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Blood, Vol. 93 No. 8 (April 15), 1999:
pp. 2605-2616
Identification and Characterization of Endothelial Glycoprotein Ib
Using Viper Venom Proteins Modulating Cell Adhesion
By
Li Tan,
M. Anna Kowalska,
Gabriel M. Romo,
Jose A. Lopez,
Zbigniew Darzynkiewicz, and
Stefan Niewiarowski
From the Department of Physiology, and Sol Sherry Thrombosis Research
Center, Philadelphia, PA; the Division of Hematology and Oncology,
Baylor College of Medicine and Veterans Administration Medical Center,
Houston, TX; and The Cancer Research Institute, New York Medical
College, Valhalla, NY.
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ABSTRACT |
The expression and function of a glycoprotein Ib (GPIb) complex on
human umbilical vein endothelial cells (HUVECs) is still a matter of
controversy. We characterized HUVEC GPIb using viper venom proteins:
alboaggregins A and B, echicetin, botrocetin, and echistatin. Echicetin
is an antagonist, and alboaggregins act as agonists of the platelet
GPIb complex. Botrocetin is a venom protein that alters von Willebrand
factor (vWF) conformation and increases its binding affinity for the
GPIb complex. Echistatin is a disintegrin that blocks  v 3.
Echistatin, but not echicetin, inhibited the adhesion to vWF of Chinese
hamster ovary (CHO) cells transfected with v3. We found the
following: (1) Binding of monoclonal antibodies against GPIb to
HUVECs was moderately increased after stimulation with cytokines and
phorbol ester. Echicetin demonstrated an inhibitory effect. (2) Both
echicetin and echistatin, an v3 antagonist, inhibited the
adhesion of HUVECs to immobilized vWF in a dose-dependent manner. The
inhibitory effect was additive when both proteins were used together.
(3) Botrocetin potentiated the adhesion of HUVECs to vWF, and this
effect was completely abolished by echicetin, but not by echistatin.
(4) CHO cells expressing GPIb/IX adhered to vWF (in the presence
of botrocetin) and to alboaggregins; GPIb was required for this
reaction. Echicetin, but not echistatin, inhibited the adhesion of
cells transfected with GPIb/IX to immobilized vWF. (5) HUVECs
adhered strongly to immobilized vWF and alboaggregins with extensive
spreading, which was inhibited by LJ1b1, a monoclonal antibody against
GPIb. The purified v3 receptor did not interact with the
alboaggregins, thereby excluding the contribution of v3 in
inducing HUVEC spreading on alboaggregins. In conclusion, our data
confirm the presence of a functional GPIb complex expressed on HUVECs
in low density. This complex may mediate HUVEC adhesion and spreading
on immobilized vWF and alboaggregins.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
VON WILLEBRAND factor (vWF) secreted by
endothelial cells is an important component of the extracellular matrix
maintaining adhesion of these cells. It is well established that the
interaction of vWF with human umbilical vein endothelial cells (HUVECs)
is mediated by  v3 integrin, a constitutively expressed protein on
the membranes of these cells.1,2 There have also been reports suggesting that vWF interaction with HUVECs could be partially mediated by an endothelial glycoprotein Ib (GPIb) complex; however, this is still controversial. Several investigators reported
immunoreactive GPIb in HUVECs.3-5 Konkle et al6
identified endothelial GPIb in tonsil sections by an immunochemical
method and subsequently isolated GPIb cDNA clones from HUVECs.
Furthermore, Beacham et al5 showed that the binding of vWF
to HUVECs could be inhibited by monoclonal antibodies (MoAbs) directed
against GPIb. Wu et al7 demonstrated that the full
GPIb/IX/V complex is expressed on the HUVEC surface. It has been shown
that before cell harvest, combined treatment with interferon-
(IFN-) and tumor necrosis factor- (TNF-) upregulates the
synthesis and surface expression of GPIb on HUVECs.5-8
Thus, both v31,2,9 and GPIb5 have been
shown to play a role in mediating HUVEC adhesion to vWF. Moreover,
Beacham et al10 also found an increased biosynthesis of
GPIb in HUVECs subjected to shear stress, a condition that can be found
in vivo in stenosed arteries. Other researchers have found
that GPIb participated in HUVEC binding to sickle cell
erythrocytes.11 Recently, Bombeli et al12 have
shown that endothelial GPIb participates in platelet bridging to
endothelial cells.
It should be noted that the biosynthesis of a GPIb complex in HUVECs
has been questioned in some recent studies. Perrault et
al13 could not identify GPIb mRNA in HUVECs by Northern
blot analysis and they could not confirm protein expression on the surface of these cells by immunologic techniques. Zieger et
al14 compared clone cDNA of GPIb precursors from human
erythroleukemia cell lines exhibiting megakaryocytic properties with
the cDNA of putative GPIb precursors from endothelial cells. They
proposed that the 45-kD protein identified as an endothelial GPIb by
Kelly et al15 may actually belong to an entirely different
class of proteins. They suggested that endothelial GPIb "results
from the incomplete polyadenylation" of an upstream gene and read
through transcription into the GIPb gene.14 In his recent
review, Ware16 suggested that GPIb gene expression in
endothelial cells was low and might require very sensitive methods to
detect. Expression of GPIb in HUVECs may be caused by the leaky
activity of a promoter typically expressed in megakaryocytic cell
lines. According to Ware,16 there are no convincing data
supporting the presence of a functional GPIb/IX/V complex anywhere
other than on the surface of megakaryocytes and platelets.
In the current study, we used the following viper venom proteins:
alboaggregin A, alboaggregin B (also called big alboaggregin with a
molecular weight of 50 kD, and small alboaggregin with a molecular
weight of 25 kD),17,18 echicetin,19-21
botrocetin,22,23 and echistatin24 for
identification and functional characterization of the endothelial GPIb
complex. Alboaggregins and echicetin compete with vWF in its binding to
the platelet GPIb complex. Botrocetin22,23 modifies vWF,
increasing the binding affinity of vWF for the platelet GPIb complex.
Echicetin19 acts as a potent inhibitor of platelet agglutination induced by bovine vWF or by human vWF and a cofactor, either ristocetin or botrocetin. Alboaggregins A and B, on the other
hand, induce agglutination of formalin-fixed platelets in the absence
of any cofactors.17,25-27 Alboaggregin A, acting on the
GPIb complex, also induces calcium-dependent signals for platelet activation, leading to platelet aggregation and the release
reaction.26,27 The platelet-activating ability of
alboaggregin A could be blocked by echicetin and by MoAbs that are
specific for the vWF binding site on the N-terminal of GPIb, but not
by antibodies that recognize other regions on the GPIb
complex.26
Alboaggregins and echicetin are highly homologous to each
other26 and to a number of other viper venom proteins,
including factor IXa/Xa binding protein,28
botrocetin,22,23 agkicetin,29 flavocetins,30 tokaracetins,31 and
yoshitobin.32 Most proteins in this group are
heterodimers,33 except for alboaggregin A, which has a
tetrameric structure. Each subunit of these proteins constitutes a
domain structure known as a carbohydrate recognition domain (CRD). This
structure was first identified as the minimum functional motif of
Ca2+-dependent animal lectins.34,35 Echistatin
is a disintegrin inhibiting IIb3 and v3.24,36
Our study using alboaggregins, echicetin, and botrocetin confirms the
presence of a GPIb complex on endothelial cells and suggests that it
may play a role, in concert with v3 receptors, in the interaction
of these cells with vWF leading to cell adhesion and spreading. We also
found that selective stimulation of HUVEC GPIb by alboaggregins results
in extensive cell spreading, suggesting cytoskeletal mobilization. In
addition, we found that Chinese hamster ovary (CHO) /IX cells and
HUVECs share similar patterns of interaction with vWF and viper venom
proteins, except that the spreading of transfected cells on immobilized
ligands was minimal.
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MATERIALS AND METHODS |
Materials.
Lyophilized snake venoms of Trimeresurus albolabris and
Echis carinatus were purchased from Latoxan (Rosans, France).
Human vWF, fibronectin, and vitronectin were purchased from Calbiochem (San Diego, CA). TNF- was purchased from Boehringer Mannheim (Indianapolis, IN), and IFN- was from GIBCO Life Technologies (Grand
Island, NY). LJIb1, an anti-GPIb MoAb that blocks vWF binding, was
kindly provided by Dr Z.M. Ruggeri (Scripps Research Institute, La
Jolla, CA). Other monoclonal GPIb binding antibodies, SZ2 and WM23,
and botrocetin were kindly provided by Dr M.C. Berndt (Baker Research
Institute, Prahran, Victoria, Australia). A MoAb recognizing v3
integrin, LM609, was purchased from Chemicon (Temecula, CA), and the
anti-v3/IIb3 integrin antibody, 7E3, was kindly provided by
Dr B.S. Coller (Mount Sinai Medical Center, New York, NY). C-18 reverse
phase columns were purchased from Vydac (Hesperia, CA). Ion exchange
Mono S and Mono Q HR 5/5 columns were from Pharmacia-LKB
(Uppsala, Sweden). Other chemicals were purchased from Fisher
Scientific (Pittsburgh, PA) and Sigma Chemical Co (St Louis, MO).
Purification of alboaggregins, echicetin, and echistatin.
Alboaggregin A and B were purified from the venom of Trimeresurus
albolabris according to a previously described
method.17,18 Echicetin and echistatin were purified from
the same sample of Echis carinatus venom. The venom was
dissolved in trifluoracetic acid and applied to a C18 column.
Protein-containing fractions were eluted with an increasing
acetonitrile gradient. Echistatin-containing fractions, eluted at 42%
acetonitrile, were collected and reloaded on reverse-phase
high-performance liquid chromatography (HPLC) using a
shallow gradient. Echicetin-containing fractions were eluted at about
70% acetonitrile on reverse-phase HPLC and were then purified by means
of cation-exchange chromatography.19 The purity of all
proteins was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with the Phast Gel System (Pharmacia) using 20% homogeneous gels and by determination of the
N-terminal sequence. Pooled pure fractions of these proteins were
further tested for their functional activities. Alboaggregins A and B
induced agglutination of formalin-fixed platelets with the same
potency. Alboaggregin A aggregated washed platelets in a dose-dependent
manner and this effect saturated at 20 nmol/L.27 At 200 nmol/L, echicetin completely inhibited the agglutination of
formalin-fixed platelets by alboaggregins and by vWF in the presence of
ristocetin. Echistatin, a disintegrin, inhibited ADP-induced platelet
aggregation in platelet-rich plasma (PRP) with an IC50 of
130 nmol/L, as previously described.37 Eristostatin
was obtained from Eristocophis macmahoni venom, as described by
McLane et al.37 To test viper venom proteins, we isolated
platelets by differential centrifugation as previously
described.38 Formalin-fixed platelets were prepared using
the method of Kirby.39
HUVEC culture.
HUVECs were isolated from freshly collected umbilical cords as
described by Jaffe et al40 and cultured in Dulbecco's
modified Eagle's medium (DMEM; GIBCO) containing 20% fetal calf
serum, 2 mmol/L penicillin/streptomycin and L-glutamine, 10 µg/mL
heparin, and 60 µg/mL endothelial cell growth supplement obtained
from porcine hypothalamus extract. The cells were incubated at 37°C in the presence of 5% CO2. HUVECs were passaged at a 1:3
ratio, and cultures from the third to sixth passages were used for flow cytometry or adhesion assays. Cell suspensions were obtained by washing
the cultures twice with cation-free Hank's Balanced Salt Solution
(HBSS) and then detaching the cells with HBSS containing 5 mmol/L EDTA,
100 µmol/L leupeptin, and 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF).
Stimulation of HUVECs with cytokines or phorbol myristate acetate
(PMA) treatment.
HUVECs were treated with combined IFN- and TNF-, as originally
described by Rajagopalan et al.41 Briefly, 10 ng/mL (final concentration) of IFN- was added to HUVECs at about 80% confluence and 72 hours before cell detachment. TNF- (100 U/mL) was added to
the culture medium 16 hours before cell detachment. The culture medium
was changed daily. Treatment with IFN- caused a characteristic elongation of the endothelial cells. The addition of TNF- resulted in an upregulation of intercellular adhesion molecule-1
(ICAM-1), which was confirmed by flow cytometry of the
cell suspension using an anti-ICAM-1 MoAb (kindly provided by Dr D. Mosser, Temple University, Philadelphia, PA).
PMA treatment has been reported to upregulate GPIb in cancer
cells.42 We therefore used a similar method to upregulate
the endothelial GPIb complex. In brief, 20 ng/mL of PMA was added to
the cell culture medium 24 hours before cell detachment. Resembling IFN-, PMA caused an elongation of individual cells. In addition, HUVECs exposed to PMA were packed more densely compared with
nonstimulated cells.
Transfected CHO cells.
CHO cells transfected with v3 receptors (VNRC3
cells)43 were kindly provided by Dr M.H. Ginsberg (Scripps
Research Institute, La Jolla, CA). Nontransfected CHO-K1 cells were
purchased from ATCC (Rockville, MD) and used as control cells. Both
VNRC3 and K1 cells were maintained in DMEM containing 10% fetal bovine
serum and supplemented with nonessential amino acids,
L-glutamine, and penicillin/streptomycin. CHO/IX cells
(CHO cells that stably express the entire GPIb/IX complex) and
CHO/IX (which express GPIb and GPIX) were maintained as
previously described.44 Briefly, CHO/IX cells were
grown in -minimal essential media (-MEM; GIBCO) containing 10%
fetal bovine serum and penicillin/streptomycin and supplemented with
geneticin, methotrexate, and hygromycin. The medium for CHO/IX cells
was supplemented only with geneticin and methotrexate. Confluent cells
were detached from plates with trypsin-EDTA for passaging or with
Versene (GIBCO) for flow cytometry and adhesion experiments to avoid
proteolysis of GPIb by trypsin. The cells were then resuspended in
-MEM containing 1% bovine serum albumin (BSA) for flow
cytometry or cell adhesion assays. Both VNRC3 cells and CHO/IX
cells were routinely checked by flow cytometry for surface expression
of the receptors. Specific MoAbs, LM609 and 7E3, were used to assess
the expression of v3. LJ1b1, SZ2, and WM23 were used to assess
the GPIb surface expression. The CHOIX cells were sorted
occasionally to retain a cell population with a high degree of receptor expression.
Cell adhesion.
Ninety-six-well microplates were coated with various concentrations of
proteins or antibodies in phosphate-buffered saline (PBS)
and left at 4°C overnight. The plates were then rinsed twice with
PBS and blocked with 3% BSA in PBS for at least 1 hour at 37°C.
After rinsing with PBS, the plates were ready for application of the
cells. HUVEC or CHO cell suspensions were prepared as described above,
the concentration was adjusted to 2 × 105 cells/mL,
and the suspensions were incubated in the absence or presence of inhibitors or antibodies for 15 minutes on ice before being
applied (at 100 µL/well) to the plates. The cells were incubated in
the microplates at 37°C in 5% CO2 for 2 hours.
Unattached cells were gently washed away with PBS. The attached cells
were then fixed for 30 minutes with 1% formaldehyde at room
temperature and stained with methylene blue according to Oliver et
al.45 The relative number of adherent cells was calculated
by lysing the stained cells with 50% ethanol and 50% hydrochloric
acid and then reading the absorbance at 630 nm on a microplate
autoreader (Bio-Tek Instruments, Winooski, VT). The percentage of cells
adhering to the plate was determined based on the linear relationship
between the absorbance reading and the number of cells counted in a
hemocytometer. The amount of nonspecific adhesion was determined by
using wells precoated with BSA only. In cell adhesion assays in which
an antibody was used as a substrate, mouse IgG was used as a control
substrate. The stained adherent cells were photographed using an
Olympus camera (Hitech Instruments Inc, Chester Pike,
Edgmont, PA) and Kodak T-Max 100 film (Eastman Kodak, Rochester,
NY). For fluorescence staining, the cells were applied to
chamber glass slides that has been precoated with various agents, fixed
with 1% formaldehyde, and dried with 100% ethanol. The slides were
stained with sulforhodamine for detection of proteins and
4,6-diamidino-2-phenylindole (DAPI) for detection of DNA. Photographs
were then taken under a fluorescence microscope.
Flow cytometry.
HUVECs and transfected CHO cells were prepared as described above and
the concentration was adjusted to 5 × 106 cells/mL.
Five hundred-microliter aliquots of each cell suspension were added to
several sample tubes. In the case of platelets, the concentration was
adjusted to 1 × 1010 platelets/mL, and 10 µL was
used in a final volume of 50 µL for each sample. The cells were then
preincubated with various proteins or antibodies for 15 minutes at room
temperature. MoAbs, prepared in PBS containing 1% BSA, were then added
to the cells for 1 hour on ice with occasional agitation. The cells
were then spun down, washed three times with PBS containing 1% BSA,
and incubated with fluorescein isothiocyanate (FITC)-conjugated goat
antimouse IgG for 1 hour on ice. After washing three times with PBS,
each cell sample was resuspended in 250 µL PBS and then fixed by the
addition of 150 µL of 3% formaldehyde. The samples were then
analyzed on a Coulter Epics Elite flow cytometer (Miami, FL). Debris
and dead cells were excluded by forward- and side-scatter gating.
Incubating the cells with FITC-labeled secondary antibody alone
assessed nonspecific binding.
Interaction of purified v3 receptor
with disintegrins and alboaggregin B.
v3 integrin was purified from VNRC3 cells as described by
Marcinkiewicz et al.46 In brief, cells were lysed by
octylglucoside. The cell lysates were then applied onto a
GRGDSPK-agarose column and receptor-containing fractions were eluted
with EDTA. Echistatin, eristostatin, or alboaggregin B were immobilized
on a 96-well enzyme-linked immunosorbent assay (ELISA)
plate in 0.05 mol/L bicarbonate buffer, pH 9.3, and by overnight
incubation at 4°C. Echistatin was used as a positive control. For a
negative control, we used eristostatin, a disintegrin that does not
interact with v3.35 The wells were blocked with 5%
nonfat milk in PBS containing 0.05% Tween. Purified v3 (0.7 µg/per well) was added to the wells and the plate was incubated for
30 minutes at 37°C. Binding of a rabbit polyclonal anti-v3
antibody (Chemicon, Temecula, CA) to the bound receptor was assessed
using a biotinylated goat antirabbit anti-body according to the Vecta
Stain ABC HRP kit (#PK-4001; Vector Laboratories, Burlingame, CA).
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RESULTS |
GPIb expression on the resting and activated HUVECs.
As shown in Fig 1, the binding of the
monoclonal anti-GPIb antibodies, LJ1b1 and SZ2, to nonstimulated
HUVECs was very low; however, it was significantly increased after
stimulation of HUVECs with IFN- and TNF- or with PMA. Echicetin
at concentrations greater than 100 nmol/L inhibited binding of these
antibodies to stimulated HUVECs (not shown). In both stimulated and
nonstimulated HUVECs, the binding of SZ2 to the cells was consistently
less pronounced than the binding of LJb1. Binding of antibody WM23 and
of nonimmune IgG to HUVEC was not significant (not shown). Both LJIb1
and SZ2 bound to washed platelets and to CHOIX cells, but not to
CHOIX cells (data not shown), confirming the specificity of these
antibodies.
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| Fig 1.
Binding of MoAbs against GPIb, LJ1b1, and SZ2 to the
suspensions of HUVECs. Resting HUVECs: After HUVEC detachment, 500 µL aliquots of 5 × 106 HUVECs/mL were
incubated with 2 µg/mL LJ1b1 or SZ2 for 1 hour on ice (shaded peak).
As a control, an aliquot of HUVECs was treated only with
FITC-conjugated goat-antimouse antibody (open peak). Flow cytometry was
conducted after washing with PBS as described in Materials and Methods.
HUVECs stimulated with cytokines: HUVECs were treated with IFN- and
TNF- before cell detachment as described by Konkle et
al.6 After HUVEC detachment, the experimental
procedure was the same as that described for the resting HUVECs. HUVECs
stimulated with PMA: The binding of LJ1b1 and SZ2 to HUVECs was
increased by pretreating HUVECs in culture with 20 ng/mL PMA for 24 hours. A maximal increase in binding was observed under these
conditions, although the increase was more pronounced for LJ1b1 than
for SZ2.
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The effect of viper venom proteins on the adhesion of HUVECs to
immobilized vWF.
Nonstimulated HUVECs adhered extensively to immobilized vWF and this
adhesion was enhanced by the presence of botrocetin during incubation.
However, cytokine-stimulated HUVECs adhered less extensively to
immobilized vWF than did resting HUVECs (not shown). This was probably
due to downregulation of v3.47 The experiments to be
described below were conducted with resting HUVECs in the presence or
absence of botrocetin.
We compared the effect of the disintegrin echistatin, an v3
inhibitor, and echicetin, a GPIb inhibitor, on the adhesion of HUVECs
to immobilized vWF (Fig 2A). Both proteins
inhibited adhesion in a dose-dependent manner with echistatin producing near complete inhibition at 200 nmol/L. Echicetin, on the other hand,
inhibited only about 35% of the adhesion at the same concentration. A
representative experiment shows that echistatin at 50 nmol/L and
echicetin at 200 nmol/L partially inhibited HUVEC adhesion to vWF and
that the effect of both inhibitors was additive (Fig 2B).
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| Fig 2.
(A) Effect of echicetin and echistatin on HUVEC adhesion
to immobilized vWF. HUVECs were preincubated with progressively
increasing concentrations of echicetin and echistatin (an v3
antagonist) for 15 minutes on ice before being applied to a 96-well
microplate previously coated with vWF as described in Materials and
Methods. OD readings of the lysed cells indicated that about 45% of
resting HUVECs adhered to immobilized vWF, and this value was used as
100% adhesion. ( ) Echicetin; ( ) echistatin. The data represent
the mean and SD of percentage of cell adhesion from four individual
experiments. (B) Additive inhibition of echicetin and echistatin on
HUVEC adhesion to immobilized vWF. HUVECs were preincubated with either
250 nmol/L echicetin (EC; lane 3), 50 nmol/L echistatin (ES; lane 4),
or both 250 nmol/L echicetin and 50 nmol/L echistatin (EC + ES; lane
5) and were then applied onto a vWF-coated plate. For a negative
control (lane 1), BSA-coated wells were used. Lane 2 represents HUVEC
adhesion to vWF in the absence of inhibitors.
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The addition of botrocetin, a venom protein that alters vWF
conformation (making it more accessible to GPIb) increased the adhesion
of HUVECs to vWF. Figure 3 shows that
echicetin (Fig 3A) abolished botrocetin-induced cell adhesion in a
dose-dependent manner, whereas echistatin (Fig 3B) did not. At a
concentration of 300 nmol/L, echicetin completely inhibited
botrocetin-enhanced adhesion of HUVECs to vWF. At concentrations
ranging from 20 to 100 nmol/L, echistatin inhibited, with an identical
pattern, the adhesion of HUVECs both to unmodulated vWF and to vWF
modulated by botrocetin. Botrocetin-enhanced cell adhesion was not
altered even in the presence of 100 nmol/L echistatin. These
experiments exclude a contribution by v3 to the
botrocetin-enhanced adhesion of HUVECs to vWF. This observation was
also confirmed with the use of MoAbs against GPIb and v3.
Table 1 shows that MoAbs directed against
v3 inhibited to the same extent HUVEC adhesion to vWF and to
botrocetin-treated vWF, whereas MoAbs against GPIb inhibited more
strongly HUVEC adhesion to botrocetin-treated vWF.
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| Fig 3.
Effect of echicetin and echistatin on botrocetin-enhanced
HUVEC adhesion to immobilized vWF. A 96-well microplate was coated with
vWF, incubated overnight at 4°C, and then blocked with 3% BSA.
HUVECs, after detachment, were applied to the microplate in the absence
() and presence () of 10 µg/mL botrocetin during the 2-hour
incubation period. Various concentrations of echicetin (A) and
echistatin (B) were added to the HUVECs before their application to the
plate. In (B), the difference between samples examined in the presence
and absence of botrocetin was significant at P < .05 for all
concentrations of echistatin studied. In (A) (for echicetin), this
statistically significant difference was not observed except for the
control sample.
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Table 1.
Inhibition of HUVEC Adhesion to Immobilized vWF in the
Presence and Absence of Botrocetin by MoAbs Against GPIb (LJIb1
and SZ2) and Against v3 (LM609 and 7E3)
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The experiments described above indicate that, besides v3, an
RGD-dependent receptor, GPIb, an RGD-independent receptor, mediates
HUVEC adhesion to vWF in the substratum.
vWF and viper venom protein interactions with HUVECs and CHO cells
transfected with GPIb/IX or
v3.
In further experiments, we compared the adhesion of HUVECs, CHOIX
cells, and VNRC3 cells to various substrates. HUVECs adhered comparably
to immobilized vWF, alboaggregins, and echicetin. The adhesion of
HUVECs to vWF did not require botrocetin
(Fig 4A). CHO/IX cells adhered to
immobilized vWF but only in the presence of botrocetin. Adhesion of
these cells to alboaggregins and echicetin did not require botrocetin
(Fig 4B). Control (nontransfected) CHO cells and CHO/IX cells did
not adhere to any of these substrates, indicating that GPIb is
critical for the interaction of CHO/IX cells with these
substrates. This finding is consistent with the previous observation
that GPIb was required for the agglutination of CHO/IX cells
in the presence of vWF.46
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| Fig 4.
(A) HUVEC adhesion to immobilized ligands. A 96-well
microplate was coated with various ligands of 1% BSA (lane 1), 15 µg/mL vWF (lane 2), 50 µg/mL echicetin (lane 3), 50 µg/mL
alboaggregin A (lane 4), and 50 µg/mL alboaggregin B (lane 5)
overnight at 4°C. After blocking with 3% BSA, 100-µL aliquots of
2 × 105 HUVECs/mL were added to each well. After 2 hours
of incubation at 37°C in the presence of 5% CO2, the
attached cells were fixed and stained with methylene blue. The cells
were then lysed and an OD reading was taken. The data represent the
mean and SD of OD readings from five individual experiments. (B)
Adhesion of transfected CHO cells to immobilized ligands. A 96-well
microplate was coated with various ligands (BSA [lane 1], vWF [lane
2], echicetin [lane 3], alboaggregin A [lane 4], and alboaggregin
B [lane 5]) and blocked with BSA. Aliquots (100 µL) of CHOIX
cells at 2 × 105 cells/mL were added to each well. Wells
coated with vWF received cells that had been treated with 10 µg/mL
botrocetin. Cells were incubated on the plate for 2 hours at 37°C
in the presence of 5% CO2. CHOIX cells and control
(untransfected) CHO cells showed baseline adhesion to immobilized vWF
that was comparable to the adhesion level of CHOIX cells to BSA
(lane 1).
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To confirm the specificity of echistatin and echicetin, we tested their
inhibitory effect on CHO/IX cells and VNRC3 cells adhering to
vWF. Echicetin (Fig 5A) had a profound
inhibitory effect on the adhesion of CHO/IX cells to vWF but
little effect on the adhesion of VNRC3 cells to immobilized vWF. At a
concentration of 50 nmol/L, echistatin (Fig 5B) inhibited the adhesion
of VNRC3 cells to vWF by approximately 80%. This percentage of
inhibition was much higher than that seen (<50%) when echistatin was
used as an inhibitor of HUVEC adhesion to
vWF. Echistatin had no significant effect
on the adhesion of CHOIX cells to vWF
(Fig 5B).
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| Fig 5.
Effect of echicetin (A) and echistatin (B) on CHO cells
transfected with either v3 or GPIb complex. A 96-well microplate
was coated with vWF. Confluent CHO cells transfected with either
v3 () or GPIbIX () were detached with Versene (GIBCO)
for 30 minutes at 37°C. After washing with PBS, the cells were
resuspended in PBS containing 1 mmol/L CaCl2 and
MgCl2. The concentration of the cell suspension was
adjusted to 2 × 105 cells/mL. Varying concentrations of
echicetin and echistatin were added to 100 µL aliquots of the cell
suspension 15 minutes before 2 hours of incubation on the plate.
Unbound cells were gently washed away with PBS. The adhesion procedure
was followed according to the legend of Fig 2.
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| Fig 6.
HUVECs adherent to various substrata examined by light
microscopy. For the adhesion assay, refer to the legend for Fig 4A.
Whenever inhibitors were used, HUVECs were preincubated with either
LJIb1 or echicetin for 15 minutes on ice before being applied, together
with the inhibitors, onto the precoated plate. This figure shows the
morphology of HUVECs adherent to different ligands observed under light
microscopy. HUVECs adherent to vWF (A), alboaggregin A (B), and
alboaggregin B (D) showed extensive cell spreading, whereas cells
attached to echicetin (C) showed cell clumping without prominent cell
spreading. The presence of 50 µg/mL LJIb1 (E) or 500 nmol/L echicetin
(F) demonstrated inhibition of cell adhesion and cell spreading on
alboaggregin B.
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| Fig 7.
HUVECs adherent to various substrata examined by
fluorescence microscopy. HUVECs were allowed to adhere to different
ligands on chamber slides. After fixation with formaldehyde and
staining with sulforhodamine and DAPI, the slides were examined by
fluorescence microscopy. Details of the nuclei and the cytoplasm were
better visualized using this dual stain. HUVECs adherent to vWF (A) and
alboaggregin B (B) demonstrated extensive cell spreading, whereas
HUVECs adherent to echicetin (C) and echistatin (D) showed minimal cell
spreading.
|
|
Effect of GPIb-binding proteins on HUVEC spreading.
HUVECs adhered comparably to immobilized vWF, echicetin, and
alboaggregins A and B as assessed by measuring the optical density of
lysed cells (Fig 4A). HUVECs adhered and spread extensively on both
immobilized vWF and alboaggregins as visualized by methylene blue
staining (Fig 6). Cells adhering to echicetin clumped with little, if
any, spreading. There was minimal adhesion to immobilized BSA or mouse
IgG (not shown). In the presence of 50 µg/mL of LJIb1, the adhesion
of HUVECs to alboaggregin A was decreased by 55% as determined by OD
reading, and spreading was also significantly inhibited. Figure 7 shows
adherent HUVECs stained with sulforhodamine and DAPI. This double
fluorescence staining visualizes in greater detail the cytoplasmic
alterations of adhering cells. There were well-defined surface
protrusions on HUVECs adherent to vWF and alboaggregins, but no such
protrusions were visible on cells adherent to echicetin and echistatin.
Because alboaggregins induced such dramatic HUVEC cell spreading and
v3 is the most abundant receptor on HUVECs mediating cell
attachment and cell shape change, we studied the interaction of
purified v3 receptor with alboaggregins, echistatin, and eristostatin (as a negative control) to evaluate the direct involvement of v3 in this adhesive phenomenon. Purified v3 bound to
immobilized echistatin in a dose-dependent manner but not to
alboaggregin B (a similar finding with alboaggregin A; data not shown)
or eristostatin (Fig 8). This experiment
strongly suggests that alboaggregins, by interacting with the
endothelial GPIb complex, might induce the cytoskeletal reorganization
that is necessary for cell spreading. It is noteworthy that there was
no significant spreading of CHOIX cells on immobilized vWF,
alboaggregins, or echicetin at least after 2 hours of incubation,
suggesting that the cellular signaling apparatus that is necessary for
GPIb-mediated cell activation in HUVECs is not available.
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| Fig 8.
Binding of purified v3 to immobilized alboaggregin
B, echistatin, and eristostatin. A 96-well microplate was coated with
various concentrations of alboaggregin B, eristostatin, and echistatin.
The plate was then blocked with PBS containing 0.05% Tween (TPBS) and
5% nonfat dry milk for at least 1 hour. Seven hundred nanograms of
purified v3 receptors was then added to each well and the plate
was then incubated for 30 minutes at 37°C. Extensive washing with
TPBS followed. Polyclonal rabbit IgG against v3 was used as
primary antibody. After 1 hour of incubation at 37°C and subsequent
washing, a biotinylated goat antirabbit IgG was used as secondary
antibody. () v3 binding to immobilized echistatin; ( )
binding to immobilized eristostatin; () binding to immobilized
alboaggregin.
|
|
| |
DISCUSSION |
The present study indicates that a GPIb complex is expressed on the
surface of HUVECs in low quantities but in sufficient quantities to
mediate the interaction of these cells with vWF. The viper venom
proteins, alboaggregins, and echicetin proved to be useful in
characterizing the endothelial GPIb complex because, in contrast to
vWF, they interact with platelet GPIb/IX without the need for
modulators. Ristocetin, or the viper venom protein botrocetin, are
required for the interaction of vWF with the platelet GPIb complex in
vitro. Alboaggregins and echicetin, on the other hand, bind platelet
GPIb/IX directly without the assistance of any cofactor. Our data
indicate that the same is true for CHO/IX cells. These cells
required botrocetin for adhesion to immobilized vWF, but they adhered
extensively to immobilized alboaggregins and echicetin without the
presence of any cofactor (Fig 4B).
The following evidence indicates that a GPIb/IX complex is expressed on
the surface of HUVECs and mediates, together with v3, HUVEC
interaction with vWF. (1) HUVECs bound two MoAbs, LJ1b1 and SZ2, that
recognize distinct epitopes in the GPIb N-terminus. (2) The adhesion
of HUVECs to immobilized vWF was inhibited by echistatin, an v3
antagonist, and by echicetin, a GPIb antagonist, in a dose-dependent
manner. Furthermore, the inhibitory effect was additive. (3) The
adhesion of HUVECs to vWF was enhanced by botrocetin, which forms a
complex with vWF and makes it more accessible to GPIb. This enhanced
adhesion was inhibited by echicetin but not by echistatin.
Botrocetin-enhanced adhesion was also inhibited by MoAbs against GPIb
but not by MoAbs against v3. (4) HUVECs adhered to immobilized
alboaggregins and subsequently spread. Cell spreading was inhibited by
the GPIb MoAb LJ1b1. Unlike vWF, alboaggregin A does not contain an
v3 binding site. Nevertheless, alboaggregin A exhibits the same
activity as vWF does in the presence of its cofactor botrocetin.
Our study is consistent with the results of Beacham et
al,8 Konkle et al,6 and Wu et
al.7 It is at variance with Perrault et al,13
who could not detect GPIb/IX on their preparations of HUVECs and
reported that this putative endothelial complex was irrelevant for cell
attachment to vWF. The reasons for these differences are not clear but
may relate to differences in cell passage number, cell preparation, or
culture techniques. Interindividual variations may also exist in the
level of GPIb expression on HUVECs, an issue that has not been
examined systematically.
The platelet GPIb complex has been extensively investigated and its
structure and function have mostly been identified.48-50 There are several differences between the platelet and the endothelial GPIb complexes. Wu et al,7 using radiolabeled MoAb L1Jb1,
found that endothelial cells and platelets each express 330,000 and 35,000 copies of a GPIb/IX complex, respectively. However, the percentage of this MoAb bound at saturation to endothelial cells was
low. They also found that a number of MoAbs reacting with platelet
GPIb/IX did not recognize the endothelial counterpart of this complex,
and we confirmed this observation in flow cytometry experiments using
the various MoAbs, LJIb1, SZ2, and WM23, of which only LJIb1 showed
pronounced binding.
The differences between platelet and endothelial GPIb may relate to
differences in posttranslational modifications in platelets and HUVECs,
respectively.48-50 WM23 recognizes an epitope in the GPIb macroglycopeptide, a region that undergoes extensive
O-glycosylation. Because protein glycosylation is a cell-type-specific
process, the epitopes for this antibody may vary with the cell type
studied. This may be a reason why WM23 does not recognize GPIb on
endothelial cells. Similarly, SZ2 binds to a region of GPIb that
undergoes posttranslational sulfation of tyrosine and the differences
in binding may relate to differences in protein sulfation in platelets and HUVECs.49,50 Despite these differences in endothelial
and platelet complexes, there are some common mechanisms in their interaction with vWF. Botrocetin enhanced vWF binding to both platelet
and endothelial GPIb. It is well established that the vWF binding site
on platelet GPIb is located on GPIb.48-53
Our studies with CHO/IX cells show that GPIb is also required
for the adhesion of these cells to immobilized alboaggregins and
echicetin. The LJ1b1 binding site is also located on GPIb. We
conclude that endothelial GPIb interacts specifically with
alboaggregins, echicetin, and vWF. It is noteworthy that echicetin acts
as an antagonist of both platelet and endothelial cell GPIb complexes,
whereas alboaggregins act as agonists. In platelets, echicetin inhibits vWF-induced agglutination, whereas alboaggregins induce platelet agglutination and aggregation. In endothelial cells, echicetin inhibited cell adhesion to vWF and HUVECs adherent to echicetin did not
spread. In contrast, HUVECs adherent to alboaggregins spread
extensively. Thus, the ability of GPIb-binding proteins to promote
HUVEC spreading correlates with their ability to activate platelets.
Results of our study on the inhibitory effect of echicetin and
echistatin on the adhesion of CHO/IX and VNRC3 cells to vWF were
in general agreement with the contention that both the GPIb binding
site (the A1 domain) and the v3 binding site (the RGD sequence in
the C1 domain) of vWF participate in the interaction between vWF and
endothelial cells.
Dong et al54 could not detect any significant intracellular
signaling after the interaction of vWF with CHO/IX cells. In our
hands, CHO/IX cells did not spread on immobilized vWF or
alboaggregins during the 2-hour incubation period. In contrast, HUVEC
adherence and spreading on alboaggregins was very rapid, suggesting
that the endothelial GPIb complex may confer a signal resulting in
reorganization of cytoskeletal structures in endothelial cells.
In conclusion, our data confirm the presence of GPIb on the surface of
the endothelial cells (Fig 9). Although
this complex is expressed in low density, it is definitely functional.
We propose that it interacts with the A1 domain of vWF, and this
interaction proceeds in parallel with v3 binding to the C1 domain
of vWF. Echicetin and MoAbs LJIb1 and SZ2 inhibit GPIb interaction with the A1 domain of vWF, and botrocetin potentiates this interaction. Echistatin and MoAbs LM609 and 7E3 inhibit interaction between v3
and the C domain of vWF. Figure 9 also shows the
possibility that endothelial GPIb and v3 may interact
independently of each other with alboaggregins and vitronectin.
Nevertheless, the cooperation of both v3 and GPIb receptors in
endothelial cell interaction with vWF and other matrix components may
significantly contribute to efficient hemostasis and tissue repair.
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| Fig 9.
Interactions of ligands with endothelial cell receptors.
This figure shows a putative scheme of the interaction between v3
and GPIb complex with vWF, vitronectin, and alboaggregins. The GPIb
complex is composed of seven molecules. One molecule of GPV is flanked
symmetrically by two molecules of GPIX, GPIb, and GPIb. vWF
interaction with v3, mediated by the C1 domain, is inhibited by
echistatin and MoAbs LJ1b and SZ2. Botrocetin alters the conformation
of the A1 domain of vWF so that it can interact with GPIb complex. This
interaction can be blocked by echicetin and by anti-GPIb MoAbs LJIb
and SZ2. The GPIb complex and v3 cooperate in binding vWF, but
each of these two receptors may independently bind specific ligands.
Alboaggregins bind to the GPIb complex, and vitronectin binds to
v3. Each of these interactions leads to cell spreading and
cytoskeletal mobilization.
|
|
| |
ACKNOWLEDGMENT |
The authors thank Dr Michael C. Berndt for carefully reading the
manuscript, Dr Dorothy Beacham and Dr Cezary Marcinkiewicz for helpful
discussions, John Gibas for performing flow cytometry, and Dr Yuqing
Wang and Mariola Marcinkiewicz for their help in preparing cultured cells.
| |
FOOTNOTES |
Submitted July 15, 1998; accepted November 23, 1998.
Supported in part by National Institutes of Health Grant No. HL 45486 (to S.N.); a Training in Thrombosis and Hemostasis Grant No. T3 HL00777
(to L.T.); a grant from the American Heart Association Grant-in-Aid,
Southeastern Pennsylvania affiliate (to S.N.); and an Established
Investigator grant from the American Heart Association (96002750; to
J.A.L.). The manuscript represents part of the dissertation (of L.T.)
submitted to Temple University as partial fullfillment for the
requirement of the degree of Doctor of Philosophy.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Stefan Niewiarowski, PhD, Department of
Physiology, Temple University School of Medicine, 3400 N Broad St, OMS
200, Philadelphia, PA 19140; e-mail, stni{at}astro.ocis.temple.edu.
| |
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TOP
ABSTRACT
INTRODUCTION