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Blood, Vol. 91 No. 7 (April 1), 1998:
pp. 2353-2359
A Fifty Percent Reduction of Platelet Surface Glycoprotein Ib
Does Not Affect Platelet Adhesion Under Flow Conditions
By
G. Henrita van Zanten,
Harry F.G. Heijnen,
Yaping Wu,
Karin M. Schut-Hese,
Pieter J. Slootweg,
Philip G. de Groot,
Jan J. Sixma, and
Rienk Nieuwland
From the Departments of Hematology and Pathology, University Hospital
Utrecht, and Graduate School of Biomembranes, Utrecht University,
Utrecht; and the Department of Clinical Chemistry, Leiden University
Medical Centre, Leiden, The Netherlands.
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ABSTRACT |
Glycoprotein (GP) Ib is an adhesion receptor on the platelet surface
that binds to von Willebrand Factor (vWF). vWF becomes attached to
collagens and other adhesive proteins that become exposed when the
vessel wall is damaged. Several investigators have shown that during
cardiopulmonary bypass (CPB) surgery and also during platelet
activation in vitro by thrombin or thrombin receptor activating peptide
(TRAP) GPIb disappears from the platelet surface. Such a disappearance
is presumed to lead to a decreased adhesive capacity. In the present
study, we show that a 65% decrease in platelet surface expression of
GPIb, due to stimulation of platelets in Orgaran anticoagulated whole
blood with 15 µmol/L TRAP, had no effect on platelet adhesion to both
collagen type III and the extracellular matrix (ECM) of human umbilical
vein endothelial cells under flow conditions in a single-pass perfusion system. In contrast to adhesion, ristocetin-induced platelet
agglutination was highly dependent on the presence of GPIb.
Immunoelectron microscopic studies showed that GPIb almost immediately
returned to the platelet surface once platelets had attached to
collagen. In a subsequent series of experiments, we showed that when
less than 50% of GPIb was blocked by an inhibitory monoclonal antibody
against GPIb (6D1), platelet adhesion under flow conditions remained
unaffected.
| |
INTRODUCTION |
GLYCOPROTEIN (GP) Ib is unique for human
platelets. It is present as a transmembrane protein in the plasma
membrane, approximately 25,000 copies per platelet. GPIb consists of
two polypeptide chains, an  and  -chain of molecular weight
(MW) 150 kD and 27 kD, respectively. The -chain is
glycosylated and sensitive to proteolysis by proteases such as
plasmin1 and cathepsin G in vitro.2 GPIb
functions as a receptor for von Willebrand Factor (vWF) and the
vWF-GPIb interaction plays a key role in the adhesion under flow
conditions to collagen,3 fibronectin,4 and
fibrinogen.5 In addition, GPIb is a high-affinity receptor
for -thrombin.6,7
When platelets are stimulated in vitro by -thrombin, or thrombin
receptor activating peptide (TRAP), the expression of GPIb on the
platelet surface decreases. This decrease is due to a
cytoskeletal-mediated redistribution to the open canalicular system
(OCS), where it is inaccessible to antibodies.8-11 The
decrease in the platelet surface expression of GPIb is reversible, as
has been shown for platelets stimulated by -thrombin, where fully
functional GPIb reappeared on the platelet surface after an initial
disappearance.12 At present, however, it is unknown whether
the diminished platelet surface expression of GPIb has any consequences
for platelet adhesion under flow.
Also the in vivo expression of GPIb on the platelet surface is
variable. Five minutes after the start of cardiopulmonary bypass (CPB),
during which time blood had contacted the artificial surface of the
extracorporeal system, the surface expression of GPIb decreased by 30%
to 40%.13,14 These findings received much attention, as
prolonged bleeding times and excessive blood loss often occur during
and after CPB. A diminished surface expression of GPIb may lead to a
reduced ability of platelets to adhere to the damaged vessel wall, and
this may result in prolonged bleeding times and excessive blood loss.
This model was supported by the finding that patients undergoing CPB
surgery who received aprotinin (trasylol), a nonspecific inhibitor of
serine proteases, showed no disappearance of GPIb and lost less
blood.13,15,16
However, this model is in contrast with the observation that carriers
of the inherited disease Bernard-Soulier Syndrome, which have half the
normal number of GPIb copies on their platelets, do not
bleed.17,18
The aim of the present study was to investigate the relationship
between platelet adhesion under flow and the presence of GPIb on the
platelet surface. Whole blood was anticoagulated with Orgaran and
stimulated with TRAP in the presence of dRGDW to avoid platelet aggregation. The advantage of platelet stimulation by TRAP was
that we were able to study the effect of GPIb downregulation in whole
blood under physiologic Ca2+ levels.
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MATERIALS AND METHODS |
Adhesive surfaces.
Human placenta collagen type III was obtained from Sigma (St Louis, MO)
and solubilized in 50 mmol/L acetic acid (1.4 mg/mL). The solubilized
collagen was sprayed onto glass coverslips (Menzel, Braunschweig,
Germany) with a retouching airbrush (Badger model 100, Badger Brush Co,
Franklin Park, IL) to a surface density of 30 µg cm-2,
supporting optimal platelet coverage.19 The amount of
collagen deposited on the coverslips was determined by weighting the
coverslips before and after spraying.
Human vascular endothelial cells derived from umbilical veins were
isolated according to Jaffe et al20 with some
modifications.21 The cells were cultured in RPMI-1640
containing 20% pooled human serum. Endothelial cells of the third
passage were used. After the cells had grown to confluence on glass
coverslips, matrices were isolated by exposing the cells to 0.1 mol/L
NH4OH.22 This step was followed by three washes
with phosphate-buffered saline (PBS: 10 mmol/L sodium phosphate, 150 mmol/L NaCl, pH 7.4).
Both the coverslips with collagen and extracellular matrix (ECM) were
subsequently blocked by incubation with a 1% human albumin solution
(Behringwerke, Marburg, Germany) in HEPES-buffered saline (HBS: 10 mmol/L Hepes, 150 mmol/L NaCl, pH 7.35), for 30 minutes at room
temperature.
Reagents.
The D-arginyl-glycyl-L-aspartyl-L-tryptophan (dRGDW) peptide was
generously provided by Dr J. Bouchaudon (Rhône-Poulenc-Rorer, Chemistry Department, Centre de Recherche de Vitry, Vitry sur Seine,
France). The preincubation period of the dRGDW peptide was 15 minutes.
The thrombin-receptor activating peptide TRAP (SFLLRN) was obtained
from Bachem Feinchemikalien AG (Bubendorf, Switzerland).
Monoclonal antibody (MoAb) 6D1, an inhibitory MoAb directed against
GPIb, was kindly provided by Dr B. Coller23 (Mount Sinai Hospital, New York, NY). The MoAb was used as ascites and was added to
the perfusate 30 minutes before perfusion.
Flow cytometry.
Fixed platelets were prepared by collecting 1 volume of blood in 5 vol
of paraformaldehyde in PBS (final concentration, 1%). Platelets were
washed twice with PBS to which 5 mmol/L EDTA had been added (PBS/EDTA)
and diluted to a concentration of 3.108/mL.
A total of 10 µL of platelet suspension was incubated with 10 µL
fluorescein isothiocyanate (FITC)-conjugated monoclonal antibody (5 µg/mL) for 30 minutes at room temperature. MoAb 6.20 (obtained from
Dr H.K. Nieuwenhuis, Department of Hematology, University Hospital
Utrecht) is a noninhibitory antibody directed against GPIb (tested in
ristocetin-induced platelet agglutination). This MoAb reacts with the
GPIb -band in a Western blot and was completely negative in
cytofluorography with platelets of a patient with the Bernard Soulier
syndrome. The occupation of GPIb by the inhibitory MoAb 6D1 was
determined by using a secondary FITC-conjugated goat antimouse antibody
and calculated as percentage of saturation.
FITC-conjugated CD62 MoAb RUU-SP 2.1724 directed against
P-selectin was used to detect P-selectin on the platelet surface after platelets were activated by TRAP.
We used control ascites or a control IgG against an antigen that is not
present on platelets.
After washing, the platelets were resuspended in 2 mL PBS for analysis.
Platelets were analyzed in a FACScan flow cytometer (Becton Dickinson,
San Jose, CA) at a wavelength of 488 nm.24 FACScan data
were analyzed with PC-LYSIS software (Becton Dickinson).
Ristocetin-induced platelet agglutination (RIPA).
For RIPA, platelets were prepared as described by Brinkhous and
Read.25 Washed platelets were incubated with 200 µmol/L dRGDW and were subsequently incubated for various periods of time (0 to
60 minutes) with 15 µmol/L TRAP at 37°C. After the incubation with TRAP, the platelets were fixed in 1.8% paraformaldehyde for 30 minutes, washed three times, and resuspended in citrated saline with
5% bovine serum albumin (BSA; Sigma) to a final concentration of 8 × 108/µL. A total of 100 µL fixed platelets was
mixed with 325 µL Tris buffer (10 mmol/L Tris, 150 mmol/L NaCl, pH
7.35) containing 0.5% BSA and 25 µL ristocetin (20 mg/mL; Diamed,
Cressier sur Morat, Switzerland), and incubated for 2 minutes at
37°C. The agglutination was triggered by the addition of 50 µL
pooled normal plasma and measured in a Chronolog lumiaggregometer at
37°C with stirring at 900 rpm.
Perfusions.
Fresh blood from healthy donors who denied having taken aspirin in the
preceding 10 days was anticoagulated with one tenth volume of 150 U/mL
Orgaran (a low molecular weight heparinoid [LMWH]; Organon, Oss, the
Netherlands). A total of 200 µmol/L of the dRGDW peptide was added to
the anticoagulated blood to avoid platelet aggregation. Perfusions were
performed in modified parallel plate perfusion chambers with a slit
height of 0.1 mm and a slit width of 2 mm,26 corresponding
with flow rates of 60 µL/min (shear rate = 300 s-1) and
320 µL/min (shear rate = 1,600 s-1). Blood was prewarmed
at 37°C for 10 minutes and was then drawn through 6 parallel
perfusion chambers by a Harvard infusion pump (pump 22, model 2400-004;
Natick, MA). At the onset of perfusion, blood samples were drawn and
immediately fixed for flow cytometry or for immunoelectron microscopy.
After each perfusion run, the coverslips were removed from the
perfusion chambers and rinsed with HBS, fixed in glutaraldehyde (0.5%
in PBS) and stained with May Grünwald/Giemsa as
described.27 Platelet adhesion was quantitated with a light
microscope (at 1,000× magnification) coupled to a computerized
image analyzer (AMS 40-10, Saffron Walden, UK). Three lines
perpendicular to the flow direction were evaluated: one line in the
center of the coverslip and two lines 3 mm to the right and 3 mm to the
left of the center. Platelet adhesion was expressed as the percentage of the surface covered with platelets.
Immunoelectron microscopy.
The expression of GPIb on the platelet surface immediately before the
start of perfusion was compared with the expression of GPIb on the
platelet surface after adhesion to collagen type III-coated coverslips.
For the latter, collagen was sprayed on melamin-coated
coverslips.28 Briefly, glass coverslips were coated with
melamin by dipping in a 1% melamin solution, containing 0.3%
paratoluene sulfonic acid, dissolved in analytical grade ethanol. The
coverslips were carefully withdrawn and immediately flamed for
polymerization.
Blood samples and perfused coverslips were fixed in a mixture of 2%
paraformaldehyde and 0.2% glutaraldehyde in 0.1 mol/L phosphate buffer
(pH 7.4). After rinsing with PBS/0.15 mmol/L glycine, the melamin foil
was removed from the glass coverslips with 0.8% hydrofluoric acid at
4°C.
The melamin foils were used for ultrathin cryosectioning and
transmission electron microscopy (JEOL, Tokyo, Japan). Immunogold labeling was performed on thin frozen sections of platelets in suspension and adhering platelets, using MoAb AK3 (generously provided
by Dr M.C. Berndt, Prahran, Australia29) followed by a
rabbit antimouse IgG (DAKOpatts, Glostrup, Denmark) intermediate step,
and finally with protein-A-gold (10 nm). The specificity of
immunolabeling was verified using an irrelevant control antibody. Quantification was performed on randomly photographed platelet profiles
by counting the number of gold particles on the plasma membrane (PM),
the membranes of the open canalicular system (OCS), -granules, and
nondefined structures. The data are expressed as percentages of the
total number of gold particles counted and represent the mean of at
least three separate immunolabeling experiments. The total numbers of
platelets counted were 70 (control) and 81 (TRAP stimulated) for the
platelets in suspension and 69 (control) and 63 (TRAP stimulated) for
the adhered platelets.
Statistical analysis.
Student's t-test was used to test for differences between
groups. P values less than .05 were considered statistically
significant.
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RESULTS |
In a preliminary set of experiments, the effect of various
concentrations of TRAP (0 to 15 µmol/L) on the disappearance of the
GPIb-antigen was analyzed by flow cytometry. Whole blood was anticoagulated with Orgaran in the presence of 200 µmol/L dRGDW. The
mean fluorescence intensity (MFI) of GPIb decreased by approximately 60% when platelets were stimulated with 10 µmol/L or 15 µmol/L TRAP, 5 minutes after the addition of the agonist
(Fig 1). The observed decrease in the
platelet surface expression of GPIb was accompanied by an increase in
P-selectin on the platelet surface. P-selectin is present on the
membranes of -granules and is expressed on the platelet surface
after platelets are activated and release their
-granules.30 The downregulation of GPIb and the surface expression of P-selectin are not coupled because the decrease in GPIb
is not dependent on the release of -granules, and the restoration of
the surface expression of GPIb can occur in a fully degranulated
platelet.12
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| Fig 1.
Effect of different concentrations of TRAP on the
platelet surface expression of GPIb and P-selectin. Orgaran
anticoagulated whole blood (+ dRGDW) was incubated (5 minutes,
37°C) with various concentrations of TRAP. After 5 minutes, the
blood was fixed in 1% paraformaldehyde, incubated with either MoAb
6.20 (against GPIb [ ]) or CD62 MoAb RUU-SP 2.17 (against
P-selectin [ ]), and analyzed by flow cytometry. Both the binding
of the anti-GPIb MoAb before the addition of TRAP and the maximal
binding of the anti-CD62 MoAb was assigned 100 arbitrary units of
fluorescence. Data represent a typical experiment.
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When blood at 37°C was stimulated with an optimal concentration of
15 µmol/L TRAP, GPIb disappeared from the platelet surface within 5 minutes. After 5 minutes, GPIb started to reappear on the cell surface,
and after 60 minutes, approximately 80% of the baseline value was
found (Fig 2). A slight increase in GPIb
was noticed in parallel experiments in which no TRAP was added. This increase probably refers to platelet activation during blood
collection. dRGDW did not interfere with the binding of the MoAbs
tested (data not shown).
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| Fig 2.
Reversibility of the TRAP-induced decrease of the
platelet surface expression of GPIb. A total of 15 µmol/L TRAP ()
was added at t = 0 minutes to Orgaran anticoagulated whole blood (+ dRGDW) at 37°C. As a control, no agonist ( ) was added. The
samples were fixed with 1% paraformaldehyde at the indicated time
points. The binding of the GPIb-specific MoAb 6.20 at t = 0 minutes
(before TRAP was added) was assigned 100 arbitrary units of
fluorescence. Data are the mean of two separate experiments.
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The TRAP-induced decrease in platelet surface expression of GPIb was
accompanied by a decrease in RIPA (Fig 3).
After 5 minutes, a time-dependent recovery in RIPA was observed. The
results presented in Fig 3 show that RIPA is highly dependent on the
platelet surface expression of GPIb.
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| Fig 3.
Effect of TRAP on RIPA. Washed platelets were incubated
with 15 µmol/L TRAP at 37°C and fixed with paraformaldehyde at
the indicated time points. The RIPA without agonist was assigned 100%. Agglutination was triggered by pooled normal plasma and 1 mg/mL ristocetin (see Materials and Methods). % agglutination (); slope of curve ( ). Data represent a typical experiment.
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To determine the effect of GPIb downregulation on platelet adhesion
under flow conditions, whole blood, anticoagulated with Orgaran was
stimulated with 15 µmol/L TRAP or vehicle at 37°C, in the
presence of dRGDW. As previously shown,31,32 dRGDW has no
inhibitory effect on platelet adhesion to collagen type III and ECM
when LMWH is used as anticoagulant.
Five minutes after the addition of TRAP, samples were removed for flow
cytometry analysis. Immediately thereafter, six parallel single-pass
perfusion runs were started at 37°C over coverslips with either
collagen type III or ECM (three runs with TRAP, three runs without
agonist). TRAP induced a disappearance of GPIb from the platelet
surface in all donors tested. The overall decrease was 65% (mean ± standard error of mean [SEM] = 35% ± 1.7% of control, n = 12).
As shown in Table 1, the adhesion of
TRAP-stimulated platelets at a shear rate of 1,600 s-1 was
not significantly different from unstimulated platelets, both to
collagen type III and ECM. Under the present conditions (ie, with the
addition of dRGDW), we did not observe differences in spreading between
TRAP-stimulated and control platelets adhered to collagen or ECM.
Similar results were obtained with collagen type III at a shear rate of
300 s-1 (data not shown), indicating that this effect was
independent of the shear stress applied to the platelets. Thus, a
decrease of approximately 65% in platelet surface expression of GPIb
has no effect on platelet adhesion under the conditions tested.
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Table 1.
Effect of 15 µmol/L TRAP on Platelet Adhesion to
Collagen Type III and to the ECM of Endothelial Cells at a Shear
Rate of 1,600 s 1
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However, it is not clear whether adherent platelets behave identical to
platelets in suspension. Once attached to an adhesive surface, it is
possible that GPIb, which had disappeared from the platelet surface,
becomes mobilized and then participates in platelet adhesion. Therefore
immunoelectron microscopic studies were performed. Platelets were
stimulated with 15 µmol/L TRAP or vehicle at 37°C. Five minutes
after the addition of TRAP, samples were removed for electron
microscopy. Immediately thereafter perfusions were started for 5 minutes over collagen type III (shear rate = 1,600 s-1). As
shown in Fig 4A, gold particles labeling
GPIb are predominantly present on the platelet surface. After
stimulation with TRAP, most gold particles are present in the OCS (Fig
4B). The relative surface expression of GPIb was quantified by counting
the gold particles associated with platelet surface membranes, OCS
membranes, -granules, and undefined structures
(Table 2). The percentage of GPIb on the
platelet surface of unstimulated platelets was 69.0% ± 2.9% (mean ± SEM, n = 4). After stimulation with TRAP, the percentage of GPIb
on the platelet surface was 35.5% ± 2.5% (mean ± SEM, n = 4),
representing a 50% decrease in platelet surface GPIb.
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| Fig 4.
Series of electron micrographs showing platelets in
suspension (A and B) and adhered platelets to collagen type III (C and D). Whole blood anticoagulated with Orgaran in the presence of dRGDW
was stimulated with 15 µmol/L TRAP (B and D) or vehicle (A and C) at
37°C. After 5 minutes, blood samples were drawn, immediately fixed,
and used for immunoelectron microscopy (A and B), and perfusions were
started for 5 minutes at a shear rate of 1,600 s-1 over
coverslips coated with collagen type III. Perfused coverslips were also
immediately fixed and used for immunoelectron microscopy (C and D).
Immunolabeling was performed on frozen thin sections with MoAb AK3 and
10 nm protein-A gold. Closed stars represent the OCS with gold
particles. Open star represents OCS without gold particles. Col,
collagen. Bar, 200 nm.
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Table 2.
Percentage of Gold Particles on the PM, the Membranes of
the OCS, -Granules, and Nondefined Structures (undefined) of
Unstimulated and TRAP-Stimulated Platelets in Suspension and After
Platelet Adhesion to Collagen Type III Under Flow Conditions
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After platelet adhesion to collagen type III, GPIb was redistributed to
the platelet surface. Both TRAP-stimulated and nonstimulated adhering
platelets showed pronounced cell surface ruffling (ie, pseudopod
formation) (Fig 4C and D). As shown in Table 2, the percentage of GPIb
on the platelet surface of vehicle-treated platelets was 64.4% ± 3.7% (mean ± SEM, n = 3). The percentage of GPIb on the
platelet surface of TRAP-treated platelets was 62.0% ± 4.5% (mean ± SEM, n = 4), which was not significantly different from
vehicle-treated platelets. In a parallel flow cytometric experiment, we
observed that the surface expression of GPIb on TRAP-stimulated
platelets present in the perfusate after perfusion was still reduced
(data not shown). This means that GPIb rapidly redistribute to the
platelet surface on adhered TRAP-stimulated platelets, but not on
TRAP-stimulated platelets in suspension. The perfusion process by
itself did not influence the time-dependent return of GPIb to the
platelet surface in circulating platelets.
To determine the critical number of GPIb receptors on the platelet
surface at which platelet adhesion becomes inhibited, we performed the
following experiments. Whole blood, anticoagulated with Orgaran (15 U/mL) in the presence of dRGDW (200 µmol/L) was preincubated for 30 minutes with increasing concentrations of MoAb 6D1, which inhibits
GPIb-mediated functions.23 After 30 minutes, the blood was
perfused for 5 minutes at a shear rate of 1,600 s-1 over
collagen type III in a single-pass perfusion system. Immediately before
the start of perfusion, blood samples were drawn for flow cytometry
analysis. As shown in Fig 5, a blockage of
44% of GPIb with 6D1 had no effect on platelet adhesion to collagen
type III. When 56% of GPIb was occupied by 6D1, adhesion was inhibited
by 18%. Adhesion decreased in a dose-dependent manner when blood was
preincubated with higher concentrations of 6D1.
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| Fig 5.
Effect of GPIb-receptor occupation by MoAb 6D1 on
platelet adhesion to collagen type III at a shear rate of 1,600 s-1. Orgaran anticoagulated whole blood (+dRGDW) was
incubated with MoAb 6D1 at room temperature for 30 minutes. The blood
was then prewarmed at 37°C for 5 minutes. After prewarming, a
sample was drawn for flow cytometry analysis and immediately thereafter
the perfusion runs were started. The amount of free GPIb () was
determined by flow cytometry. The platelet coverage () is expressed
as percent control (without MoAb 6D1). Data are obtained in three
independent experiments with blood from three different donors (mean ± SEM, n = 3). * Significantly different from control.
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To determine the total amount of GPIb present on the surface, a
constant concentration of FITC-conjugated MoAb 6.20 was added after
platelets were fixed and washed for flow cytometry. FITC-conjugated MoAb 6.20 binds to GPIb, but has no effect on GPIb-mediated functions. The total amount of GPIb present on the platelet surface, as detected by FITC-conjugated MoAb 6.20, remained constant, independent of the
binding with MoAb 6D1.
| |
DISCUSSION |
Earlier flow cytometric studies with MoAbs against GPIb-IX have shown
that GPIb is downregulated on the platelet surface when platelets are
stimulated in vitro by plasmin, -thrombin, or
TRAP.8,33-35 Downregulation of GPIb was also shown in vivo
during CPB surgery13,14 in blood emerging from
bleeding-time wounds9 and in patients with severe
atherosclerosis.36 As a consequence of this decreased number of GPIb on the platelet surface and because of the central role
of GPIb in platelet adhesion, the platelet adhesion to the vessel wall
may be impaired.
Despite the substantial number of publications about the reduced
surface expression of GPIb,8-12,33-36 it is still not known whether a decrease of GPIb on the platelet surface has any functional consequences for the adhesive capacity of platelets. For this reason,
we studied platelet adhesion under flow conditions using platelets with
a reduced number of GPIb on the surface. In the present study, we show
that a 50% reduction of platelet surface GPIb has no effect on the
GPIb-mediated platelet adhesion under flow conditions. To reduce the
amount of functional GPIb on the platelet surface, two independent
strategies were followed.
First, platelets were stimulated in Orgaran anticoagulated blood with
15 µmol/L TRAP. dRGDW was added to avoid platelet aggregation. The
effectivity of TRAP stimulation was shown by flow cytometry, ristocetin-induced platelet agglutination, and electron microscopy. In
flow cytometric studies, we showed that stimulation of platelets with
TRAP decreased the amount of GPIb present on the platelet surface by
approximately 65%. The TRAP-induced disappearance of GPIb was maximal
approximately 5 minutes after the addition of TRAP and was followed by
a time-dependent return of GPIb to the platelet surface. Immunoelectron
microscopy on frozen thin sections of TRAP-stimulated platelets
confirmed the decrease in GPIb on the platelet surface observed in flow
cytometry. The decrease of GPIb on the platelet surface was accompanied
by an increase of GPIb within the OCS and is in agreement with previous
immunoelectron microscopy studies in which platelets were activated by
thrombin.8,10 As previously observed with
thrombin,10,37 the TRAP-induced disappearance of GPIb in
our present study resulted in a decreased RIPA.
The results from our perfusion experiments clearly showed that a 65%
reduction of GPIb on the platelet surface had no effect on the
GPIb-mediated platelet adhesion to collagen type III and ECM under flow
conditions. As previously shown by Nurden et al,38 the
collagen receptor GPIaIIa does not show a reduction in surface expression on the platelet and therefore may contribute to normal adhesion.
Immunoelectron microscopy showed, however, that TRAP-stimulated
platelets adhering to collagen type III had almost the same surface
expression of GPIb as unstimulated platelets. These findings suggest
that on adhesion GPIb rapidly returns to the platelet surface. The
number of GPIb on the surface of TRAP-stimulated platelets still
present in the perfusate after perfusion did not differ from that of
TRAP-stimulated platelets before perfusion, suggesting that the
redistribution of GPIb to the platelet surface is induced when
platelets adhere. From these experiments however, it is not clear to
what extent GPIb returning from the OCS to the platelet surface
contributes to platelet adhesion to collagen.
To further investigate this issue, we followed a second approach and
determined the critical number of GPIb receptors necessary for platelet
adhesion under flow. We used a MoAb against GPIb to inhibit the
function of GPIb on the platelet surface. Using this approach, we could
show that inhibition of platelet adhesion only occurs when more than
50% of platelet surface GPIb is blocked.
The critical percentage of platelet surface GPIb necessary for platelet
adhesion under flow varied between the different approaches followed
and probably also by the different antibodies used.11 Because a rapid return of GPIb to the platelet surface was observed in
adhering platelets, the critical 50% reduction found by blocking the
GPIb receptor is probably more realistic.
Apart from the controversial studies with respect to the clearance of
GPIb from the platelet surface when platelets are stimulated with
thrombin,11,39 we show here that only 50% of platelet surface GPIb is necessary for platelet adhesion under flow conditions. These results are in agreement with previous studies with carriers of
the inherited disease Bernard-Soulier syndrome, who lack about 50% of
GPIb, but have no bleeding complications.17,18 Our present findings further suggest that the observed disappearance of GPIb during
CPB by itself, which is usually less than 50%, is unlikely to affect
platelet adhesion.
In conclusion, a 50% reduction of platelet surface GPIb has no effect
on platelet adhesion under flow conditions. GPIb is not only essential
for platelet adhesion, but also for platelet-platelet interaction at
high shear rates.40-42 At present, it is not clear why GPIb
is reversibly expressed on the platelet surface. It is quite possible
that the reversible surface expression of GPIb represents a refined
mechanism to control thrombus formation rather than adhesion at high
shear rates.
| |
FOOTNOTES |
Submitted August 11, 1997;
accepted November 19, 1997.
Supported by Grant No. 93.112 from The Netherlands Heart Foundation.
Address reprint requests to Jan J. Sixma, MD, University Hospital
Utrecht, Department of Hematology, PO Box 85500, 3508 GA Utrecht, The
Netherlands.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
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Abstract
Introduction
Abstract