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Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3684-3693
Shear-Dependent Rolling on von Willebrand Factor of Mammalian Cells
Expressing the Platelet Glycoprotein Ib-IX-V Complex
By
Becky J. Fredrickson,
Jing-Fei Dong,
Larry V. McIntire, and
José A. López
From the Cox Laboratory for Biomedical Engineering, Rice University,
Houston, TX; and the Department of Medicine, Division of
Hematology/Oncology, and the Department of Molecular and Human
Genetics, Baylor College of Medicine and Veterans Affairs Medical
Center, Houston, TX.
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ABSTRACT |
Mural thrombi form on exposed arterial subendothelium by a two-step
process of platelet adhesion and aggregation. At high shear stresses
such as are found in stenotic arteries, both steps are mediated by von
Willebrand factor (vWF). Platelets initially adhere on vWF affixed to
the subendothelial matrix through the glycoprotein (GP) Ib-IX-V
complex. To examine the role of the GP Ib-IX-V complex under dynamic
conditions, we modeled initial platelet adhesion at shear stresses
ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides,
mammalian cells expressing full or partial GP Ib-IX-V complexes, and a
parallel plate flow chamber with phase contrast video microscopy and
digital image processing. Mammalian cells expressing the full complex
tethered and rolled on the vWF substrate, whereas control cells did
not. The rolling was completely inhibited by the monoclonal GP Ib
antibody, AK2, or the vWF antibody, 5D2, both shown previously to block
vWF-dependent platelet aggregation. Other GP Ib antibodies, WM23 and
SZ2, did not significantly change the number or mean velocity of
rolling cells. At low levels of GP Ib surface expression, cells
expressing the full complex rolled slower than cells expressing the
complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells
into the chamber did not significantly change the number or the mean
velocity of rolling cells. Inhibiting sulfation of the tyrosine
residues within the GP Ib subunit reduced the number but did not
change the mean velocity of the rolling cells. Our results indicate
that, under the conditions of these experiments, bonds between vWF and
GP Ib constantly form and break under fluid shear stress.
Additionally, our results suggest that GP Ib-IX-V complexes behave like
selectin receptors in their ability to mediate smooth rolling while
cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood
vessel wall. The system described provides a valuable approach for
investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
PATHOLOGIC ARTERIAL thrombosis is
mediated by platelets and depends on the local fluid environment in the
vessel.1-4 In the presence of elevated shear stresses, such
as occur in stenotic arteries, mural thrombus formation is initiated
when the platelet glycoprotein (GP) Ib-IX-V complex binds to von
Willebrand factor (vWF) immobilized on the exposed subendothelium of a
vessel injured as the result of angioplasty or atherosclerotic plaque
rupture.2,5-7 It has been demonstrated that the integrin GP
IIb-IIIa complex also has a role in the adhesion of platelets to the
subendothelial matrix.8 Previous studies using surfaces
coated with purified vWF suggest that vWF-GP Ib complex binding
mediates both the initial tethering of platelets from the bulk fluid to
the surface and the transient platelet translocation that
follows.9 These studies also indicate that firm adhesion of
platelets is supported by activation of the GP IIb-IIIa complex, which
then binds the immobilized vWF.9,10 Firm adhesion occurs
rapidly, therefore, platelet translocation is most readily observed
under specific conditions that inhibit platelet activation or GP
IIb-IIIa complex binding.9 Under arterial shear conditions,
aggregation to the initial and subsequent layers of platelets is
mediated by soluble vWF binding to GP IIb-IIIa complexes on adjacent
platelets.2,5,11,12 In contrast to thrombosis occurring in
high shear conditions, thrombosis under low shear conditions is
supported mainly by fibrinogen-platelet rather than by vWF-platelet
interactions.9,13 Currently, it is unknown whether a change
in vWF itself or a change in the platelet complex enhances vWF-GP
Ib-IX-V mediated adhesion under high shear conditions.
High shear conditions also impact vWF-GP Ib-IX-V binding by limiting
the contact time between this ligand-receptor pair. A fast on-rate is
required to support binding during the relatively short contact time
available under high shear conditions. Additionally, a higher bond
strength is necessary to support firm adhesion in the presence of
elevated shear stresses. Compared with vascular adhesion processes in
the venous system, such as selectin-mediated neutrophil binding to
activated endothelial cells, binding between vWF and the GP Ib-IX-V
complex, must withstand shear stresses that are greater by at least an
order of magnitude.
The GP Ib-IX-V complex is of significant research interest because of
its role in pathologic thrombosis as well as in hemostasis. This
complex contains four polypeptide subunits, GP Ib, GP Ib , GP IX,
and GP V, present in a stoichiometry on the platelet surface of
2:2:2:1.14 The first three polypeptides are required for expression of the complex on the cell surface15 and GP V is necessary to form a high-affinity site for thrombin on the
complex.16 GP Ib contains the regions that bind both
thrombin and vWF, within a 300 amino acid domain at its
N-terminus.14 This region is separated from the platelet
plasma membrane by an elongated mucin-like sequence called the
macroglycopeptide, whose function may be to position the ligand-binding
domain above the surrounding molecules on the platelet
surface.17
We have developed an experimental model to isolate the vWF-GP Ib-IX-V
interactions that mediate initial platelet adhesion to exposed
subendothelium. To evaluate these interactions under dynamic
conditions, we used vWF-coated glass coverslips, mammalian cells
expressing full or partial GP Ib-IX-V complexes, and a parallel plate
flow chamber with phase contrast video microscopy and digital image
processing. Our data demonstrate that purified vWF supports tethering
and rolling of cells expressing the GP Ib-IX-V complex and that this
interaction is dependent on vWF-GP Ib binding. Furthermore, the
data suggest an unexpected role for GP V in enhancing the avidity of
interaction between GP Ib-IX and vWF independent of its effect on the
degree of GP Ib-IX expression. Additionally, our data indicate that
exposing immobilized vWF to high shear stresses does not enhance
vWF-GP Ib-IX-V interactions. Finally, our results suggest that, under
dynamic conditions, sulfation of the tyrosine residues within the GP
Ib subunit contributes to the formation of GP Ib-vWF bonds but
may not affect the off-rate of bond formation.
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MATERIALS AND METHODS |
Cell lines.
The transfected mammalian cell lines used in our studies are summarized
in Table 1. L cell lines were grown in
Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc,
Grand Island, NY). The Chinese hamster ovary (CHO) cell lines were
grown in -minimal essential medium (-MEM; Life Technologies).
Both media were supplemented with 10% heat-inactivated fetal bovine
serum (FBS) and the selection drugs indicated in Table 1. For each assay, a portion of the cells to be used in flow chamber experiments was stained with fluorescein isothiocyanate (FITC)-conjugated GP Ib
antibody and analyzed for surface expression by flow cytometry (see
below). With the exception of experiments specifically investigating differences between L and CHO cell lines, all experiments used L cells
expressing full or partial GP Ib-IX-V complexes. Control cells included
L and CHO cells expressing only GP Ib and GP IX.
Inhibition of tyrosine sulfation.
Inhibition of sulfation of the tyrosine residues within the
ligand-binding region of GP Ib was completed as described
previously.18 L cells expressing full GP Ib-IX-V complexes
were grown in 100-mm culture dishes to 80% confluence and then
switched to sulfate-free DMEM medium with 2% of the normal
concentration of methionine and cysteine and supplemented with 10%
dialyzed FBS. Sodium chlorate and guaiacol were then added to the
medium at final concentrations of 5 and 0.2 mmol/L, respectively. Cells
were maintained under these conditions for 24 hours before further
assays. Control cells were grown in complete medium.
Flow cytometry.
Surface expression of GP Ib was determined by flow cytometry after
surface labeling GP Ib-IX and GP Ib-IXV expressing cells with the
FITC-conjugated monoclonal GP Ib antibody, AN51 (DAKO, Carpinteria,
CA). Before labeling, the cells were harvested with 0.54 mmol/L EDTA.
The cells were then washed with phosphate-buffered saline (PBS) and
incubated for 30 minutes in culture medium containing 1% bovine serum
albumin (BSA; fraction V; Sigma Chemical Co, St Louis, MO) to block
nonspecific antibody binding. The cells were then incubated with the
FITC-AN51, at a concentration of 1.4 µg/mL for 1 hour at room
temperature. After the cells were washed twice with PBS, the geometric
mean fluorescence of each sample was determined using a FACScan flow
cytometer (Becton Dickinson, San Jose, CA) that stimulates the
fluorescent dye with an argon-ion laser at 488 nm and collects the
light emitted above 530 nm. Nonspecific binding was determined by the
background fluorescence from LIX and CHOIX cells stained with the
same antibody. The data were analyzed using Cellquest software from
Becton Dickinson. All experiments except those specifically
investigating the effect of receptor density evaluated L and CHO cells
with comparable GP Ib surface expression.
Monoclonal antibodies (MoAbs).
The MoAbs AK2, SZ2, 5D2, and WM23 were generously provided by Dr
Michael Berndt (Baker Medical Research Institute, Prahran, Victoria,
Australia). AK2 and SZ2 bind to GP Ib, AK2 within the first 275 residues and SZ2 between residues 276-282.19 WM23 is
directed against the macroglycopeptide region of GP Ib and does not
interfere with vWF-GP Ib binding.20 5D2 was raised against a 39/34-kD dispase fragment of vWF (residues 480-718 of the
mature protein) that contains the GP Ib binding site.21 Before introduction into the flow chamber, cells were incubated with
one of the four antibodies: 6 µg/mL AK2, 25 µg/mL SZ2, 50 µg/mL
5D2, and 25 µg/mL WM23. All of these antibodies saturate at a
concentration of less than 5 µg/mL, based on previous studies (M. Berndt, personal communication, June 26, 1998).19-22 After 15 to 20 minutes of
incubation, the cells were resuspended in Dulbecco's PBS (Sigma
Chemical Co) to a final concentration of 500,000 cells/mL.
Preparation of vWF coverslips.
Purified vWF was obtained from normal human cryoprecipitate by glycine
and NaCl precipitation23,24 and then purified on a
Sepharose 4B column (2.5 × 50 cm with a bed volume of 3,000 ml;
Pharmacia, Inc, Piscataway, NJ). Using an enzyme-linked immunoassay (Spectro vWF Catalog No. V-46; Ramco Laboratories, Inc, Houston, TX),
the vWF concentration of the pooled peak fractions was determined relative to normal vWF plasma concentration. For each flow chamber experiment, the required amount of vWF was diluted to 30%, 250%, 500%, or 750% of normal plasma concentration in Dulbecco's PBS (Sigma Chemical Co). For this concentration range, it has been shown
that the amount of vWF absorbed on glass coverslips will increase as
the concentration of vWF in solution increases.25 Therefore, we used the concentration of the vWF solution coated on the
coverslip as an index of vWF surface density. Glass coverslips (No. 1, 24 × 50 mm; Corning, Inc, Corning, NY) were coated with 200 µL
of the desired vWF solution and incubated for a minimum of 45 minutes
in a humid chamber. When 30% vWF was used, after the initial 45 minutes of incubation, the coverslips were rinsed and then coated with
400 µL of PBS containing 1% bovine serum albumin (Boehringer
Mannheim, Indianapolis, IN) for a minimum of 20 minutes. To aid in
handling the coverslips, a 15-mm edge was left uncoated. Immediately
before using each coverslip, excess vWF or bovine serum albumin was
rinsed off with 5.0 mL of 0.9% NaCl. The parallel plate flow chamber
was then assembled with the coverslip forming the bottom of the
chamber.26-28 All experiments were completed on surfaces
prepared using a 500% vWF solution, with the exception of those
experiments specifically investigating the effect of vWF surface
density.
Parallel plate flow chamber and digital image processing.
The number and velocity of rolling cells were determined using a
parallel plate flow chamber and phase contrast video microscopy. The
parallel plate flow chamber consists of a polycarbonate slab, a silicon
gasket, and a vWF-coated glass coverslip
(Fig 1). These three components are held
together by vacuum. The thickness of the silicon gasket determines the
height of the gap between the coverslip and the polycarbonate slab. A
syringe pump connected to the outlet port draws fluid across this gap
through the chamber. The wall shear stress depends on the height of the
gap, the width of the chamber, the fluid viscosity, and the flow rate
through the chamber.29,30
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| Fig 1.
Schematic of the parallel plate flow chamber. The chamber
consists of a polycarbonate slab (A), a silicon gasket (B), and a
vWF-coated glass coverslip (C) held together by vacuum. During
experiments, the parallel plate flow chamber is mounted on an
inverted-stage microscope connected to a video camera and a video-
cassette recorder.
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During experiments, the parallel plate flow chamber was mounted on an
inverted-stage microscope (DIAPHOT-TMD; Nikon, Garden City, NY)
equipped with a ×20 phase objective (Nikon), a ×5
projection lens (Nikon), and a silicon-intensified target video camera
(Model C2400; Hammatsu, Waltman, MA) connected to a video cassette
recorder. In some experiments after the chamber was assembled and
mounted, vWF immobilized on the glass coverslip was sheared by
perfusing Dulbecco's PBS through the chamber for 5 minutes at a
constant flow rate to produce a constant wall shear stress of 40 dyn/cm2. In most experiments, 0.6 mL of either L or CHO
cells at a concentration of 500,000 cells/mL was transferred into the
parallel plate flow chamber and allowed to settle on the immobilized
vWF for 1 minute. Perfusion of Dulbecco's PBS was then initiated and
continued for 4 minutes at wall shear stresses ranging from 5 to 40 dyn/cm2. In other experiments, after the immobilized vWF
was presheared, a cell suspension was continuously perfused through the
chamber for 5 minutes without an initial settling period. In these
experiments, cells were perfused through the chamber at a concentration
of 100,000 cells/mL and a wall shear stress of 2 dyn/cm2.
Throughout all experiments, the parallel plate flow chamber and PBS or
cell solution were maintained at 37°C by a thermostatic air bath
(Model 279; Laboratory Products, Boston, MA). Cell rolling in a single
field of view was recorded in real time for 4 or 5 minutes on
videotape. The video data were analyzed off-line using Inovision
imaging software (IC-300 Modular Image Processing; Workstation Inovision Corp, Durham, NC) to quantify the number of rolling cells and
the mean velocity of the cells.31,32 Mean velocity was
calculated by overlapping sequential maximization images snapped at 30 frames per second and determining the distance the cells rolled during
the time period of the snaps. An average of 30 to 50 cells was used to
determine the mean velocity in each experimental run. For each
condition investigated, three to eight experimental runs were
completed, so final velocity calculations were based on measurements
from approximately 100 to 350 cells. The number of rolling cells was
determined by focusing on a single field of view and counting each cell
that rolled on the vWF surface during the entire 4- or 5-minute flow
period. Only cells that rolled maintaining continuous surface contact
were included in the determination of the number and velocity of
rolling cells.
All experiments were conducted after vWF had been sheared for 5 minutes
at 40 dyn/cm2 before the introduction of cells, with the
exception of the experiments completed to investigate the potential
functional change in immobilized vWF after exposure to elevated shear
conditions. With the exception of experiments to investigate the
tethering ability of cells, all experiments were completed by injecting
the cells into the flow chamber and allowing the cells to settle for 1 minute before the initiation of flow.
Statistics.
Results are reported as the mean ± SEM. The statistical
significance of the difference between means was determined by ANOVA using the Fischer's protected least significant difference (PLSD) test.
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RESULTS |
vWF-GP Ib-IX-V-mediated cell rolling.
Previous studies investigating the interaction of platelets with
purified vWF under dynamic conditions have demonstrated a sequential
process in which the initial tethering and translocation of the
platelets involves the GP Ib-IX-V complex, whereas firm adhesion
requires binding of vWF by GP IIb-IIIa.9 To examine the
vWF-GP Ib-IX-V interactions that initiate platelet adhesion, we first
isolated these interactions using L cells expressing the GP Ib-IX-V
complex and immobilized vWF in a parallel plate flow chamber. In the
presence of shear, L cells expressing the full GP Ib-IX-V complex
rolled on immobilized vWF (Fig 2C and D).
At a shear stress of 10 dyn/cm2, greater than 700 LIXV cells rolled on the immobilized vWF in a single field of
view during the 4-minute flow period (Fig 3B). The average velocity of the rolling LIXV cells was 80 µm/s (Fig 3A). In contrast, not a single LIX cell rolled on the
immobilized vWF after flow was initiated (Fig 2A and B). Instead, the
resulting shear force caused these cells to be lifted from the vWF
surface and carried toward the center of the flowstream, suggesting
that rolling was dependent on the presence of the GP Ib subunit. Our results also suggest that cell tethering from the flowstream to immobilized vWF depends on the presence of the GP Ib subunit. At a
wall shear stress of 2 dyn/cm2, greater than 190 LIXV
cells tethered to the vWF surface and rolled with an average velocity
of 33 µm/s when a cell suspension was perfused through the chamber
for 5 minutes (Fig 4). When LIX cells
were perfused through the chamber, no rolling cells were observed (Fig
4).
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| Fig 2.
Video images of L-cell rolling on purified vWF at a wall
shear stress of 10 dyn/cm2. Images were created using a
digital image processing system to snap frames of previously recorded
experiments. LIX cells (A) and LIXV cells (C) were introduced
into the chamber and allowed to incubate for 1 minute before the
initiation of flow. After 30 seconds of flow, LIX cells (B) had been
swept away from the vWF surface, whereas LIXV cells (D) rolled
maintaining continuous surface contact. Images of rolling cells were
created by snapping 30 frames per second and overlapping all 30 frames.
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| Fig 3.
Effect of MoAbs on LIXV cell rolling. Before
introduction into the parallel plate flow chamber, LIXV cells
were incubated with one of four MoAbs: SZ2 (anti-GP Ib), WM23
(anti-GP Ib), 5D2 (anti-vWF), or AK2 (anti-GP Ib). The mean
velocity (A) was calculated on a digital image processing system using
1-second maximization images. The number of rolling cells (B) was
determined by counting the total number of cells rolling on the vWF
surface in a single field of view over a 4-minute flow period. During
experimental runs, the entire system was maintained at 37°C.
Dulbecco's PBS was pumped through the chamber at a constant flow rate,
producing a constant shear stress of 10 dyn/cm2. Values are
the mean ± SEM, n = 3.
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| Fig 4.
Shear-dependent cell tethering and rolling. The velocity
of cells that tethered and rolled on the vWF surface was determined at
a wall shear stress of 2 dyn/cm2. Over a 5-minute flow
period, LIXV cells tethered to the vWF surface and rolled
maintaining continuous surface contact. In contrast, LIX cells did
not tether and roll on the vWF surface. Values are the mean ± SEM, n
= 3 to 4.
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To confirm the specificity of LIXV cell rolling on immobilized
vWF, flow chamber experiments were performed in the presence of
monoclonal GP Ib and vWF antibodies. Four MoAbs were used in our
studies: AK2, 5D2, SZ2 and WM23. AK2, directed against an epitope in
the N-terminal 275 residues of GP Ib,19 and 5D2, directed against the A1 domain of vWF,21 are both strong
inhibitors of ristocetin- and botrocetin-induced binding of vWF to
platelets as well as asialo-vWF-dependent platelet
aggregation.19-21,33 SZ2, directed against the sulfated
tyrosine-containing sequence of GP Ib (residues 276 to 282),
inhibits botrocetin-induced binding of vWF to platelets and
asialo-vWF-induced platelet aggregation, but is only a weak inhibitor
of ristocetin-induced binding of vWF to platelets.19 WM23
is directed against the macroglycopeptide core of GP Ib and has no
effect on vWF-GP Ib-IX-V interactions.20,33 LIXV
cell rolling on immobilized vWF was completely inhibited by both AK2
and 5D2 (Fig 3). The two other GP Ib antibodies, WM23 and SZ2, did
not significantly change the number or the mean velocity of rolling
cells (Fig 3).
Comparison of cell lines.
L and CHO cells expressing the GP Ib-IX complex (no GP V) or a complex
of GP Ib and GP IX (no GP V, no GP Ib) were used to evaluate
potential differences attributable to the use of different cell lines.
No significant difference between the number or velocity of rolling
cells was observed between the two cell lines expressing GP Ib-IX
(Fig 5). L and CHO cells lacking GP Ib
did not roll on the immobilized vWF after flow was initiated.
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| Fig 5.
Comparison of L and CHO cell lines. The mean velocity (A)
and the total number (B) of rolling LIX and CHOIX cells
were determined at a wall shear stress of 10 dyn/cm2.
Values are the mean ± SEM, n = 4 to 7.
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Influence of wall shear stress.
To gain insight into the strength and rate of vWF-GP Ib-IX-V binding,
the influence of wall shear stress on rolling LIXV cells was
determined. Shear stresses ranging from 5 to 40 dyn/cm2
were investigated by varying the flow rate of Dulbecco's PBS through
the chamber. The mean velocity of rolling cells increased slightly as
shear stress increased from 5 to 20 dyn/cm2, but did not
change between 20 and 40 dyn/cm2
(Fig 6A). In contrast to this modest effect
on velocity, increasing the shear stress from 5 to 40 dyn/cm2 decreased the number of rolling cells by 88% (Fig
6B).
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| Fig 6.
Influence of shear stress on LIXV cell rolling. The
mean velocity (A) and the total number (B) of rolling cells were
determined at wall shear stresses ranging from 5 to 40 dyn/cm2. Values are the mean ± SEM, n = 3 to 6. *Differences between means are statistically significant, P < .05.
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Effect of vWF surface density.
To determine the effect of vWF surface density on LIXV cell
rolling, glass coverslips were coated with solutions of varying vWF
concentration. The velocity of LIXV cells decreased by 50%, whereas the number of rolling cells increased by 240% when the concentration of vWF coated on the glass coverslip was increased from
30% to 250% (Fig 7). No significant
differences were observed between the number or the velocity of rolling
LIXV cells on coverslips coated with solutions containing 250%,
500%, or 750% vWF (Fig 7).
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| Fig 7.
Influence of vWF surface density on LIXV cell
rolling. Mean velocity (A) and the total number (B) of rolling cells
were determined on surfaces prepared using solutions with vWF
concentrations ranging from 30% to 750% of normal plasma
concentration. Values are the mean ± SEM, n = 4 to 8. *Statistically significant from 30% surface, P < .05.
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Effect of receptor density.
To examine the effect of receptor density on cell rolling, populations
of LIXV cells with different levels of GP Ib surface expression were evaluated in the parallel plate flow chamber. Before
beginning flow chamber experiments, an aliquot of cells from each
sample was removed and used to assess the level of GP Ib surface
expression by FITC-AN51 staining and flow cytometry. The geometric mean
fluorescence of each cell sample was used as an index of receptor
density. A wide range of receptor densities was present in the
different cell samples as indicated by the greater than 100-fold
variation in geometric mean fluorescence. The velocity of the rolling
LIXV cells appeared to decrease as GP Ib surface expression
increased over this very broad range of receptor densities (y = 122.42  24.191 log (x), R = .57;
Fig 8). To determine whether the
apparent decrease in velocity was statistically significant, the cells
were divided into three groups: low receptor density cells
(10 < geometric mean fluorescence < 60 fluorescent units), medium
receptor density cells (60 < geometric mean fluorescence < 800),
and high receptor density cells (geometric mean fluorescence > 800).
LIXV cells with low receptor density rolled significantly faster
than those cells with medium or high receptor density
(Fig 9A). No significant difference in
rolling velocities was observed between LIXV cells with a medium
receptor density and those with a high receptor density (Fig 9A). The
number of rolling cells was independent of receptor density (Fig 9B).
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| Fig 8.
Effect of receptor density on LIXV cell rolling.
The mean velocities were calculated based on an average of 30 to 50 cells from an experimental run. Each datapoint represents a single
experimental run. Values are the mean ± SEM.
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| Fig 9.
Effect of receptor density and role of the GP V subunit.
Mean velocity (A) and the total number (B) of rolling LIXV and
LIX cells with different receptor densities were determined at a
wall shear stress of 10 dyn/cm2. Values are the mean ± SEM, n = 6 to 14. *, §,  , #Differences between means are
statistically significant, P < .05.
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Role of GP V.
L cells expressing either the GP Ib-IX complex or the full complex
containing GP V were evaluated to determine the influence of GP V on
cell rolling. Cells with a similar receptor density were compared: the
geometric mean fluorescence of samples from either cell line ranged
from 10 to 60 fluorescent units (low receptor density group) or from
800 to 3,500 fluorescent units (high receptor density group). In the
low receptor density group, the average velocity of LIX cells (no
GP V) was 61% greater than that of LIXV cells (Fig 9A).
Additionally, within the low receptor density group, the number of
rolling LIX cells was 50% lower than the number of rolling
LIXV cells (Fig 9B). When L cells with a high receptor density
were evaluated, the number and velocity of rolling LIX cells was
not significantly different from that of LIXV cells (Fig 9).
Additionally, as observed using fully transfected cells, LIX
cells with a low receptor density rolled significantly faster than
LIX cells with a high receptor density (Fig 9A). LIX cells
with a high receptor density were also evaluated on surfaces with
different vWF densities. Similar to the fully transfected cells, the
number and velocity of high receptor density LIX cells did not
significantly change as the concentration of vWF coated on the glass
coverslip varied from 250% to 750% (Fig
10).
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| Fig 10.
Influence of vWF surface density and the role of the GP
V subunit. LIXV and LIX cells with a high receptor density
(geometric mean >800 fluorescent units) were evaluated on surfaces
prepared using vWF solutions with concentrations ranging from 250% to
750% of normal vWF plasma concentration. The mean velocity (A) and the
total number (B) of rolling cells were determined at a wall shear
stress of 10 dyn/cm2. Values are the mean ± SEM, n = 4 to 8.
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Functional effect of shear on immobilized vWF.
It has been demonstrated that high shear forces change the structure of
immobilized vWF from a globular conformation to an open, extended
conformation.34 Therefore, we investigated whether exposing
vWF to high shear before perfusing LIXV cells might influence
cell adhesion and rolling. Preshearing of vWF was accomplished by
perfusing Dulbecco's PBS through the flow chamber for 5 minutes at 40 dyn/cm2 before introducing cells. Preshearing the vWF did
not significantly change the number of rolling cells or the mean
velocity of the cells (Fig 11).
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| Fig 11.
Effect of preshearing vWF. The mean velocity (A) and the
total number (B) of rolling LIXV cells were determined at a wall
shear stress of 10 dyn/cm2. In preshear experiments, before
cells were introduced into the chamber, vWF immobilized on the glass
coverslip was exposed to a shear stress of 40 dyn/cm2 for 5 minutes by perfusing Dulbecco's PBS through the chamber. Values are
the mean ± SEM, n = 4 to 7.
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Functional effect of GP Ib tyrosine sulfation.
Previous studies investigating the structure of the GP Ib-IX-V complex
have determined that the GP Ib subunit is modified by the sulfation
of three tyrosine residues (Y276, Y278, and Y279).18,19 To
examine whether sulfate modification of the tyrosine residues impacts
GP Ib-vWF interactions under dynamic conditions, we compared rolling
of sulfated LIXV cells with that of LIXV cells lacking sulfation. The velocity of LIXV cells lacking sulfation was not
significantly different than the velocity of sulfated LIXV cells
(Fig 12A). In contrast, the number of
rolling LIXV cells decreased by 58% when tyrosine sulfation was
inhibited (Fig 12B).
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| Fig 12.
Effect of sulfation of the tyrosine residues within the
GP Ib subunit. The mean velocity (A) and the total number (B) of
rolling cells were determined at a wall shear stress of 10 dyn/cm2. Sulfation inhibition experiments were completed
using LIXV cells grown in complete or sulfate-depleted
media. Values are the mean ± SEM, n = 4. *Differences between means are statistically significant,
P < .05.
|
|
| |
DISCUSSION |
The platelet GP Ib-IX-V complex initiates pathologic arterial
thrombosis by binding to vWF immobilized on the subendothelium of a
denuded vessel. It has been shown previously that blocking this initial
step of platelet adhesion decreases formation of thrombi and reduces
long-term restenosis of narrowed vessels after injury.2,35
To better understand the specific molecular mechanisms that support
platelet adhesion, we have developed a dynamic experimental model that
isolates vWF-GP Ib-IX-V interactions. Our system consists of
vWF-coated glass coverslips, mammalian cells expressing full or partial
GP Ib-IX-V complexes, and a parallel plate flow chamber with phase
contrast video microscopy and digital image processing. We have
demonstrated that, in the presence of shear, L and CHO cells expressing
the GP Ib-IX-V complex will tether and roll on immobilized vWF, whereas
cells expressing subcomplexes lacking GP Ib do not exhibit any
strong interactions. These studies indicate that the GP Ib-IX-V complex
supports cell tethering and rolling on immobilized vWF independent of
other platelet adhesive components.
In support of this, we have also shown that cell rolling is completely
inhibited by the monoclonal GP Ib antibody AK2 or the vWF antibody
5D2. These antibodies have been shown previously to block asialo-vWF-,
ristocetin-, and botrocetin-induced platelet aggregation.19-22,33 The GP Ib antibody SZ2 did not
significantly change the number or mean velocity of rolling cells. SZ2
has been shown previously to block asialo-vWF- and botrocetin- but not ristocetin-induced platelet aggregation.19,22 These
contrasting results suggest that different vWF binding sites may be
involved in platelet aggregation induced by botrocetin and asialo-vWF
versus aggregation induced by ristocetin and platelet adhesion under flow, even though all of these processes require a vWF-GP Ib interaction. Thus, these findings suggest that ristocetin-dependent vWF
binding to the GP Ib-IX-V complex may better reflect the binding reaction that occurs under shear in vivo.
Our results indicate that, under the conditions of these experiments,
bonds between vWF and GP Ib continually form and break under fluid
shear stress. When the wall shear stress was increased from 5 to 40 dyn/cm2, the number of rolling LIXV cells
significantly decreased, whereas no significant change in velocity was
observed. We selected a wall shear stress of 10 dyn/cm2 to
use in all other experiments. Although this shear stress is lower than
shear stresses present in the arterial system, it is important to
consider the resultant shear force acting on platelets in the blood
stream relative to the shear force acting on the mammalian cells in our
system. The shear force acting on cells near a vessel wall depends on
both the fluid shear stress and the cell size.36,37
Neglecting differences in shape, the resultant shear force used in our
experiments with L and CHO cells corresponds to the shear force a
platelet would experience in the presence of a wall shear stress of 110 dyn/cm2. Physiologically, shear stresses of this magnitude
are only present in stenosed arteries, where platelet adhesion requires
vWF-GP Ib-IX-V binding. At wall shear stresses of 10 dyn/cm2, the average velocity of the rolling cells was only
2% of the calculated velocity of a neutrally buoyant, rigid sphere of
the same size flowing near a plane wall.36
In platelets, there are approximately 25,000 copies of GP Ib, GP
Ib, and GP IX38 and half as many copies of GP
V,39 indicating that the number of complexes may be either
12,000 or 6,000, depending on the numbers of each polypeptide present
in the complex. These complexes are anchored to the cytoskeleton, with
this anchorage providing for an orderly spatial distribution on the
plasma membrane.14 In the mammalian cells used in our study, the number of GP Ib-IX-V complexes present on the surface varied, but, as in platelets, they were anchored to the cytoskeleton and evenly distributed throughout the cell's smooth, spherical membrane.15 In our studies, we found that cells with a low
GP Ib receptor density rolled significantly faster than cells with a
high receptor density. We also observed that, beyond a certain level of
GP Ib expression, cell velocity was independent of receptor density.
Similarly, significant differences in cell velocity were observed on
surfaces coated with high and low vWF concentrations, but, once a
threshold level of vWF surface density was reached, cell velocity
remained nearly constant. Recent studies with whole blood in a similar
system have shown that platelet translocation velocity is also
independent of vWF surface density above a threshold level.25
Our results suggest that GP Ib-IX-V complexes behave like selectin
receptors in their ability to mediate smooth rolling while cells
maintain continuous surface contact. Such a mechanism, in vivo, will
allow platelets to slow down and eventually arrest on the blood vessel
wall. Selectin-mediated rolling has been studied in parallel plate flow
chamber systems similar to the one presented here. In those studies,
neutrophils were perfused through the chamber over activated
endothelial cells expressing P-selectin. Previous work31
has demonstrated that optimal selectin-mediated neutrophil rolling
occurs at wall shear stresses of 1 to 2 dyn/cm2. At higher
shear stresses, the number of rolling neutrophils rapidly decreases and
is nearly zero at 10 dyn/cm2.31 In contrast, we
found that LIXV cells roll on immobilized vWF in significant
numbers at shear stresses as high as 40 dyn/cm2. Because
neutrophils and the mammalian cells used in our study are similar in
size, this suggests that either vWF GP Ib-IX-V bonds are stronger or
that these bonds are able to form more rapidly than the bonds between
P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin.
We have used this new system to evaluate the role of GP V in vWF-GP
Ib-IX-V mediated cell rolling. Previous studies suggest that GP V aids
in establishing the topology of the GP Ib-IX-V complex.40
Additional work using transfected mammalian cells has shown that the GP
V subunit is not required for ristocetin-41 or
botrocetin-induced aggregation (unpublished data). In
contrast to these modulator-induced interactions, we observed that the shear-dependent cell rolling in our system is influenced by the presence of GP V. When GP Ib surface expression was low, LIX cells (no GP V) rolled 61% faster and the total number of rolling cells decreased by 50% relative to cells expressing the full complex. These differences were not observed when cells with a higher receptor density were compared. The results using low receptor density cells
suggest that GP V may function to maintain the optimal conformation of
the complex required for its interaction with surface-bound vWF under
dynamic conditions. The lack of impact of GP V at high receptor
densities suggests that, under these conditions, the ability of GP V to
assist in optimal spacing of the full complex is diminished.
Alternatively, as has been suggested for its role in thrombin binding,
GP V may juxtapose adjacent GP Ib subunits such that they can more
efficiently interact with the two A1 domains in the vWF dimer. This
effect would be less pronounced at high receptor densities. As with
other functions of the GP Ib-IX-V complex, further studies of the role
of GP V are needed.
We have also used this new system to investigate the effect of shear on
immobilized vWF function. Past studies using atomic force microscopy
have demonstrated that exposure to elevated shear conditions results in
a structural change in immobilized vWF.34 It has been
suggested that this observed structural change enhances vWF-GP Ib
binding under high shear conditions. We investigated the functional
effect of shearing vWF by conducting parallel plate flow chamber
experiments with and without exposing immobilized vWF to high shear
immediately before use. We found that preshearing vWF did not
significantly change the number of rolling cells or the mean velocity
of the cells, suggesting that shear does not change the function of
immobilized vWF with respect to GP Ib-IX-V complex binding. However,
although the shear stress used in non-preshear experiments was well
below the established critical shear stress required to alter
vWF,34 it is possible that the shear conditions present in
the non-preshear system did change the conformation of the immobilized
vWF. It is also possible that the vWF conformation induced by
preshearing could have reverted within the incubation time before
reinitiating flow.
Finally, we have used this new system to evaluate the effect of
sulfation of GP Ib tyrosine residues on cell rolling. Past evidence
suggests that sulfation of these residues may be required for optimal
GP Ib-vWF binding.18,19,42 The velocity of LIXV cells lacking sulfation was not significantly different than the velocity of sulfated LIXV cells. These results are consistent with those obtained in our system using the MoAb SZ2, which binds to
the region of the GP Ib subunit containing the sulfated tyrosine residues.19 Although cell velocity did not change, the
number of rolling LIXV cells decreased by 58% when tyrosine
sulfation was inhibited. This decrease in the number of rolling cells
suggests that sulfation impacts the on-rate of GP Ib-vWF bond
formation, that is, sulfation impacts GP Ib-vWF interactions by
altering the ability of GP Ib to form bonds with immobilized vWF
under dynamic conditions. In contrast, the off-rate of bond formation, indicated by cell velocity, appears to be independent of sulfation.
To summarize, we have developed an experimental model of the vWF-GP
Ib-IX-V interactions that mediate initial platelet adhesion to an
injured vessel wall. This system provides a valuable tool for
investigating the roles of individual receptors and ligands in the
process of platelet adhesion and thrombosis. Our results with this new
system suggest the novel finding that optimal binding between
immobilized vWF and GP Ib requires the presence of the GP V subunit
within the GP Ib-IX-V complex. Additionally, our results indicate that
the function of immobilized vWF with respect to GP Ib-IX-V binding does
not change after exposure to high shear. Finally, our results
demonstrate that, under dynamic conditions, sulfation of the GP Ib
tyrosine residues contributes to the ability of GP Ib-vWF bonds to
form but does not affect the off-rate of bond formation.
This system can now be used to further analyze mutant receptor
complexes under conditions that approximate more closely the in vivo
situation than do the currently used methods of inducing GP
Ib-IX-V-vWF interactions with the modulators ristocetin and botrocetin. Basic mechanism studies such as these will lead to an
improved understanding of the pathophysiology of arterial thrombosis and accelerate the development of novel, more potent antithrombotic agents.
| |
ACKNOWLEDGMENT |
The authors thank Nancy A. Turner and Leticia H. Nolasco for their
assistance in preparing the purified vWF and Shan Gao for her technical
assistance with the cells. The authors also thank Dr Michael Berndt for
providing reagents and for helpful discussions about the manuscript.
| |
FOOTNOTES |
Submitted February 12, 1998;
accepted July 8, 1998.
Supported by National Institutes of Health (NIH) Grants No. HL-18672,
NS-23327, HL-02463, and HL-46416; the Robert A. Welch Foundation Grant
No. C-938; a Grant in Aid from the American Heart Association-Texas
Affiliate; the NIH Medical Scientist Training Program at Baylor College
of Medicine; and a Graduate Fellowship from the Whitaker Foundation.
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 Larry V. McIntire, PhD, Cox Laboratory for
Biomedical Engineering, Rice University, PO Box 1892, Houston, TX
77251.
| |
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V. Afshar-Kharghan, R. Diz-Kucukkaya, E. H. Ludwig, A. J. Marian, and J. A. Lopez
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Genetics of Arterial Prothrombotic Risk States
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J.-F. Dong, M. C. Berndt, A. Schade, L. V. McIntire, R. K. Andrews, and J. A. Lopez
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M. Gu, X. Xi, G. D. Englund, M. C. Berndt, and X. Du
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G. M. Romo, J.-F. Dong, A. J. Schade, E. E. Gardiner, G. S. Kansas, C. Q. Li, L. V. McIntire, M. C. Berndt, and J. A. Lopez
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Glycoprotein (GP) Ib-IX-transfected Cells Roll on a von Willebrand Factor Matrix under Flow. IMPORTANCE OF THE GPIb/ACTIN-BINDING PROTEIN (ABP-280) INTERACTION IN MAINTAINING ADHESION UNDER HIGH SHEAR
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Tyrosine Sulfation of Glycoprotein Ibalpha . ROLE OF ELECTROSTATIC INTERACTIONS IN VON WILLEBRAND FACTOR BINDING
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J.-f. Dong, A. J. Schade, G. M. Romo, R. K. Andrews, S. Gao, L. V. McIntire, and J. A. Lopez
Novel Gain-of-function Mutations of Platelet Glycoprotein Ibalpha by Valine Mutagenesis in the Cys209-Cys248 Disulfide Loop. FUNCTIONAL ANALYSIS UNDER STATIC AND DYNAMIC CONDITIONS
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Top
Abstract
Introduction