors was performed in low sulfate and low leucine
medium for 48 h with L-[3H]leucine (20 µCi/ml) and 35SO42
(50 µCi/ml) (both from Amersham Pharmacia Biotech).
Extraction and Purification of Sulfated PG--
After incubation
with radioactive precursors, the culture medium was decanted and the
cell monolayer washed 3 times with 20 ml of ice-cold phosphate-buffered
saline (PBS)/dish. Cells were extracted overnight at 4 °C with 4 M guanidinium chloride (GdmCl) and 1% Triton X-100,
containing proteinase inhibitors (10 mM
N-ethylmaleimide, 1 mM
diisopropylphosphofluoridate, 10 mM EDTA). The extract was centrifuged at 30,000 × g for 30 min, and the
supernatant was subjected to isopycnic density gradient centrifugation
in CsCl. The extract was adjusted to a density of 1.35 g/ml in 4 M GdmCl, 1% Triton X-100 by adding solid CsCl, Triton
X-100, and water (25). After centrifugation for 48 h at
100,000 × g in an International IEC/B-60
ultracentrifuge, fractions were collected from the bottom of the tube
(1-ml fractions) and assayed for radioactivity and density. PGs were
pooled from the high density fractions (fractions 1-6) (
= 1.45-1.3 g/ml), which showed high 35S and low
3H radioactivity (26). The pooled fractions were then
dialyzed against 4 M GdmCl and chromatographed on Sepharose
CL-2B in 4 M GdmCl. The dual-labeled material was pooled,
and fractions of 100 µl were precipitated with at least 4 volumes of
95% ethanol at 4 °C overnight and recovered by centrifugation at
4,000 × g for 60 min. The resulting pellets were
air-dried and then solubilized in 200 µl of the appropriate buffer
prior to digestion with chondroitinase ABC from Proteus
vulgaris or heparitinase from Flavobacterium heparinum
(Seikagaku Kogyo Co., Tokyo, Japan). Either the ethanol precipitates of
crude preparations or the digested samples were used in affinity
chromatography experiments.
Preparation of GAGs from Cell Cultures--
After incubation for
48 h with 50 µCi/ml
35SO42
, cell monolayers
were washed twice with 20 ml/dish of phosphate-buffered saline and
removed from the culture dish by trypsinization. The trypsinate and the
cells were incubated overnight at 37 °C with Pronase (Calbiochem, B
grade) and papain (Merck) at a final concentration of 1 mg/ml each.
After addition of trichloroacetic acid (5% final concentration), the
mixture was kept at 4 °C for 60 min and centrifuged 10 min at
15,000 × g. The sulfated GAG side chains of PGs were
precipitated from the supernatant fraction by addition of 2.5 volumes
of cold ethanol. The samples were maintained for 24 h at 4 °C,
centrifuged at high speed, and washed twice with ethanol, and the
pellet was dissolved in distilled water (27). Sulfate-labeled GAGs were subjected to affinity chromatography as such or after treatment with
chondroitinase ABC or heparitinase.
Treatment of Cells with Sodium Chlorate or GAG Lyases--
HSF
or CHO monolayers were incubated with 30 mM sodium chlorate
for 12 days to inhibit ATP sulfurylase and hence the production of
3'-phosphoadenosine 5'-phosphosulfate, the active sulfate donor for
sulfotransferases (28). To control the effectiveness of such a
treatment, parallel cell monolayers were incubated in the presence of
35SO42
(50 µCi/ml) added
on days 1 and 5 and then processed for GAGs extraction as described
above. Sulfate-labeled GAGs were then incubated with bacterial
heparitinase (2.5 units/ml, 12 h, 37 °C, pH 6.2).
Sulfate-labeled HS degradation products were analyzed by gel filtration
on Sepharose 6B. Chlorate-treated cell monolayers were used for binding
studies with labeled u-PA. For some experiments, HSF or CHO monolayers
were treated with heparitinase or chondroitinase ABC. Cell monolayers
were incubated with 2.5 units/ml F. heparinum heparitinase (Seikagaku Kogyo Co. Ltd., Japan) or 1 unit/ml of P. vulgaris chondroitinase ABC (EC 4.2.2.4) (Seikagaku Kogyo Co Ltd., Japan) in PBS containing 1 mg/ml bovine serum albumin (BSA)
for 2 h at room temperature. At the end of incubation cells were
washed twice with cold PBS and used for binding experiments.
Preparation of HS Subfamilies and HS Oligomers from HP
By-products--
HP by-products from beef lung (HS) were supplied by
Glaxo Operations Ltd. (Runcorn, UK) and were fractionated according to Rodén et al. (29) and Fransson et al. (30).
Briefly, crude material (1 g) was treated with alkaline copper sulfate
to remove dermatan sulfate. The soluble material was fractionated with
ethanol as the calcium salt to remove chondroitin sulfate. GAGs that
precipitated between 18 and 36% (v/v) ethanol were recovered and
fractionated as quaternary ammonium complexes with cetylpyridinium
chloride. The complexes were dissociated and dissolved by increasing
concentrations of NaCl. The following fractions were obtained: HS-1
(0.2-0.4 M NaCl), HS-2 (0.4-0.6 M), HS-3
(0.6-0.8 M), HS-4 (0.8-1.0 M), and HS-5
(1.0-1.2 M). The amino sugar and uronic acid composition of each fraction was reported (31). Oligosaccharides were prepared from
HS-4 by periodate oxidation/alkaline elimination (32, 33). Briefly,
HS-4 (50 mg) was solubilized in 25 ml of 0.05 formate buffer, pH 3.0, 0.02 M NaIO4 for 24 h at 4 °C in the
dark. Oxidation at pH 3.0 selectively destroys glucuronic acid (GlcUA)
in GlcNAc-containing block regions. Such oxyglycans were dialyzed and
cleaved by alkaline elimination (5 mg/ml solutions were adjusted to pH
12 with 2 M NaOH and kept at room temperature for 30 min at
20 °C). The solutions were adjusted to pH 5 with 2 M
acetic acid and subjected to gel chromatography on Sephadex G-50
superfine (size 20 × 2500 mm; eluent 0.2 M pyridine
acetate, pH 5.0; elution rate 30 ml/h). The structure of the fragments
obtained may be described by the following general formula: GlcN
(HexUA
GlcN)n
R (32, 33); where HexUA is
a periodate-resistant uronic acid (glucuronic or iduronic acid); GlcN
is glucosamine, and R is a C3 fragment of the structure
-O-C(CHO)=CH2. Each glucosamine may be substituted with -NSO3 (N-linked sulfate group), -NAc
(N-linked acetyl group), -OSO3
(O-linked sulfate group). Fragments with n = 1-4 and >5 were obtained, as evaluated by coelution with molecular
standards commercially available (Seikagaku Kogyo Co., Ltd., Japan). It is interesting that the reported analysis of HS4 degradation products (33) indicates that oligomers with n = 5 and
n >5 (which account for more than 50% of degradation
products) are compatible with the presence of alternating repeats
containing glucuronic acid (GlcUA) and iduronic acid (IdoUA). Such
oligomers are rich in heparin-like repeats, i.e.
GlcN2-NSO3(6-OSO3)-IdoUA(2-OSO3).
HS subfamilies and oligomers from HS-4 were used in ligand binding competition experiments on cell monolayers and in affinity chromatography.
Iodination of u-PA and Soluble u-PAR, u-PA Binding Studies, u-PAR
Assay, and Phospholipase C Treatment of Cell Monolayers--
Human
two-chain u-PA was provided by Lepetit Research Center, Varese, Italy,
courtesy of Dr. M. L. Nolli and was also purchased from Serono
(Rome, Italy). Human u-PA was purified by affinity chromatography on
Sepharose CH-4B (Amersham Pharmacia Biotech) substituted with
p-aminobenzamidine (34), as described previously (35). u-PA
was pure on polyacrylamide gel electrophoresis performed after the
purification step (not shown). Binding studies were performed as
described previously (36), using 125I-u-PA iodinated with
IODO-GEN (Pierce), with a specific activity of 25 µCi/µg. Briefly,
confluent monolayers of cells cultured in 24-well multiple plates
(Nunc-Intermed, Denmark), maintained in serum-free conditions for
24 h after reaching confluence, were washed 4 times with ice-cold
PBS. One ml of ice-cold PBS containing increasing amounts of
125I-u-PA was then added to each well. Each ligand
concentration was added in triplicate wells, and the plates were
incubated on ice for 60 min. After 4 washes of the unbound
radioactivity with ice-cold PBS, the monolayers were solubilized with
0.3 ml of NaOH 1.0 M and counted in a LKB-Minigamma
Counter. Specific binding was determined by measuring the difference of
cell-bound radioactivity in the presence and absence of 5 µg of
unlabeled u-PA. The results of binding experiments were analyzed by the
Scatchard plots to obtain both the receptor number and the
Kd values of receptor/ligand interaction. Before
binding, the cells were also subjected to acid wash to uncouple
endogenous u-PA from u-PAR (37). Low molecular weight (33,000 Da) u-PA (LMW u-PA), a kind gift of Dr. M. L. Nolli (Lepetit
Research Center, Varese, Italy) was also iodinated and used in affinity
chromatography experiments (specific activity, 23 µCi/µg). A
soluble recombinant form of human u-PAR (su-PAR), a gift
from Dr. U. H. Weidle (Roche Molecular Biochemicals), was
iodinated with the same method, for use in affinity binding experiments
(specific activity 28 µCi/µg). su-PAR, which lacks the
glycosylphosphatidylinositol (GPI) anchor linking u-PAR to the cell
surface, was released into the culture medium of a CHO cell line
transfected with the GPI-defective human u-PAR (namely
CHOdhfr
cell line), was purified on u-PA-substituted
affinity columns, and retained the property of binding human u-PA.
IMUBIND total u-PAR enzyme-linked immunosorbent assay kit (American
Diagnostica) and was also used to measure u-PAR number. In this case,
cell monolayers were lysed with lysis buffer, as suggested by the
manufacturer, and an aliquot of cell lysate was used for u-PAR
determination. In some experiments, we also measured the
ratio between externally and internally located u-PAR. Cell monolayers
were treated with GPI-specific phospholipase C (GPI-PLC) as described (36) to cleave the GPI anchor linking u-PAR to the cell surface. The
quantity of receptor was then determined on aliquots of the cell lysate
of GPI-PLC-treated cells (internal u-PAR) and of the GPI-PLC incubation
medium (external u-PAR).
Affinity Chromatography--
Various HS subfamilies were
prepared from beef lung HP by-products as described above (29, 30); HP
from beef intestinal mucosa was a kind gift from Laboratorio Derivati
Organici (Vercelli, Italy); DS was obtained as the copper salt from the
same procedure on the material of the same source (beef lung) and then
transformed into the sodium salt; HA was from human umbilical cord; C6S
was from shark cartilage, and C4S from bovine trachea was of commercial origin (Sigma). GAGs were immobilized on Sepharose 4B (Amersham Pharmacia Biotech) activated with CNBr and substituted with adipic acid
dihydrazide, as previously described (38); C4S, C6S, and HA were
partially periodate-oxidized for 5 min at 37 °C in 0.02 M NaIO4, 0.05 M sodium phosphate
buffer, pH 7.0; HP, various HSs, and DS were oxidized in 0.02 M NaIO4, 0.05 M formate buffer, pH 3.0, at 4 °C for 5 min. Both reactions were stopped by the addition of 10% mannitol. The partially periodate-oxidized chains were dialyzed
and then coupled to adipic acid-substituted agarose. The resulting
aldimines were stabilized by reduction with NaBH4. The
incubation mixture (5 mg of GAGs/ml agarose) was washed in distilled
water, and amino sugars were measured in an amino acid analyzer. The
binding efficiency was 90%. Affinity chromatographies were performed
at room temperature on columns (10 × 150 mm) containing 12 ml of
gel. The columns were equilibrated with 0.1 M Tris/HCl buffer, pH 7.9, containing 0.1% BSA. Either one of
125I-labeled u-PA or su-PAR (10 ng in 1 ml of the same
buffer) was applied and drained into the column. 5 ml (void volume) was
then eluted to distribute the ligand throughout the whole bed of the gel. 2 h later the column was washed with the same buffer at a rate of 40 ml·cm
2·h
1. 5 column volumes (50 ml) were eluted, until radioactivity decreased to
the background value. Elution was then performed with a linear gradient
of 0.0-1.5 M NaCl in 0.1 M Tris/HCl buffer, pH
7.9, containing 0.1% BSA, at a flow rate of 40 ml·cm
2·h
1. The
slope of the gradient was checked by conductivity measurements. Affinity chromatographies of labeled cell PGs and GAGs were performed on u-PA- or u-PAR-substituted CNBr-activated Sepharose 4B, derivatized with the ligands according to the manufacturer's instructions (Amersham Pharmacia Biotech). Gel loading and washing were as described
above, but the amount of gel was 6 ml (10 × 75 mm column), and
the flow rate 20 ml·cm
2·h
1. The
same flow rate was used in NaCl gradient elution, which was performed
with the same buffer described above.
 |
RESULTS |
Affinity Chromatography of u-PA and u-PAR on GAGs-derivatized
Sepharose--
Fig. 1a shows
the binding profile of 125I-u-PA on HP-substituted agarose.
No binding was observed to HA, C4S, C6S, whereas DS exhibited only a
weak binding (data not shown). HP-bound u-PA eluted between 1 and 1.2 M NaCl. Copolymeric oversulfated HS-5, whose composition is
very close to HP (molar ratio of uronic acid (carbazole) to
hexosamine above 1, like that of HP) and displays anti-coagulant
activity (31), was not used in the preparation of affinity gels. The
other HS subfamilies (fractions 1-4, where the same ratio was close to
1), devoid of anti-coagulant activity, showed increasing u-PA binding
along with increasing complexity of the GAG molecule. In fact, u-PA
eluted from HS-4 at 0.8-0.9 M NaCl, whereas low sulfated
HS-1/3 released u-PA at NaCl concentrations below 0.5 M
(Fig. 1b). Fig. 1c shows u-PA affinity
chromatography on HS-4-substituted agarose, where 125I-u-PA
binding was competed with HS-4-derived oligomers. The ligand (10 ng of
125I-u-PA) was preincubated with 100 µg of each HS-4
degradation product at 37 °C for 1 h before application to the
affinity gel and then loaded on the top of the column, as described
under "Experimental Procedures." Affinity binding patterns clearly
indicate that only oligomers with n >5 (namely oligomers
with more than five dimeric repeats of uronic acid and glucosamine)
were able to compete efficiently with u-PA/HS-4 binding (Fig.
1c). When LMW 125I-u-PA was used as ligand in
affinity chromatographies, no binding was measured (data not shown).
These results indicate that u-PA/GAGs interactions mainly occur through
a sequence present on the u-PA ATF, which is not present in LMW u-PA.
No binding was observed when 125I-su-PAR was used in
affinity chromatography experiments on GAG-substituted gels (data not
shown), indicating that the receptor does not exhibit any affinity for
HS and other GAGs.

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
Affinity chromatography of
125I-u-PA on Sepharose substituted with HP (Sepharose-HP)
(a) or with subfamilies of HS with increasing degrees
of sulfation (b): HS-1 (filled black
circles); HS-2 (filled black squares); HS-3
(empty triangles); HS-4 (empty
circles). The columns were equilibrated with 0.1 M Tris/HCl buffer, pH 7.9, containing 0.1% BSA. 1 ml of
125I-labeled ligand (1.5 ng/ml, corresponding to about
75 × 103 cpm) was applied and drained into the
column, and the affinity chromatography was performed as described
under "Experimental Procedures." The whole volume of each fraction
(1 ml) was counted. The recovery of radioactivity was 87% for
Sepharose-HP and ranged from 79 to 84% for various HS subfamilies;
NaCl gradient was evaluated by measuring conductivity of each fraction.
c shows u-PA elution from Sepharose-HS-4 in the presence of
100 µg each of HS-4-derived oligomers added with u-PA at the moment
of gel loading. Also in these experiments the recovery of the applied
radioactivity (75 × 103 cpm) ranged from 78 to 84%
for various oligomers.
|
|
Affinity Chromatography of Labeled Cellular PGs and GAGs on
u-PA-substituted Agarose--
Fig.
2a shows the isopycnic density
gradient centrifugation in CsCl, 4 M GdmCl, 1% Triton
X-100 of a crude dual-labeled PG preparation from HSF
monolayers. Highly sulfated fractions (
= 1.45-1.3 g/ml) were
pooled, dialyzed against 4 M GdmCl, and chromatographed on
Sepharose CL-2B (Fig. 2b). Fractions containing highly
sulfated material were pooled as indicated in Fig. 2b and ethanol-precipitated, to be used in affinity chromatography
experiments. The crude dual-labeled PG preparation was affinity
chromatographed on u-PA-substituted agarose (Fig.
3a). The elution pattern
showed that highly sulfated material elutes at 1.0 M NaCl.
Fig. 3b shows that chondroitinase ABC treatment, able to
cleave C4S, C6S, DS, and HA, leaves unaltered the PG/u-PA affinity,
which is completely abolished by heparitinase treatment, that
specifically degrades HSs. These results indicate that neither the
protein nor the chondroitin-sulfate moiety affect PG/u-PA affinity,
which relies solely on u-PA-HS side chain interactions. To confirm
these observations, purified 35S-labeled GAGs extracted
from cultured HSF were chromatographed on the same gels. The
undigested GAGs mixture eluted at 1.0 M NaCl (Fig.
3c), whereas the treatment with heparitinase abolished the
binding, which was unaffected by chondroitinase ABC treatment (Fig.
3d), thus giving further support to the data indicating HS
as the only u-PA-binding GAG. Affinity chromatography of PGs and GAGs
on u-PAR-substituted agarose gave negative results (not shown), thus
confirming data obtained with 125I-u-PAR on
GAGs-substituted columns.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 2.
PG preparation from monolayer culture of
HSF. a, isopycnic density gradient centrifugation in
CsCl, 4 M GdmCl, 1% Triton X-100 of detergent extract of
HSF monolayers and collection of double-labeled molecules from cell
lysates of HSF (black column, 35S radioactivity;
hatched columns, 3H radioactivity). 1-ml
fractions were collected, and 20-µl aliquots of each fraction were
counted in a liquid scintillation counter. Total 35S
radioactivity of fractions 1-6 was 3.73 × 106 cpm.
These highly sulfated fractions (corresponding to densities of
1.45-1.30 g/ml) were pooled and dialyzed against 4.0 M
GdmCl. About 1.0 × 106 cpm (35S) were
chromatographed in 4.0 M GdmCl on Sepharose Cl-2B column
(b) and fractions of 1 ml each were collected. 50-µl
aliquots were counted, with a total 35S recovery of about
0.5 × 106 cpm. Filled squares indicate
35S radioactivity, and open circles indicate
3H radioactivity. Highly sulfated fractions were pooled, as
shown in b (fractions 39-51). Aliquots of 100 µl, containing about 50 × 103 35S cpm,
were precipitated in ethanol and stored for use in affinity
chromatography experiments.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 3.
Affinity chromatography of PGs and GAGs from
HSF monolayer cultures, on Sepharose u-PA. Before application to
affinity columns, aliquots of double-labeled ethanol-precipitated PGs,
pooled from Sepharose Cl 2B as described in Fig. 2, were solubilized in
distilled water (40 × 103 35S cpm were
applied). a, chromatography of PGs (only 35S
labeling is shown) on Sepharose-u-PA (filled squares). Each
1-ml fraction was counted, and 84% of total applied 35S
radioactivity was recovered. A Sepharose-albumin gel was used as
negative control (open circles). b, affinity
pattern of PGs to u-PA-substituted gels following chondroitinase ABC
treatment (filled squares, 53% recovery of total applied
35S radioactivity) and affinity after heparitinase
treatment (open squares, 9.25% recovery of total applied
35S radioactivity). c, affinity chromatography
of purified GAGs (35S-labeled) on Sepharose u-PA (15.0 × 103 35S cpm applied, 79.3% recovered).
d, affinity chromatography of purified labeled GAGs on
Sepharose-u-PA, following chondroitinase ABC treatment (filled
squares, recovery 54.2%) and heparitinase treatment (open
squares, recovery 14.2%).
|
|
Binding of u-PA on Cells Treated with Sodium Chlorate and
Heparitinase--
In 12-day cultures in the presence of 30 mM sodium chlorate, HSF showed a decrease of sulfated GAGs,
as shown in Fig. 4a. We were
unable to obtain the complete inhibition of sulfated GAGs expression
either by increasing sodium chlorate concentration to 60 mM
or by extending the treatment. Similar results were obtained with
heparitinase treatment. The amount of u-PAR, determined by enzyme-linked immunosorbent assay in control, untreated cells and
following each treatment, did not show appreciable differences: 4.0 ± 0.5 ng of u-PAR/106 cells under control
conditions (n = 6); 4.1 ± 0.7 and 4.15 ± 0.7 ng/106 cells after treatment with heparitinase
(n = 8) or sodium chlorate (n = 8),
respectively. Since the chemical change produced in membrane PGs may
have caused a change in the traffic of u-PAR between the inside and
outside of the cell, we also measured the selective release of membrane
u-PAR by phospholipase C cleavage (36) to exclude that chlorate caused
a change in the external display of functional u-PAR on the cell
surface. In untreated control cells, about 60% u-PAR (2.5 ± 0.4 ng/106 cells) was released from the cell membrane. Similar
values were obtained in heparitinase-treated HSF (2.6 ± 0.4 ng/106 cells) and in chlorate-treated HSF (2.5 ± 0.5 ng/106 cells), indicating that the treatments did not
produce a redistribution of u-PAR between the inside and the outside of
the cell. On the contrary, the Scatchard elaboration of ligand binding
studies on sodium chlorate and heparitinase-treated cells (Fig.
4b) indicated a 30% reduction of bound u-PA and a parallel
decrease of u-PA/u-PAR affinity. The values of the slope of the
regression lines were
4.0 × 10
7 (with
a confidence interval (CI)
5.9 × 10
7/
1.2 × 10
7, and a Kd of 1.0 × 10
9 M) in control untreated
HSF and
1.5 × 10
7 (CI,
2.5 × 10
7/
5.2 × 10
8, and a Kd of 2.7 × 10
9 M) in chlorate-treated
cells. The slopes were compared by the Student's t test for
parallelism, which gave a p = 0.05, indicating that the
two lines diverged significantly. The number of bound u-PA
molecules/cell varied from 61.4 ± 9 × 103 in
control HSF to 41.0 ± 7 × 103 in
chlorate-treated HSF. Following heparitinase treatment, the slope of
the regression line was
1.9 × 10
7,
and p = 0.08, indicating that the lines of the
Scatchard plot of control and heparitinase-treated HSF did not diverge
significantly (not shown in the figure). Nevertheless, the number of
bound u-PA molecules/cell was 44.6 ± 11 × 103.
Binding experiments were performed on cell monolayers pre-washed with
acidic buffer, to release u-PAR-bound u-PA, as described under
"Experimental Procedures" (37). The decrease of u-PA binding and
the decrease of its affinity for u-PAR may be likely related to the
presence of small amounts of HS on the cell membrane or in the ECM,
still able to favor a specific u-PA/u-PAR interaction. No variation of
u-PA binding was observed after treatment of cell monolayers with
chondroitinase ABC from P. vulgaris. In the case of
heparitinase treatment the decrease of binding could depend on the
release of ECM-associated FGF-2, which is known to induce fibroblasts
to secrete u-PA (39) that, in turn, could saturate free u-PAR on the
cell surface. Thus, some experiments with heparitinase were performed
in the presence of anti-FGF-2 antibodies to prevent interaction of
FGF-2 with its receptor. The results of u-PA binding were similar to
those observed in the absence of antibodies (not shown).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of chlorate on sulfate incorporation
and u-PA binding. a, HSF were cultured in the absence
(filled circles) or in the presence (empty
circles) of 30 mM chlorate and incubated with
35SO4, as described under "Experimental
Procedures." After 12 days HSF were processed for GAGs extraction.
Sulfate-labeled ethanol-precipitable material was digested with
heparitinase for 12 h at 37 °C, pH 6.2, and analyzed by gel
filtration on Sepharose 6B to check low molecular weight HS degradation
products released upon heparitinase treatment. The gel was loaded with
12.0 × 103 35S cpm, and about 89.5% cpm
were recovered, in 0.9-ml fractions. b, typical Scatchard
plots of 125I-u-PA ligand binding assay on control HSF
(filled circles) and chlorate-treated HSF (open
circles). The experiments shown in this figure were performed with
a 125I-u-PA preparation with a specific activity of 20 µCi/µg. 10 data points for each binding curve were obtained
(n = 10), where each point indicates the difference
between total and nonspecific radioactivity bound to cell monolayers.
Each point is the result of the difference between the mean of 3 different determinations in 3 different wells for each condition (total
cell-bound radioactivity and nonspecific radioactivity). Each plot also
shows the 95% CI, the confidence interval
|
|
u-PAR Assay and u-PA Binding in CHO Cells Transfected with
u-PAR--
Wild type CHO fibroblasts do not express human u-PAR (Table
I). Both parental CHO cell clones (GAGs
expressing CHO K1 and mutant CHO pgsA 745 which do not express any GAG)
were stably transfected with human u-PAR cDNA by the method of
calcium phosphate. Many clones were obtained, expressing human u-PAR at
various extents, as reported in Table I. To check whether u-PAR was
expressed on the cell surface or within the transfected cells, CHO
monolayers were treated with GPI-PLC, as described (36), to cleave the GPI anchor linking u-PAR to the cell surface. u-PAR determination was
then performed in aliquots of the GPI-PLC incubation medium (external
u-PAR). Internal u-PAR was calculated as the difference between total
u-PAR and the GPI-PLC-removable fraction. Reproducible results were
obtained, indicating that u-PAR was mainly exposed on the external cell
surface of transfected cells. Table I also shows u-PAR distribution
measured in all the transfected clones of CHO K1 and CHO pgsA. Typical
125I-u-PA binding assays were performed on both parental
clones and on all the clones obtained by u-PAR transfections. The
Scatchard analysis of binding data (shown in Fig.
5 for 5 parental and 5 GAGs-defective
clones of human u-PAR-transfected CHO fibroblasts) indicated that only
transfected wild type CHO K1 cells were able to bind specifically and
saturably u-PA (u-PAR number and Kd value of
u-PA/u-PAR interaction are reported in the figure legends, together
with the statistical analysis of data), whereas mutant transfected CHO
pgsA could bind u-PA only unspecifically (Fig. 5, a and
b). No specific u-PA binding was obtained in mutant CHO pgsA
when binding assays were performed in the presence of externally added
beef lung HS-4 (0.1-10 mg/ml) (data not shown). This observation may
indicate that only solid phase-associated HS (cell surface-associated or ECM-entrapped) can confer specificity on u-PA/u-PAR interaction, as
further addressed under the "Discussion."

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
Scatchard plots of binding data in parental
and GAGs-defective CHO fibroblasts. All the transfected CHO clones
were subjected to ligand binding experiments. 10 data points for each
binding curve were obtained (n = 10), where each point
indicates the difference between total and nonspecific radioactivity
bound to cell monolayers. As reported in the legend to Fig. 4, each
point is the result of the difference between the mean of 3 different determinations in 3 different wells for
each condition (total cell-bound radioactivity and nonspecific
radioactivity). Each plot also reports b, the slope of the
regression line, and 95% CI, its confidence interval. The following
values of p (which indicates the probability to obtain the
measured value of b, given a theoretical slope = 0)
were obtained. For parental CHO K1: clone 5, p < 0.0005 (Kd = 1.5 × 10 9 M; 17.8 × 103 molecules of bound u-PA/cell); clone 6, p = 0.004 (Kd = 2.4 × 10 9 M; 39.6 × 103 molecules of bound u-PA/cell); clone 9, p = 0.071 (Kd = 2.0 × 10 9 M; 15.3 × 103 molecules of bound u-PA/cell); clone 11, p = 0.083 (Kd = 4.0 × 10 9 M; 53.4 × 103 molecules of bound u-PA/cell); clone 12, p = 0.010 (Kd = 1.8 × 10 9 M; 33.5 × 103 molecules of bound u-PA/cell). For GAGs-defective CHO
pgsA: clone 1, p = 0.485; clone 3, p = 0.048; clone 4, p = 0.005; clone 5, p < 0.0005; clone 6, p = 0.005. To compare b
values of CHO K1 and of CHO pgsA, the two-sample Wilcoxon rank-sum test
for unpaired samples has been used. The results indicated a significant
difference between the medians of the slopes of the two groups:
p = 0.0062.
|
|
Contribution of Surface HS to u-PA-specific Binding in CHO
Cells--
Since HS shows u-PA affinity, we planned experiments to
evaluate the contribution of surface HS to the total specific binding. We therefore performed 125I-u-PA ligand binding on parental
CHO that express HS, but not receptors for human u-PA, and on parental
CHO transfected with human u-PAR (CHO K1 clone 11), extensively treated
with GPI-PLC to clear u-PAR from the cell membrane. In binding
experiments only specific binding was evaluated, namely the difference
between total bound 125I-u-PA and nonspecific
125I-u-PA (that is the binding that occurs in the presence
of an excess of unlabeled u-PA, added to the binding solution
containing increasing amounts of labeled ligand), as previously
described (37). In both cases the Scatchard analysis of binding data
revealed the presence of specific u-PA-binding sites different from
u-PAR. The Kd of the interaction was ~1.9
nM, the same order of magnitude of u-PA/u-PAR affinity
observed in these cells and reported in the literature (7). The number
of specific cell-associated u-PA molecules/cell was 9.4 ± 2.3 × 103. Parallel parental CHO K1 monolayers were
also treated with chondroitinase ABC or with heparitinase and then
subjected to u-PA binding. The treatment with chondroitinase ABC did
not show appreciable variations of u-PA binding (8.7 ± 3.0 u-PA
molecules/cell; n = 4). Following heparitinase
treatment, the Scatchard plots of binding data did not allow us to
calculate specific binding (not shown). The same experiments, performed
with parental GAGs-defective mutants CHO pgsA 745 or the same cells
transfected with human u-PAR extensively treated with GPI-PLC, did not
reveal the presence of any specific binding.
Activity of HS-4, HS-4 Oligomers on u-PA Binding to
u-PAR-transfected CHO Cells--
To compete for specific u-PA binding
to cell surface HS, binding assays were performed by addition to cell
monolayers of fixed amounts of 125I-u-PA in the presence of
increasing concentrations of either one of the following compounds:
HS-4 or HS-4 fragments corresponding to oligomers with the general
formula GlcN
(HexUA
GlcN)n
R, as described
under "Experimental Procedures." Fragments with n = 1-4 and >5 were used. Experiments were performed on HSF and on CHO K1
clone 11 that express sulfated GAGs and human u-PAR. Fig.
6a shows that in CHO cells the
addition of exogenous HS-4 decreased u-PA binding in a
dose-dependent fashion. Only HS-4-derived oligomers with
n > 5 were able to displace efficiently u-PA in binding competition experiments in the same cell line (Fig.
6b). The same experiments, performed in HSF monolayers, gave
similar results (not shown).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
u-PA binding in normal CHO cells transfected
with human u-PAR with HS-4 (a) and with HS-4 oligomers
(b). 125I-u-PA (100 ng/ml) was incubated for 60 min with confluent CHO monolayers in the absence and in the presence of
cold u-PA to obtain the specific binding, as described in the text
(control conditions). Parallel triplicate wells were incubated in the
same conditions in the presence of increasing amounts of HS-4
(a) or HS-4 oligomers (b), and the specific
binding was calculated as usual. For HS-4 oligomers the symbols are as
follows: up triangle, n = 1; down
triangle, n = 2; diamond,
n = 3; square, n = 4;
circle, n = 5 and n >5. Each
value represents the mean ± S.D. of 6 determinations.
|
|
 |
DISCUSSION |
In this study, we demonstrated that fibroblast HS, the most
ubiquitous HP-like GAG, strongly affects the interaction of u-PA with
its cellular receptor u-PAR. Such an activity is mediated by affinity
interactions of HS with u-PA, which occurs through binding of u-PA to a
sequence of more than 5 repeating dimeric units of a highly sulfated
copolymeric HS, rich in HP-like repeats with the following general
formula: GlcN2
NSO3(6-OSO3)
IdoUA(2-OSO3), where IdoUA may alternate with GlcUA.
The binding that resulted between u-PA and u-PAR was reinforced
with regard to both the affinity and the number of interacting
molecules. In either the presence (treatment of cells with sodium
chlorate or with heparitinase) or the absence (CHO cells deficient for
the synthesis of sulfated GAGs) of low amounts of HS in the
extracellular environment, specific u-PAR saturation by exogenously
added u-PA was reduced or completely absent. On this basis, we suggest
that extracellular HS (cell-associated or ECM-associated) could serve
at least two functions in the regulation of u-PA/u-PAR interaction as
follows: (a) as an adaptor molecule, which promotes the
interaction of u-PA with its receptor; (b) as a source of a
ready-to-use u-PA, an optimal site to endow rapidly u-PAR with its
specific ligand. In our experience, and on the basis of all the data
reported in the literature on a large variety of cell lines, the
Scatchard analysis of u-PA/u-PAR binding data is in agreement with a
model of a single class of receptors. By taking into consideration the
close similarity between the Kd values of u-PA/u-PAR
and of the u-PA/HS interaction (both in the range of 1.5-2.0
nM), the impossibility of identifying two different binding
sites makes sense. Moreover, the capacities of u-PA/uPAR and of u-PA/HS
compartments are also similar.
The structural background of the HS-binding capacity of u-PA, which
does not contain the typical HP-binding motif
(XBBXBX and/or
XBBBXXBX, where B is the probability
of a basic residue and X is a hydropathic residue) (15), is
not well known. Stephens et al. (18) have shown that a
synthetic decapeptide Arg52-Trp62 from the
kringle sequence of the u-PA ATF, rich in basic residues, is effective
in competitive assays of u-PA binding to HP and dextran sulfate.
Therefore, the u-PA kringle is endowed with affinity for polyanion
binding. Highly sulfated copolymeric HS-4, rich in HP-like repeats, may
provide an optimal substrate for such interactions.
u-PA binding to the wild type CHO cells transfected with human
u-PAR was strongly inhibited by exogenously added HS-4 or
decasaccharides and larger HS-4 saccharides. However, no specific
binding of u-PA was observed in mutant CHO pgsA cells transfected with
human u-PAR when binding was performed in the presence of exogenously
added HS-4. Therefore, we suggest that only solid phase-associated HS can confer specificity to the u-PA/u-PAR interaction. We think that
this apparent discrepancy may be explained on the basis of the
privileged location of membrane- or ECM-associated HS. Indeed, on the
cell surface and within the ECM, HP-like repeats of HS have the chance
to interact not only with u-PA, but also with
1
integrins (40-44), leukocyte integrin Mac-1 (45), fibronectin and
laminin (46), collagens (47), and VN (48). Many of these proteins are
essential components of the focal adhesions (49), where also u-PAR has
been shown to be preferentially located (50). The resulting
multimolecular complex may possibly cooperate to increase the affinity
of solid phase-associated HS for u-PA. Such cooperations are likely to
be unfavored for liquid phase HS and HS-u-PA complexes, as also
suggested by the high concentrations of HS-4 or its decasaccharides and
larger saccharides required for the displacement of about 50% u-PA
specifically bound to CHO cells. There is the additional possibility
that soluble HS-u-PA complexes are unable to bind the receptor, as
described for soluble GAGs-chemokines complexes (51).
Interest in HS PGs stems from the increasing evidence of the functional
implications of their interactions with growth factors and ECM
molecules (52). Of particular importance is their role in the retention
of growth factors in the ECM (53) and their participation as
coreceptors at the cell surface together with tyrosine kinase FGF
receptors (54-56). A possible explanation for the failure of FGF to
signal in the absence of HS is that the receptor binding cannot occur.
The sulfate residues, which may be present in four different positions
of the GAG backbone, have been shown to determine the specificity in
HS/proteins interaction (57). All the members of the FGF family, which
consists of nine structurally related polypeptides, bind HP and HS with
relatively high affinity and are thus referred to as "heparin-binding
growth factors." Since also u-PA is endowed with an intrinsic growth factor activity in many cell lines (7), interacts with HS, and requires
HS to bind u-PAR, we propose to enclose it among "heparin-binding
growth factors."
The importance of HS/u-PA interaction is likely to be many fold as
follows: 1) regulation of functional interactions of u-PA/u-PAR with
ECM. At the level of focal contacts of cultured cells, u-PAR and u-PA
colocalize with vinculin at the intracellular side and with VN and
other ECM components at the extracellular side, thereby connecting ECM
with the cytoskeleton (for a review see Ref. 58). Interaction of u-PAR
domains 2 and 3 with VN is 10-fold enhanced upon u-PAR interaction with
native u-PA or u-PA derivatives devoid of catalytic activity (59). VN,
in turn, binds to integrin receptors
v
3
or
v
5, and the simultaneous interactions
between u-PAR/u-PA, VN, and integrins are required for cell spreading
and migration, which also requires cell detachment mediated by u-PA
bound to u-PAR domain 1. A particular form of HS PG (syndecan 4) is
selectively localized at focal contacts in several cell lines,
including fibroblasts (for a review see Ref. 52). Since HS is
indispensable for the specific binding between u-PAR and u-PA, one may
likely infer that HS is essential for u-PAR-dependent cell
spreading and migration. It is possible that the reported stimulation
of tumor cell invasion into fibrin gels by HS and HP (22) relies on
HP-like GAGs-dependent promotion of u-PA/u-PAR interaction.
It is noteworthy that in melanomas a predominance of HS PG at the cell
surface is a marker of a more aggressive phenotype (60). 2) Coreceptors
of u-PA at the cell surface are therefore able to promote transduction of u-PA/u-PAR interaction. Some evidence indicates that u-PAR is
structurally and functionally coupled with a variety of integrins. Such
coupling inhibits the adhesive properties of integrins and stimulates
u-PAR-dependent adhesion to VN (61). There are indications that the u-PA/u-PAR/PAI-1 system modulates the affinity of integrins for their ECM ligands also by generating intracellular signals (62).
Since u-PAR is a GPI-anchored protein, the only possible mechanism of
transducing u-PA/u-PAR interaction relies on the presence of an
"adaptor" protein able to sense u-PA/u-PAR binding and to transfer
the signal to other membrane transduction effectors. In some cell lines
the multimolecular cell surface u-PAR transducing apparatus seems to be
localized at the level of specific sites of cell membrane, referred to
as caveolae, microinvaginations of the plasma membrane whose role is
unknown (63). The most specific protein of these specialized areas of
membrane is caveolin, which is in close contact with u-PAR (64), so
that it can be coprecipitated with anti-u-PAR antibodies. This suggests
that caveolae promote plasmin generation by recruitment of u-PA and u-PAR within a very restricted area of the cell membrane (65). Within
caveolae, caveolin oligomerizes with integrins and co-clusters with
GPI-anchored proteins (66). Interestingly, such GPI-anchored proteins
include u-PAR and a particular HS PG (67), whose structural characteristics suggest association with caveolar-signaling components, as extensively discussed by Mertens et al. (68). Caveolin is phosphorylated on tyrosine residues and physically interacts with a
number of signal transduction effectors, including G proteins, Ras, and
nonreceptor tyrosine kinases (7). By regulating u-PA/u-PAR interaction,
HS may also promote caveolin-generated signaling. Taken together, our
results and other observations suggest that the GAG moiety of HS PG, by
regulating the affinity of u-PA for its cellular receptor, has the
possibility to post-translationally regulate cell surface plasmin
generation-dependent events, such as
u-PA-dependent invasion and plasmin generation-independent ones (recently reviewed in Ref. 7), involving signal transduction upon
u-PA/u-PAR interaction. In this respect, the activity of HS in the u-PA
system does not substantially differ from the activity of HP-like
molecules in the regulation of the activity of FGF-2. Indeed, the GAG
moiety of HS PG is necessary for binding of FGF-2 to its high affinity
receptor by inducing a conformational change of FGF-2. Such a change is
likely to support a high affinity receptor binding in a manner similar
to the "induced-fit" mechanism often implied in enzyme-substrate
interactions (55).
 |
FOOTNOTES |
*
This work was supported by Grants from MURST, Fondi per la
Ricerca di Ateneo, and Telethon Project 1073.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence may be addressed: Dept. of Experimental
Pathology and Oncology, University of Florence, Viale G.B. Morgagni
50-50134 Florence, Italy. Tel.: 39-55-414814; Fax: 39-55-416908; E-mail: delrosso@unifi.it.
Published, JBC Papers in Press, November 20, 2000, DOI 10.1074/jbc.M005993200
 |
ABBREVIATIONS |
The abbreviations used are:
u-PAR, u-PA
receptor;
ATF, amino-terminal fragment of u-PA;
BSA, bovine serum
albumin;
C4S, chondroitin 4-sulfate;
C6S, chondroitin 6-sulfate;
CHO, Chinese hamster ovary fibroblasts;
DS, dermatan sulfate;
ECM, extracellular matrix;
FCS, fetal calf serum;
GAGs, glycosaminoglycans;
GlcN, D-glucosamine which may or may not have an
N-substituent;
GlcNAc, N-acetylated glucosamine;
GPI-PLC, glycosylphosphatidylinositol-phospholipase C;
GdmCl, guanidinium chloride;
HA, hyaluronic acid;
HexUA, glycuronic acid;
HP, heparin;
HS, heparan sulfate;
HSF, human skin fibroblasts;
IdoUA, iduronic acid;
LMW u-PA, low molecular weight u-PA;
PBS, phosphate
buffered saline;
PG, proteoglycan;
su-PAR, soluble u-PAR;
u-PA, urokinase-type plasminogen activator;
VN, vitronectin;
GPI, glycosylphosphatidylinositol;
FGF, fibroblast growth factor;
PBS, phosphate-buffered saline;
pgs, proteoglycan
synthesis-defective.
 |
REFERENCES |
1.
|
Andreasen, P. A.,
Kjoller, L.,
Christensen, L.,
and Duffy, M. J.
(1997)
Int. J. Cancer
72,
1-22[CrossRef][Medline]
[Order article via Infotrieve]
|
2.
|
Plesner, T.,
Behrendt, N.,
and Ploug, M.
(1997)
Stem Cells
15,
398-408[Abstract/Free Full Text]
|
3.
|
Vassalli, J.-D.,
and Pepper, M. S.
(1994)
Nature
370,
14-15[CrossRef][Medline]
[Order article via Infotrieve]
|
4.
|
Fibbi, G.,
Caldini, R.,
Chevanne, M.,
Pucci, M.,
Schiavone, N.,
Morbidelli, L.,
Parenti, A.,
Granger, H. J.,
Del Rosso, M.,
and Ziche, M.
(1998)
Lab. Invest.
78,
1109-1119[Medline]
[Order article via Infotrieve]
|
5.
|
Duffy, M. J.
(1993)
Fibrinolysis
7,
295-302
|
6.
|
Besser, D.,
Verde, P.,
Nagamine, Y.,
and Blasi, F.
(1996)
Fibrinolysis
10,
215-237
|
7.
|
Mignatti, P.,
and Rifkin, D.
(2000)
Adv. Cancer Res.
78,
103-157[Medline]
[Order article via Infotrieve]
|
8.
|
Wang, H.,
Skibber, J.,
Juarez, J.,
and Boyd, D.
(1994)
Int. J. Cancer
58,
650-657[Medline]
[Order article via Infotrieve]
|
9.
|
Wang, G. J.,
Collinge, M.,
Blasi, F.,
Pardi, R.,
and Bender, J. R.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6296-6301[Abstract/Free Full Text]
|
10.
|
Cubellis, M. V.,
Wun, T. C.,
and Blasi, F.
(1990)
EMBO J.
9,
1079-1085[Abstract]
|
11.
|
Høyer-Hansen, G.,
Ploug, M.,
Behrendt, N.,
Ronne, E.,
and Danø, K.
(1997)
Eur. J. Biochem.
243,
21-26[Abstract]
|
12.
|
Møller, L. B.,
Pöllänen, J.,
Rønne, E.,
Pedersen, N.,
and Blasi, F.
(1993)
J. Biol. Chem.
268,
11152-11159[Abstract/Free Full Text]
|
13.
|
Andrade-Gordon, P.,
and Strickland, S.
(1986)
Biochemistry
25,
4033-4040[Medline]
[Order article via Infotrieve]
|
14.
|
Kjellen, L.,
and Lindahl, U.
(1991)
Annu. Rev. Biochem.
60,
443-475[CrossRef][Medline]
[Order article via Infotrieve]
|
15.
|
Cardin, A. D.,
and Weintraub, H. J.
(1989)
Arteriosclerosis
9,
21-32[Abstract]
|
16.
|
Fears, R.
(1988)
Biochem. J.
249,
77-81[Medline]
[Order article via Infotrieve]
|
17.
|
Stephens, R. W.,
Pollanen, J.,
Tapiovara, H.,
Woodrow, G.,
and Vaheri, A.
(1991)
Semin. Thromb. Haemostasis
17,
201-209[Medline]
[Order article via Infotrieve]
|
18.
|
Stephens, R. W.,
Bokman, A. M.,
Myohanen, H. T.,
Tapiovara, H.,
Pedersen, N.,
Grondahl-Hansen, J.,
Llinas, M.,
and Vaheri, A.
(1992)
Biochemistry
31,
7572-7579[Medline]
[Order article via Infotrieve]
|
19.
|
Rijken, D. C.,
de Munk, G. A.,
and Jie, A. F.
(1993)
Thromb. Haemostasis
70,
867-872[Medline]
[Order article via Infotrieve]
|
20.
|
Edelberg, J. M.,
Weissler, M.,
and Pizzo, S. V.
(1991)
Biochem. J.
276,
785-791[Medline]
[Order article via Infotrieve]
|
21.
|
Bertolesi, G. E.,
Farias, E. F.,
Alonso, D. F.,
Bal de Kier Joffe, E.,
Lauria de Cidre, S.,
and Eijan, A. M.
(1997)
Blood Coagul. & Fibrinolysis
8,
403-410[Medline]
[Order article via Infotrieve]
|
22.
|
Brunner, G.,
Reimbold, K.,
Meissauer, A.,
Schirrmacher, V.,
and Erkell, L. J.
(1998)
Exp. Cell Res.
239,
301-310[CrossRef][Medline]
[Order article via Infotrieve]
|
23.
|
Okayama, H.,
and Berg, P.
(1982)
Mol. Cell. Biol.
2,
161-170[Medline]
[Order article via Infotrieve]
|
24.
|
Roldan, A. L.,
Cubellis, M. V.,
Masucci, M. T.,
Behrendt, N.,
Lund, L. R.,
Danø, K.,
Appella, E.,
and Blasi, F.
(1990)
EMBO J.
9,
467-474[Abstract]
|
25.
|
Schmidtchen, A.,
Carlstedt, I.,
Malmstrom, A.,
and Fransson, L. A.
(1990)
Biochem. J.
265,
289-300[Medline]
[Order article via Infotrieve]
|
26.
|
Carlstedt, I.,
Coster, L.,
Malmstrom, A.,
and Fransson, L. A.
(1983)
J. Biol. Chem.
258,
11629-11635[Abstract/Free Full Text]
|
27.
|
Del Rosso, M.,
Cappelletti, R.,
Vannucchi, S.,
Romagnani, S.,
and Chiarugi, V.
(1979)
Biochim. Biophys. Acta
586,
512-517[Medline]
[Order article via Infotrieve]
|
28.
|
Miao, H.-Q.,
Ishai-Michaeli, R.,
Atzmon, R.,
Peretz, T.,
and Vlodavsky, I.
(1996)
J. Biol. Chem.
271,
4879-4886[Abstract/Free Full Text]
|
29.
|
Rodén, L.,
Baker, J.,
Cifonelli, J. A.,
and Mathews, M. B.
(1972)
Methods Enzymol.
28,
73-140
|
30.
|
Fransson, L. A.,
Havsmark, B.,
and Sheehan, J. K.
(1981)
J. Biol. Chem.
256,
13039-13043[Abstract/Free Full Text]
|
31.
|
Fransson, L. A.,
Sjoberg, I.,
and Havsmark, B.
(1980)
Eur. J. Biochem.
106,
59-69[Abstract]
|
32.
|
Fransson, L. A.
(1978)
Carbohydr. Res.
62,
235-244[CrossRef]
|
33.
|
Fransson, L. A.,
Malmstrom, A.,
Sjoberg, I.,
and Huckerby, T. N.
(1980)
Carbohydr. Res.
80,
131-145[CrossRef]
|
34.
|
Holmberg, L.,
Bladh, B.,
and Åstedt, B.
(1970)
Biochim. Biophys. Acta
445,
215-222
|
35.
|
Del Rosso, M.,
Dini, G.,
and Fibbi, G.
(1985)
Cancer Res.
45,
630-636[Abstract]
|
36.
|
Del Rosso, M.,
Pedersen, N.,
Fibbi, G.,
Pucci, M.,
Dini, G.,
Anichini, E.,
and Blasi, F.
(1992)
Exp. Cell Res.
203,
427-434[Medline]
[Order article via Infotrieve]
|
37.
|
Anichini, E.,
Fibbi, G.,
Pucci, M.,
Caldini, R.,
Chevanne, M.,
and Del Rosso, M.
(1994)
Exp. Cell Res.
213,
438-448[CrossRef][Medline]
[Order article via Infotrieve]
|
38.
|
Del Rosso, M.,
Cappelletti, R.,
Viti, M.,
Vannucchi, S.,
and Chiarugi, V.
(1981)
Biochem. J.
199,
699-704[Medline]
[Order article via Infotrieve]
|
39.
|
Saksela, O.,
Moscatelli, D.,
and Rifkin, D. B.
(1987)
J. Cell Biol.
105,
957-963[Abstract]
|
40.
|
Battaglia, C.,
Aumailley, M.,
Mann, K.,
Mayer, U.,
and Timpl, R.
(1993)
Eur. J. Cell Biol.
61,
92-99[Medline]
[Order article via Infotrieve]
|
41.
|
Couchman, J. R.,
and Woods, A.
(1999)
J. Cell Sci.
112,
3415-3420[Abstract/Free Full Text]
|
42.
|
Klass, C. M.,
Couchman, J. R.,
and Woods, A.
(2000)
J. Cell Sci.
113,
493-506[Abstract/Free Full Text]
|
43.
|
Hershkoviz, R.,
Schor, H.,
Ariel, A.,
Hecht, I.,
Cohen, I. R.,
Lider, O.,
and Cahalon, L.
(2000)
Immunology
99,
87-93[CrossRef][Medline]
[Order article via Infotrieve]
|
44.
|
Kusano, Y.,
Oguri, K.,
Nagayasu, Y.,
Munesue, S.,
Ishihara, M.,
Saiki, I.,
Yonekura, H.,
Yamamoto, H.,
and Okayama, M.
(2000)
Exp. Cell Res.
256,
434-444[CrossRef][Medline]
[Order article via Infotrieve]
|
45.
|
Diamond, M. S.,
Alon, R.,
Parkos, C. A.,
Quinn, M. T.,
and Springer, T. A.
(1995)
J. Cell Biol.
130,
1473-1482[Abstract]
|
46.
|
Couchman, J. R.,
Austria, M. R.,
and Woods, A.
(1990)
J. Invest. Dermatol.
94,
7S-14S[Abstract]
|
47.
|
Bork, P.
(1992)
FEBS Lett.
307,
49-54[CrossRef][Medline]
[Order article via Infotrieve]
|
48.
|
Dawes, J.
(1993)
Haemostasis
23,
212-219[Medline]
[Order article via Infotrieve]
|
49.
|
Critchley, D. R.
(2000)
Curr. Opin. Cell Biol.
12,
133-139[CrossRef][Medline]
[Order article via Infotrieve]
|
50.
|
Pöllänen, J.,
Hedman, K.,
Nielsen, L. S.,
Danø, K.,
and Vaheri, A.
(1988)
J. Cell Biol.
106,
87-95[Abstract]
|
51.
|
Kuschert, G. S.,
Coulin, F.,
Power, C. A.,
Proudfoot, A. E.,
Hubbard, R. E.,
Hoogewerf, A. J.,
and Wells, T. N.
(1999)
Biochemistry
38,
12959-12968[CrossRef][Medline]
[Order article via Infotrieve]
|
52.
|
Carey, D. J.
(1997)
Biochem. J.
327,
1-16[Medline]
[Order article via Infotrieve]
|
53.
|
Vlodavsky, I.,
Bar-Shavit, R.,
Ishai-Michaeli, R.,
Bashkin, P.,
and Fuks, Z.
(1991)
Trends Biochem. Sci.
16,
268-271[CrossRef][Medline]
[Order article via Infotrieve]
|
54.
|
Rapraeger, A. C.,
Krufke, A.,
and Olwin, B. B.
(1991)
Science
252,
1705-1708[Medline]
[Order article via Infotrieve]
|
55.
|
Yayon, A.,
Klagsbrun, M.,
Esko, J. D.,
and Ornitz, D. M.
(1991)
Cell
64,
841-848[Medline]
[Order article via Infotrieve]
|
56.
|
Olwin, B. B.,
and Rapraeger, A.
(1992)
J. Cell Biol.
118,
631-639[Abstract]
|
57.
|
Guimond, S.,
Maccarana, M.,
Olwin, B. B.,
Lindahl, U.,
and Rapraeger, A. C.
(1993)
J. Cell Biol.
268,
23906-23914
|
58.
|
Preissner, K. T.,
and Seiffert, D.
(1998)
Thromb. Res.
89,
1-21[CrossRef][Medline]
[Order article via Infotrieve]
|
59.
|
Chang, A. W.,
Kuo, A.,
Barnathan, E. S.,
and Okada, S. S.
(1998)
Arterioscler. Thromb. Vasc. Biol.
18,
1855-1860[Abstract/Free Full Text]
|
60.
|
Timar, J.,
Ladanyi, A.,
Lapis, K.,
and Moczar, M.
(1992)
Am. J. Pathol.
141,
467-474[Abstract]
|
61.
|
Wei, Y.,
Lukashev, M.,
Simon, D. I.,
Bodary, S. C.,
Rosenberg, S.,
Doyle, M. V.,
and Chapman, H. A.
(1996)
Science
273,
1551-1555[Abstract]
|
62.
|
Chapman, H. A.
(1997)
Curr. Opin. Cell Biol.
9,
714-724[CrossRef][Medline]
[Order article via Infotrieve]
|
63.
|
Parton, R. G.
(1996)
Curr. Opin. Cell Biol.
8,
542-548[CrossRef][Medline]
[Order article via Infotrieve]
|
64.
|
Chapman, H. A.,
Wei, Y.,
Simon, D. I.,
and Waltz, D. A.
(1999)
Thromb. Haemostasis
82,
291-297[Medline]
[Order article via Infotrieve]
|
65.
|
Stahl, A.,
and Mueller, B. M.
(1995)
J. Cell Biol.
129,
335-344[Abstract]
|
66.
|
Stahl, A.,
and Mueller, B. M.
(1994)
Cancer Res.
54,
3066-3071[Abstract]
|
67.
|
Filmus, J.,
Shi, W.,
Wong, Z. M.,
and Wong, M. J.
(1995)
Biochem. J.
311,
561-565[Medline]
[Order article via Infotrieve]
|
68.
|
Mertens, G.,
Van der Schueren, B.,
van del Berghe, H.,
and David, G.
(1996)
J. Cell Biol.
132,
487-497[Abstract]
|
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.