|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 514-522
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Different mechanisms define the antiadhesive function of high
molecular weight kininogen in integrin- and urokinase
receptor-dependent interactions
Triantafyllos Chavakis,
Sandip M. Kanse,
Florea Lupu,
Hans-Peter Hammes,
Werner Müller-Esterl,
Robin A. Pixley,
Robert W. Colman, and
Klaus T. Preissner
From the Institute for Biochemistry and the Department of Internal
Medicine, Justus-Liebig-University, Giessen, Germany; Weston Center for
Experimental Research, Thrombosis Research Institute, Manresa Road,
London, England; Institute for Physiological Chemistry,
Johannes-Gutenberg-Universität, Mainz, Germany; and the Sol
Sherry Thrombosis Research Center, Temple University School of
Medicine, Philadelphia, PA.
 |
Abstract |
Proteolytic cleavage of single-chain high molecular weight kininogen
(HK) by kallikrein releases the short-lived vasodilator bradykinin and
leaves behind 2-chain high molecular weight kininogen (HKa) that has
been previously reported to exert antiadhesive properties as well as to
bind to the urokinase receptor (uPAR) on endothelial cells. In this
study we defined the molecular mechanisms for the antiadhesive effects
of HKa related to disruption of integrin- and uPAR-mediated cellular
interactions. Vitronectin (VN) but not fibrinogen or
fibronectin-dependent  v 3 integrin-mediated adhesion
of endothelial cells was blocked by HKa or its isolated domain 5. In a
purified system, HKa but not HK competed for the interaction of VN with
v3 integrin, because HKa and the isolated domain 5 but not HK bound to both multimeric and native VN in a
Zn2+-dependent manner. The interaction between HKa or
domain 5 with VN was prevented by heparin, plasminogen activator
inhibitor-1, and a recombinant glutathione-S-transferase (GST)-fusion
peptide GST-VN (1-77) consisting of the amino terminal portion of VN
(amino acids 1-77), but not by a cyclic arginyl-glycyl-aspartyl
peptide, indicating that HKa interacts with the amino terminal portion of VN ("somatomedin B region"). Furthermore, we have confirmed that HKa but not HK bound to uPAR and to the truncated 2-domain form of
uPAR lacking domain 1 in a Zn2+-dependent manner. Through
these interactions, HKa or its recombinant His-Gly-Lys-rich domain 5 completely inhibited the uPAR-dependent adhesion of myelomonocytic U937
cells and uPAR-transfected BAF-3 cells to VN and thereby promoted cell
detachment. By immunogold electron microscopy, both VN and HK/HKa were
found to be colocalized in sections from human atherosclerotic coronary
artery, indicating that the described interactions are likely to take
place in vivo. Taken together, HK and HKa inhibit different
VN-responsive adhesion receptor systems and may thereby influence
endothelial cell- or leukocyte-related interactions in the vasculature,
particularly under inflammatory conditions.
(Blood. 2000;96:514-522)
© 2000 by The American Society of Hematology.
| |
Introduction |
Cell-to-cell and cell-to-extracellular matrix
interactions determine morphogenetic processes during development,
vascular remodeling, or inflammation. The dynamics of cellular contacts require the presence of pro- and anti- or counter-adhesive components as well as different proteolytic systems that need to be regulated in a
spatiotemporal manner.1,2 In particular, the pattern of
expression of adhesion receptors such as selectins or integrins in the
vasculature as well as changes in the composition of the extracellular
matrix are directly related to alterations of vascular cell
interactions and relate to the pathophysiology of cardiovascular diseases.3
The multifunctional proteins fibrinogen (FBG)/fibrin and vitronectin
(VN) accumulate at extracellular matrix sites associated with wound
healing,4,5 malignant tumors,6 or
angiogenesis.7,8 These and other adhesive glycoproteins are
recognized by integrins of the  v subfamily, which are prominently
expressed on migrating and proliferating vascular cells and whose
functions can effectively be blocked by synthetic antagonists such as
cyclic arginyl-glycyl-aspartyl (cRGD) peptides.9,10
Moreover, the plasminogen activation system contributes to cell
invasion by mediating not only pericellular proteolysis but also by
participating in the modulation of cell adhesion in a nonproteolytic
fashion.11,12 Through direct interactions particularly
expressed at vitronectin-rich matrix sites, urokinase (uPA) acts as
proadhesive factor, whereas plasminogen activator inhibitor-1 (PAI-1)
abrogates both integrin- and urokinase receptor (uPAR)-dependent cell
adhesion.13-17 In the latter system, uPAR also serves as
high-affinity cell surface-associated binding protein for
VN.18,19 A third ligand for uPAR is high molecular weight kininogen (HK)20; however, the functional relationships
among these components in cellular contacts remain to be defined.
HK is composed of 6 domains and is present in plasma at a concentration
of 0.67 µmol/L. It serves a nonenzymatic cofactor role in the
initiation of the contact phase,21-23 associated with vascular injury, inflammation, or activation of complement in humoral
immune defense. In particular, kallikrein can liberate the short-lived
vasodilator peptide bradykinin from HK,24 thereby generating HKa (2-chain kinin-free HK), which lacks most of its domain
4. Domain 5 in HKa is rich in His, Gly, and Lys, which enables HKa to
bind to anionic surfaces, zinc, or heparin.25-28 Moreover,
HK/HKa binding to cells is mediated by domains 3 and 5,29,30 contributing to the regulation of pericellular
plasmin generation by modulating plasma kallikrein-dependent formation of uPA,31 a reaction dependent on the binding of plasma
prekallikrein to HK domain 6.22 On granulocytes, HK and FBG
compete for binding to the integrin
M2,32,33 whereas on
endothelial cells the binding proteins for globular C1q (denoted gC1qR)
as well as cytokeratin-134-36 were identified as binding
proteins for HK. This role for gC1qR has been disputed in light of the
recent finding that gC1qR appears to be a mitochondrial
protein.37 On endothelial cells, HKa is recognized by uPAR,
and this Zn2+-dependent binding can be inhibited by
VN.20
These diverse observations, together with a recent report on
antiadhesive properties of HKa,38,39 prompted us to
investigate the underlying mechanisms for the contribution of
kininogens in adhesive interactions involving different adhesion
receptors on blood and vessel wall cells. Our results indicate that
direct binding of HKa to matrix-associated VN competes for integrin- and uPAR-dependent cell adhesion uncovering a plausible mechanism for
the antiadhesive properties of HKa in tissue remodeling.
| |
Materials and methods |
Reagents
Recombinant Gly158scuPA (noncleavable mutant of high
molecular weight uPA) was produced in Chinese hamster ovary
cells40 and was provided by Dr H. Roger Lijnen
(Leuven, Belgium). uPA was from Medac (Hamburg, Germany); VN was
purified from human plasma and converted to the multimeric form as
previously described41,42; FBG was purchased from
Kabivitrum (Munich, Germany); fibronectin was from Sigma (Munich,
Germany); cRGD-containing peptide (cRGDfV) was from Bachem (Heidelberg,
Germany); and recombinant v3 integrin was kindly
provided by Dr Simon Goodman (Merck KGaA, Darmstadt, Germany). Recombinant soluble uPAR (suPAR) was obtained from Dr Niels Behrendt (Finsen Laboratory, Copenhagen, Denmark).
Single- and 2-chain high molecular weight kininogen (HK and HKa) were purchased from Enzyme Research Laboratories (South Bend,
IN). The purified HK and HKa (more than 95%) appeared as a major band of 140 kd and 110 kd, respectively, on nonreduced sodium dodecyl sulfate gels. HK had been digested with plasma kallikrein (HK to
kallikrein = 100:1, mol/mol) for 20 minutes at 37°C. The
resulting HKa was composed of 2 bands of 62 and 46 kDa when analyzed by reduced sodium dodecyl sulfate gel electrophoresis.
Glutathione-S-transferase (GST) fused to domains 3, 5, and 6 of HK were
produced as previously described.43,26 GST was N-terminally
attached to the following sequences of HK: G235 to M357 (domain 3),
K420 to S513 (domain 5), and T503 to S626 (domain 6). Likewise in
GST-VN (1-77), GST was fused to the amino terminal portion (amino acids
1-77) of VN and produced as previously described.44 Active
PAI-1 was from Astra Hässle AB (Mölndal, Sweden). Murine
monoclonal antibody (mAb) 13H1 against human VN42,45 was
kindly provided by Dr Paul Declerck (Leuven, Belgium); anti-uPAR mAb
(R3)46 was provided by Dr Gunilla Hoyer-Hansen
(Finsen Laboratory). ZnCl2 was from Sigma; vitamin
D3- was from Biomol (Hamburg, Germany); transforming growth
factor- was from R & D Systems (Boston, MA); and interleukin-3 was
from PBH (Hannover, Germany). The characteristics of mAb HKH13 against
domain 3 and HKH18 against domain 1 of HK were previously described.47
Cell culture
Monocytic cells (U937) and MG63 human osteosarcoma cells were from
American Type Culture Collection (ATCC, Rockville, MD) and cultured as
described by the supplier in RPMI-1640 medium or Dulbecco's modified
Eagle's medium (DMEM), respectively, containing 10% (vol/vol) fetal
calf serum. BAF-3 cells were from ATCC and cultured in RPMI-1640 medium
containing 10% fetal calf serum and 2 ng/mL interleukin-3. Bovine
retinal endothelial cells (BRECs) were kindly provided by Dr Sigrid
Zink (Diabetes Research Institute, University of
Düsseldorf, Germany) and cultivated as described.48 Bovine adrenocortical endothelial cells (BAECs) were kindly provided by
Dr Mathias Clauss (Max-Planck-Institute, Bad Nauheim,
Germany) and cultivated in DMEM containing 10% (vol/vol) fetal
calf serum. All culture media were from Gibco (Eggenstein, Germany).
Construction of uPAR-transfected BAF-3 cells
BAF-3 cells (interleukin-3-dependent mouse B-cell line) were
transfected by electroporation with uPAR complementary DNA in the sense
and antisense orientation using the expression vector pCDNA3. Cells
were selected in the presence of G418 (1.2 mg/mL) and found to express
uPAR by fluorescence-activated cell sorter analysis, Northern blotting,
and uPAR enzyme-linked immunosorbent assay (ELISA) (details to be
published elsewhere).
Radiolabeling of vitronectin
Multimeric VN (100 µg) was labeled with 18.5 MBq
Na125I (Amersham, Braunschweig, Germany) using Iodogen
(Pierce, Oud Beijerland, The Netherlands) according to a previously
outlined procedure.49,50 After separation on a Sephadex
G-25 column (Pharmacia, Freiburg, Germany) suspended in Tris-buffered
saline (TBS) (20 mmol/L Tris base; 0.15-mol/L NaCl, pH 7.4; TBS)
containing 0.1% (wt/vol) bovine serum albumin (BSA; Sigma), the
labeled protein was dialyzed against TBS. The specific activity was
185-370 kBq/µg VN.
Binding interactions in a purified system
Polystyrene microtiter wells (high binding, type I, Costar,
Badhoevedorp, The Netherlands) were coated at a concentration of 5 µg/mL with purified uPAR in 15 mmol/L Na2CO3,
35 mmol/L NaHCO3 (pH 9.6), with 5 µg/mL
v3 integrin in TBS containing 1 mmol/L CaCl2 and 1 mmol/L MgCl2, or with 5 µg/mL of
HK or its isoforms in TBS, and subsequently blocked with 3% (wt/vol)
BSA. Binding of 125I-VN to the immobilized components was
performed at 4°C for 18 hours in a final volume of 50 µL in the
absence or presence of competitors as indicated in the figure legends:
Binding to v3 integrin was carried out in TBS
containing 0.05% (wt/vol) Tween 20, 0.3% (wt/vol) BSA, 1 mmol/L
CaCl2, and 1 mmol/L MgCl2, while binding to
uPAR or HK/HKa was performed in the absence of added divalent cations.
Thereafter, the wells were washed and counted in a  counter.
Nonspecific binding was measured in the presence of more than 100-fold
molar excess of unlabeled VN to estimate specific binding. Nonspecific
binding of 125I-VN to BSA-coated wells (in the absence of
the immobilized receptor) was used as an additional control in all
experiments and was subtracted in calculating the specific binding.
ELISA for ligand-receptor interactions
Maxisorp plates (high binding capacity; Nunc, Roskilde,
Denmark) were coated with uPAR (5 µg/mL) or different
forms and fragments of HK (HKa, D3, D5, D6; each at 5 µg/mL),
respectively. After blocking with 3% (wt/vol) BSA in TBS, 2 µg/mL
native or multimeric VN in TBS containing 0.3% BSA and 0.05% Tween 20 was added to the wells. After incubation for 2 hours at 22°C, mAb
VN-745 against VN at a concentration of 125 ng/mL was
added, followed by addition of a secondary goat antimouse
immunoglobulin G (Dako, Hamburg, Germany) and the substrate ABTS, and
binding was quantitated at 405 nm in a Thermomax Reader (Molecular
Devices, Menlo Park, CA). The identical protocol was used when the
binding of HK/HKa in the presence or absence of 50 µmol/L
ZnCl2 to immobilized multimeric or native VN, uPAR, or
v3 integrin (each at 5 µg/mL) was tested, except
that mAb HKH13 or HKH18 were used for the detection of bound kininogen.
Nonspecific binding to BSA-coated wells was used as blank and was
subtracted to calculate the specific binding. In case of binding of
HK/HKa to immobilized VN, uPAR, or v3 integrin,
binding to noncoated wells and to BSA-coated wells in the absence or
presence of Zn2+ was also estimated to calculate
nonspecific binding. These values were also subtracted to estimate the
specific binding of HK/HKa.
Cell adhesion assays
Cell adhesion to VN-, FBG-, or fibronectin-coated plates
(and to BSA-coated wells as control) was tested according to a
previously described protocol.19,51 Briefly, multiwell
plates were coated with 2 µg/mL native or multimeric VN or 10 µg/mL
FBG or fibronectin (dissolved in bicarbonate buffer, pH 9.6),
respectively, and blocked with 3% (wt/vol) BSA. BAF-3 or U937 cells,
which had been differentiated for 24 hours with vitamin
D3 (100 nmol/L) and transforming growth factor- (2 ng/mL), were washed in serum-free RPMI and plated onto the precoated
wells for 60 to 90 minutes at 37°C in the absence or presence of
competitors in serum-free RPMI. Proteolytic conversion of HK to HKa in
the absence and the presence of monocytic cells as described in the
same adhesion assay was performed and, after the incubation period, the
supernatant was collected and analyzed for HK or HKa in a Western blot
using the antibody HKH18.47
Confluent BREC, BAEC, or MG63 osteosarcoma cells were detached
with trypsin, which was subsequently neutralized with soybean trypsin
inhibitor (Sigma), washed, and plated onto precoated wells as described
above. After the incubation period for the adhesion assay in serum-free
DMEM, the wells were washed and the number of adherent cells were
quantified by crystal violet staining at 590 nm.
Electron microscopy
Secondary antibodies conjugated to 15 nm
colloidal gold were from BioCell Research Labs (Boston, MA),
and protein A coupled to 10 nm gold was from the Department of Cell
Biology, University of Utrecht, The Netherlands. All other reagents
used for electron microscopy were from TAAB Laboratory Equipment Ltd,
Reading, England.
Sections of human atherosclerotic coronary arteries were
obtained within 2 hours of surgery, cut into approximately
1-µm pieces, and fixed in 4% (wt/vol)
paraformaldehyde/phosphate-buffered saline (PBS) on ice for 30 minutes.
Specimens were dehydrated through a graded series of ethanol solutions
with progressive lowering of temperature for 1 hour each (30% at
0°C, 50% at  20°C, 70% at 30°C, 90% at
30°C, 100% ethanol at 30°C) and then infiltrated with 50% (vol/vol) K4M Lowicryl (TAAB) in ethanol at
30°C for 16 hours followed by 100% K4M Lowicryl at
30°C for 2 × 12 hours. Finally, tissues were
embedded in K4M Lowicryl at 30°C for 16 hours under UV
light, and polymerization was completed at room temperature under UV
light. Blocks were cut on an Ultracut microtome, and
80-nm-thick sections were taken onto 400 mesh copper/rhodium electron
microscopy grids (TAAB).
Double immunogold labeling of VN and kininogen was performed
by sequentially staining both sides of tissue sections. These were
initially treated with blocking buffer (10% [vol/vol] fetal calf
serum/0.02 mol/L glycine or PBS) for 30 minutes to quench the
unspecific binding sites. For detection of VN, the sections were
incubated with rabbit antihuman VN antibodies41 diluted 1:50 in blocking buffer for 1 hour at room temperature. Thereafter, the
sections were washed in PBS for 3 × 5 minutes and subsequently treated with protein A conjugated to 10 nm gold particles diluted 1:50
in blocking buffer for 1 hour.52 Grids were then washed in PBS for 3 × 5 minutes to remove any unbound protein A
gold, fixed in 2.5% glutaraldehyde or PBS for 5 minutes, and finally washed in double-distilled water for 30 minutes. For the detection of
the second antigen kininogen, the grids were turned over and incubated
in blocking buffer as before. Kininogen antigen was detected by
incubation for 1 hour with sheep antihuman kininogen antiserum
(I107, 10 µg/mL) diluted 1:25 in blocking buffer. Unbound antibodies
were washed off as mentioned above. Specifically bound antibodies were
detected by incubation for 1 hour with rabbit antigoat
immunoglobulin G coupled to 15 nm gold particles diluted 1:50 in
blocking buffer. As negative controls, tissue sections were used that
were similarly treated except that the primary antibodies were omitted.
After immunostaining, the grids were coated with Formvar film, and
sections were contrasted using uranyl acetate and lead citrate.
Sections were analyzed using a Philips 201 transmission electron microscope.
| |
Results |
Inhibition of v3 integrin-dependent adhesion to
VN by kininogen
Endothelial cells adhere via different integrins to matrix proteins
such as VN, FBG, or fibronectin. The effect of HK or HKa on the
adhesion of BREC, BAEC, and MG63 human osteosarcoma cells to these
proteins was tested. Adhesion to VN was v3
integrin-mediated, as it was abolished by cRGDfV.
Similar to PAI-1, which can block this v3
integrin-VN interaction, HKa reduced endothelial cell adhesion to VN
by 70% to 80%, whereas HK had a much weaker effect. Neither HK nor
HKa affected the adhesion of these cells to FBG or fibronectin (Figure
1A). To further define the specificity of
the antiadhesive properties of HKa for the VN substrate, 2 adhesion
protocols were compared: (1) cells and competitors were added
simultaneously to the VN-coated wells, or (2) prior to the adhesion
assay, the VN-coated wells were incubated with the competitors for 1 hour, followed by extensive washing. In both cases, HKa inhibited cell
adhesion in a pattern similar to PAI-1 or mAb 13H1 directed against VN,
which both block VN-dependent adhesion due to direct binding to VN. In
contrast, the cRGDfV inhibited adhesion when added simultaneously to
the cells, but it had no effect when it was preincubated on the VN
substrate. The latter was expected, because cRGDfV abolishes adhesion
by binding specifically to the v3 integrin but not by
binding to VN (Figure 1B). Adhesion to FBG was not blocked even by high
concentrations of kininogens, when added simultaneously to the seeded
cells, whereas partial inhibition of cell adhesion was observed after
preincubation of HKa with immobilized FBG prior to the cell adhesion
assay. This reduction in cell adhesion was due to the Vroman
effect,53 because HKa displaced FBG from the adhesion plate
(data not shown), while VN was resistant to the Vroman
effect38,39,54 (data not shown). When endothelial cell
adhesion was compared with both multimeric VN or native VN, there was
hardly any difference between both substrates. HKa but not HK could
block cell adhesion to multimeric VN and native VN to the same extent
(not shown).
View larger version (33K):
[in this window]
[in a new window]
| Fig 1.
The role of kininogen on integrin-mediated endothelial
cell adhesion.
(A) The adhesion of BRECs to VN-, fibronectin (FN)- and FBG-coated
wells was analyzed in the absence () or in the presence of the
competitors HK, HKa, or cRGDfV (10 µg/mL each), 100 nmol/L PAI-1, or
10 mmol/L ethylenediaminetetraacetic acid as indicated. The extent of
cell adhesion is presented as absorbance at 590 nm. Data are mean ± SEM (n = 3) of a typical experiment, and similar results were
obtained in at least 3 separate experiments. (B) The effect of HK, HKa,
cRGDfV (10 µg/mL each), or 20 µg/mL anti-VN mAb 13H1 on the
adhesion of BREC to VN was studied according to 2 different protocols:
(a) Cells and competitors were added simultaneously to the
VN-coated wells (filled bars), and (b) VN-coated wells were
blocked and then incubated for 1 hour with the different competitors
followed by extensive washing and addition of cells (hatched bars). The
extent of cell adhesion is presented as absorbance at 590 nm. Data are
mean ± SEM (n = 3) of a typical experiment; similar results were
obtained in at least 3 separate experiments.
|
|
In subsequent experiments, the effect of different forms of kininogen
on adhesion of endothelial cells to VN was investigated (Figure
2A). HKa was by far the most effective
component (inhibitory concentration of 50% = 25 nmol/L) and could
completely block cell adhesion at a concentration of 10 µg/mL (about
85 nmol/L). While domain 5 but not 3 could also reproduce most of the
HKa effect, higher concentrations of HK were needed to provide a
maximal inhibition of 50%. In contrast to cRGDfV, which was able to
reverse the adhesion process partially (Figure 2B), HK or HKa did not
promote detachment of endothelial cells that had adhered onto the VN
substrate for 1 hour. Similar results as with BREC and BAEC were also
obtained with MG63 human osteosarcoma cells (not shown).
View larger version (19K):
[in this window]
[in a new window]
| Fig 2.
The effect of different kininogen forms on endothelial
cell adhesion to VN.
(A) BRECs were allowed to adhere to VN in the absence or presence of
various concentrations of HK (filled circles), HKa (open circles),
domain 3 (filled squares), domain 5 (open squares), and domain 6 (filled triangles), and the extent of adhesion is presented as
percentage of control. Data are mean ± SEM (n = 3) of a typical
experiment, and similar results were observed in at least 3 different
experiments. (B) After cells had adhered to VN for 1 hour, HK (open
circles), HKa (filled circles), or cRGDfV (filled squares) (10 µg/mL
each) was added, and remaining adherent cells after various times were
quantitated (presented as absorbance at 590 nm). Data are mean ± SEM (n = 3) of a typical experiment, and similar results were
obtained in 3 separate experiments.
|
|
Binding of kininogen to VN
The specificity of the antiadhesive effect of HKa in the
v3 integrin-VN interaction can be explained by
direct blockade of either the integrin v3 or the
substrate VN by HKa. The latter was demonstrated by the fact that
preincubation of the VN substrate with HKa but not with HK could
inhibit cell adhesion. HKa blocked the binding of 125I-VN
to isolated v3 integrin (Table
1), whereas HK had minimal inhibitory
activity. While HKa at 20 µg/mL (170 nmol/L) reduced specific binding
by 60%, PAI-1 at 200 nmol/L diminished binding by 85%; however, these
concentrations of HKa and PAI-1 induced a similar inhibition of
endothelial cell adhesion to VN. Because both HKa and VN are
Zn2+-binding proteins,51 the binding of
different concentrations of VN to immobilized HK or HKa was tested in
the absence or presence of ZnCl2 in an ELISA for direct
ligand-receptor interactions. Zn2+-dependent specific
binding of VN to only HKa but not to HK was observed. In addition to
HKa, VN could specifically interact with immobilized domain 5 in a
Zn2+-dependent manner, whereas no binding to domains 3 or 6 was found (Figure 3A). In the reverse
situation, specific binding of soluble HKa and domain 5 but not HK to
immobilized multimeric or native VN was noted, where more efficient
binding was seen with immobilized multimeric VN
(Table 2). Also,
multimeric VN bound more efficiently to immobilized HKa or domain 5 than did native VN (Table 3). Heparin
almost completely abolished the binding of multimeric VN to HKa
or domain 5 but only partially interfered with the binding of
native VN (Table 3). Because the interaction between VN and HKa or domain 5 was prevented by PAI-1 or by the fusion product GST-VN (1-77) (Figure 3B), kininogen forms can compete with
PAI-1 for an overlapping binding site on VN, which is located within the amino terminal portion of the adhesive protein.
In contrast, the cRGDfV had no effect on the kininogen-VN
interaction (Figure 3B). No substantial differences in the binding
pattern between VN and kininogen were observed regardless whether ELISA
(Maxisorp) or cell adhesion plates were used (not shown).
View larger version (30K):
[in this window]
[in a new window]
| Fig 3.
Binding of VN to different immobilized kininogen
isoforms.
(A) The binding of 2-µg/mL VN to immobilized HK, HKa,
GST-D3, GST-D5, GST-D6, or GST (each 5 µg/mL) in the absence (filled
bars) or the presence (hatched bars) of 50 µmol/L ZnCl2
was carried out, and specific binding is presented as
absorbance at 405 nm. Data are mean ± SEM (n = 3) of a typical
experiment, and similar results were obtained in 3 separate
experiments. (B) The binding of VN to immobilized HKa (filled bars) or
domain 5 (hatched bars) was performed in the absence
() or presence of 10 µg/mL heparin, 10 µg/mL cRGDfV, 200 nmol/L PAI-1, 10 µg/mL GST-VN (1-77), or 10 µg/mL GST in buffer
containing 50 µmol/L ZnCl2. Specific
binding of VN is presented as percentage of control (binding of VN to
HKa or D5 in the absence of any competitor). Data are mean ± SEM
(n = 3) of a typical experiment, and similar results were obtained in
3 separate experiments.
|
|
Finally, no direct binding of HKa or HK to v3
integrin was observed (not shown), indicating that the interaction
between HKa and VN prevented its ligation to the v3
integrin. This observation is consistent with the failure of a blocking
antibody (7E3) to v3 integrin or of FBG to inhibit
the binding of HK/HKa to endothelial cells.20
Inhibition of leukocyte adhesion to VN by kininogen
As previously established, the adhesion of myelomonocytic U937 cells
(differentiated with transforming growth factor- [2 ng/mL] and
vitamin D3 [100 nmol/L] for 24 hours) to immobilized VN
is predominantly mediated by uPAR.18,55 Moreover, uPA
augments this adhesion by increasing the affinity of the uPAR-VN
interaction.19 The same characteristics were found for
uPAR-transfected BAF-3 (sense-uPAR) but not for control cells
(antisense-uPAR). The addition of HK or HKa resulted in a complete
inhibition of adhesion of uPAR-transfected BAF-3 cells and U937 cells
also in the presence of uPA, reminiscent of the blocking effect of
PAI-1 in this system.16 An identical pattern of inhibition
with HKa, HK, or PAI-1 was obtained for the adhesion of human
peripheral blood monocytes (data not shown). HKa but not HK was also
antiadhesive when these substances were preincubated onto the VN
substrate prior to the cell adhesion step (data not shown).
To characterize the involved domains of HKa responsible for
antiadhesion, uPA-stimulated adhesion of U937 cells was tested in the
presence of increasing concentrations of HK, HKa, and the recombinant
GST-fusion domains 3, 5, or 6, respectively (Figure 4A). HKa, HK, and domain 5 but not domain 3 or 6 abolished cell adhesion, indicating that domain 5 contains most of
the antiadhesive activity also for the uPAR-dependent system. To
differentiate between the varying antiadhesive activities of HK and
HKa, the kinetics of cell adhesion in the presence of both kininogen
forms was studied. When cells were plated in the presence of uPA and HK
or HKa, respectively, HKa could immediately block the augmenting effect
of uPA on cell adhesion, whereas only after 1 hour could HK
significantly reduce adhesion (Figure 4B). In contrast to the v
integrin-dependent endothelial cell adhesion, HKa, similar to PAI-1,
promoted the immediate detachment of U937 cells that had adhered to VN
for 1.5 hours under the influence of uPA (Figure 4C), whereas about 20 minutes of lag-phase was needed for HK-induced cell dissociation. These
findings indicate that HKa was directly interfering with the uPAR-VN
interaction, whereas HK needed proteolytic activation by cell-derived
proteases as deduced from Western blot analysis (Figure 4D).
View larger version (146K):
[in this window]
[in a new window]
| Fig 4.
Inhibition of uPA-induced cell adhesion by kininogen.
(A) uPA-induced U937 cell adhesion to VN was studied in the absence or
presence of various concentrations of HK (open circles), HKa (filled
circles), isolated domain 3 (filled triangles), domain 5 (open
squares), or domain 6 (open triangles). (B) On a VN substrate, seeding
of U937 cells together with uPA was performed in the absence (filled
circles) and in the presence of 10 µg/mL HK (open circles) or 10 µg/mL HKa (filled squares), and the extent of cell adhesion was
analyzed after various times as indicated. (C) After allowing the
adhesion of U937 cells to VN in the presence of 50 nmol/L uPA for an
incubation period of 90 minutes, 10 µg/mL HK (open circles), 10 µg/mL HKa (filled circles), or 100 nmol/L PAI-1 (open squares),
respectively, was added, and the residual extent of cell adhesion was
measured. Data are expressed as percentage of control, which is
represented by the adhesion of cells in the absence of any stimulus or
competitor. Data are mean ± SEM (n = 3) of a typical experiment;
similar results were obtained in 3 separate experiments. (D) An
adhesion assay with U937 myelomonocytic cells was performed in the
absence () or presence of HK or HKa. After the incubation period
for the adhesion assay (90 minutes), the supernatant was collected,
centrifuged, and analyzed in a Western blot with the antibody HKH18,
which is directed against domain 1 of kininogen and detects both HK and
HKa. As a control, parallel wells without cells were incubated with the
adhesion medium and HK or HKa. Molecular weight markers are indicated
at the right margin. Similar results were observed in 3 separate
experiments.
|
|
Binding of kininogen to uPAR
Subsequent experiments revealed that HKa and the isolated
domain 5 inhibited the binding of 125I-VN to immobilized
uPAR in a Zn2+-dependent manner, whereas HK, domain 3, or
domain 6 were ineffective (Figure 5). In
the absence of Zn2+, HKa and domain 5 presented only a weak
inhibition of the uPAR-VN interaction (data not shown).
This property of kininogen could be explained by the direct binding of
HKa and the domain 5 to VN as described above. Because uPAR has been
identified as a binding site on endothelial cells for HKa but not for
HK,20 direct binding of HKa to suPAR was tested. As shown
in Figure 6A, HKa but not HK bound
specifically to immobilized suPAR in a Zn2+-dependent
fashion. Moreover, HKa also interacted with the truncated 2-domain form
of uPAR that lacks domain 1. The HKa-uPAR interaction was blocked by VN
and also heparin, whereas uPA had no inhibitory effect (Figure 6B). The
binding of HKa to uPAR could be attributed to domain 5, because the
recombinant GST-D5 but not GST-D3 or GST-D6 inhibited the interaction
(data not shown).
View larger version (20K):
[in this window]
[in a new window]
| Fig 5.
The effect of different kininogen forms on the
binding of 125I-VN to immobilized uPAR.
The binding of 125I-VN to immobilized uPAR together
with 20 nmol/L uPA and 50 µmol/L ZnCl2 was studied in the
absence or presence of HK (open circles), HKa (filled circles), domain
3 (open squares), domain 5 (filled squares), or domain 6 (open
triangles). Specific binding is expressed as percentage of control,
which represents the binding of 125I-VN to uPAR in the
absence of uPA. Data are mean ± SEM (n = 3) of a typical
experiment, and similar results were obtained in 3 separate
experiments.
|
|
View larger version (24K):
[in this window]
[in a new window]
| Fig 6.
Binding of HK or HKa to immobilized uPAR.
(A) The binding of HK or HKa (each 2 µg/mL) to immobilized suPAR or
the truncated 2-domain uPAR (D2 + 3) (each 5 µg/mL) was studied in
the absence (filled bars) or presence (hatched bars) of 50 µmol/L
ZnCl2. Specific binding is shown as absorbance at 405 nm.
Data are mean ± SEM (n = 3) of a typical experiment,
and similar results were obtained in 3 separate experiments. (B) The
binding of 2 µg/mL HKa together with 50 µmol/L
ZnCl2 to immobilized suPAR was studied in the absence
() or presence of 10 µg/mL heparin, 20 µg/mL VN, or 50 nmol/L uPA. Specific binding is expressed as absorbance at 405 nm. Data
represent mean ± SEM (n = 3) of a typical experiment, and similar
results were obtained in 3 separate experiments.
|
|
Colocalization of VN and HK in sections of human atherosclerotic
plaques
Consequences for the above-mentioned molecular relations in
(patho)physiology depend on the availability of the different components at sites of tissue remodeling. As an example, the
colocalization of VN with PAI-1 or uPAR could be demonstrated in
diseased vessel sections (Lupu et al and Chavakis et al, unpublished
observations), implying direct functional interactions.
Double-labeling immunogold electron microscopy was performed in
sections from human atherosclerotic coronary arteries obtained from
cardiac surgery. VN was localized both bound to plasma membranes of
cells, particularly smooth muscle cells, as well as associated with
different fibrillar or amorphous structures of the extracellular
matrix. HK was found in similar locations, and frequently VN and HK
were localized in close proximity or direct apposition (Figure
7). These data indicate that the above-described close molecular interaction could also take place in
vivo.
View larger version (191K):
[in this window]
[in a new window]
| Fig 7.
Localization of vitronectin and kininogen in human
advanced atherosclerotic lesions by double-labeling immunogold electron
microscopy.
Vitronectin (10 nm gold particles, arrowheads) is detected both bound
to plasma membranes of smooth muscle cells (SMC) (A and B) as well as
in association with different fibrillar or amorphous extracellular
matrix structures (ECM) (A, B, and C). Kininogen, labeled by 15 nm gold
particles (arrows), was found in similar locations. Frequently, the 2 proteins were localized in close proximity, occasionally in direct
apposition, indicating close molecular interaction (arrows and
arrowheads); bars = 0.5 µm.
|
|
| |
Discussion |
Apart from its role as precursor of bradykinin that contributes
important vasodilator functions in vascular homeostasis, high molecular
weight kininogen exhibits antiadhesive properties38,39,55 especially in the 2-chain, kinin-free form (HKa). In the present study,
we continued to define the underlying mechanisms of the antiadhesive
function of HKa. The characterization of the binding interactions to
different adhesion receptors as well as to VN-rich matrices revealed
that HKa is responsible for the disruption of uPAR and
v3 integrin-dependent cellular contacts. HKa was
shown to directly bind to VN and uPAR, to abrogate endothelial and
leukocytic cell adhesion, and thereby could play a regulatory role in
tissue remodeling of the vasculature. Binding of HKa to VN was blocked by recombinant uPAR and heparin as well as by active PAI-1 but not by
cRGDfV, indicating that HKa does not interfere with the cell attachment
sequence of VN but competes with PAI-1 or uPAR for binding to the amino
terminal "somatomedin B" domain of VN, which is proximal to the
integrin recognition motif.56 This hypothesis is also
strengthened by the fact that the kininogen-VN interaction was blocked
by a GST-fusion peptide consisting of the amino terminal portion of VN
(amino acids 1-77). The antiadhesive function of HKa is particularly
expressed in the His-Gly-Lys-rich domain 5, which contains the major
cell binding domain and resembles the activity of PAI-1 as a prominent
counter-adhesive factor in VN-dependent cell adhesion mediated by both
integrins and uPAR.
Our observations further define the requirements for this function of
HKa, conferred by its domain 5. HKa or domain 5 but not domains 3 or 6 inhibited the v3 integrin-dependent adhesion of
endothelial cells to VN but did not promote detachment of cells that
had already adhered to VN. HK/HKa did not block endothelial cell
adhesion to fibronectin and, in contrast to Asakura and
coworkers,38 we could not demonstrate HKa-dependent
blockade of adhesion of endothelial cells to FBG, because HKa did not
directly bind to the v3 integrin or this integrin
ligand (data not shown). However, HKa preincubated onto FBG-coated
surfaces prior to the adhesion step resulted in partial inhibition of
cell adhesion. This was due to the Vroman effect53 namely,
the displacement of FBG from the plate by HKa. This effect did not take
place when the surface was coated with VN, which is in accordance with
the known "resistance" of VN to the Vroman effect.54
In addition, HKa or domain 5 completely abolished the uPAR-dependent
adhesion to VN of differentiated U937 myelomonocytic cells as well as
of uPAR-transfected BAF-3 cells. As a consequence and in contrast to
the effect on integrin-mediated adhesion with endothelial cells, HKa
promoted the detachment of adherent U937 cells, because HKa competed
with both uPAR and the substrate VN. Thus, the inhibitory effect of HKa
appears to be specific for VN-rich tissue matrices such as in
provisional wounds and can be attributed to direct blockade of the
substrate and of uPAR.
Instantaneously, HKa was antiadhesive when added together with the
cells or when preincubated onto the VN substrate, whereas HK, which
does not bind VN, was antiadhesive after a lag-phase only in the first
case. These observations can be best explained by the fact that HK is
converted to HKa by proteases in the cell cultures to become a potent
antiadhesive factor, and data supporting this hypothesis were
presented. Cell surface-dependent conversion of HK to HKa was recently
described,57 implying that HK itself appears to be required
for prekallikrein activation and thereby promotes its own conversion
into HKa.
The consequences of the antiadhesive properties of kininogen are
several. After tissue damage, leukocytes are recruited to the injured
site, and neutrophil and monocyte adhesion to a provisional matrix is
pivotal for this process. In the inflamed or injured tissue, the
release of bradykinin from HK is enhanced, which also leads to a
localized infiltration of additional inflammatory cells. Moreover,
through inhibition of v integrin-dependent adhesion, HKa could
contribute to angiogenesis-regulating activities, because the
v3 integrin-VN interaction appears to be crucial
during neovascularization in different tissues.58 Peptides
derived from kininogen's domain 5 inhibit new vessel formation in the chicken chorioallantoic membrane,59 and work is in progress to define the role of HKa or fragments thereof in angiogenic processes in malignancies or in diabetic retinopathy.
The binding of HKa to both uPAR and VN on the cell surface may
approximate kallikrein and prourokinase, thereby leading to potent
plasmin formation. In addition, HKa binds directly to
plasminogen,60 which could further amplify plasmin
generation. Indeed, we observed colocalization of VN and kininogen at
sites of tissue remodeling such as in the vasculature and, by
immunogold electron microscopy, HK/VN-colocalization was demonstrated
in human atherosclerotic coronary arteries. These data indicate that
the described interactions between kininogen and VN may take place in
vivo. Depending on its local concentration, however, HKa could also
interfere with the assembly of the ternary uPA/uPAR/VN complex on the
surface of vascular cells or the extracellular matrix, which
augments plasmin generation.61 These interactions not only
define HKa as an intrinsic regulator of the fibrinolytic system but,
through its functions as antiadhesive component, HKa also may play a
regulatory role in angiogenesis, atherogenesis, or cancer metastasis.
Prior to the findings that HKa binds to VN and uPAR, other kininogen
binding proteins were identified that include cellular receptors such
as Mac-1 on neutrophils33 or glycoprotein Ib on unactivated
platelets62 as well as cell-associated thrombospondin on
activated platelets.63 Moreover,
cytokeratin-134 and gC1qR were identified as binding
proteins for HK on endothelial cells,35,36 the latter also
serving as binding protein for multimeric VN.64 Interestingly, VN is found on the surface of several other cell types
when cultivated in serum and can possibly account for an appreciable
degree of binding sites for kininogen and other cellular ligands.20 In particular, specific binding of both VN and
kininogen tp various bacterial strains could play a role in the
infection process and the host response toward bacterial
entry.65,66
Finally, recent studies with VN knockout mice67 and with
kininogen-deficient rats68 indicated a similar
phenotype in both cases that was more sensitive to thrombosis. Thus,
the proposed antithrombotic properties of HK and its fragments could
be mediated at least in part by interactions with VN,
because loss of VN in mice is expected to influence the functional
status of its ligands such as PAI-1 and kininogen as well. Because
several ligands of VN exhibit opposing functional activities in
hemostasis and cell adhesion, a detailed dissection of transgene animal
models can lead to further information on the pathophysiologic role of
this multifunctional adhesive protein.
| |
Acknowledgments |
The excellent technical assistance of U. Schubert and T. Schmidt is
gratefully acknowledged. We appreciate the contribution of Dan Johnson
and Dr Yen Lin in the preparation and characterization of the
recombinant HK domains and of Thomas Renné for providing monoclonal and polyclonal antibodies to HK. We also acknowledge the
generous gift of reagents from Drs H. Roger Lijnen, Gunilla Hoyer-Hansen, and Niels Behrendt.
| |
Footnotes |
Submitted October 1, 1999; accepted March 1, 2000.
This work is part of the MD/PhD thesis of T.C. at the Institute for
Biochemistry, Medical Faculty, Justus-Liebig-University, Giessen,
Germany. Supported in part by a grant (Pr 327/1-4) from the Deutsche
Forschungsgemeinschaft (Bonn, Germany) to K.T.P. and by National
Institutes of Health grants HL56914 and CA63938 to R.W.C.
Reprints: Klaus T. Preissner, Institut für Biochemie,
Fachbereich Humanmedizin, Justus-Liebig-Universität,
Friedrichstrasse 24, D-35392 Giessen, Germany; e-mail:
klaus.t.preissner{at}biochemie.med.uni-giessen.de.
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.
| |
References |
1.
Pöllänen J, Stephens RW, Vaheri A.
Directed plasminogen activation at the surface of normal and malignant cells.
Adv Cancer Res.
1991;57:273[Medline]
[Order article via Infotrieve].
2.
Lin CQ, Bissell MJ.
Multi-faceted regulation of cell differentiation by extracellular matrix.
FASEB J.
1993;7:737[Abstract].
3.
Carlos TM, Harlan JM.
Leukocyte-endothelial adhesion molecules.
Blood.
1994;84:2068[Abstract/Free Full Text].
4.
Gailit J, Clark RAF.
Wound repair in the context of extracellular matrix.
Curr Opin Cell Biol.
1994;6:717[Medline]
[Order article via Infotrieve].
5.
Preissner KT, Pötzsch B.
Vessel wall-dependent metabolic pathways of the adhesive proteins, von-Willebrand-factor and vitronectin.
Histol Histopathol.
1995;10:239[Medline]
[Order article via Infotrieve].
6.
Loridon-Rosa B, Vielh P, Cuadrado C, Bur |
Top
Abstract
Introduction