© 1999 American Heart Association, Inc.
Thrombosis |
Studies of Adhesion-Dependent Platelet Activation
Distinct Roles for Different Participating Receptors Can Be Dissociated by Proteolysis of Collagen
From the Wihuri Research Institute (P.S., R.L.) and the Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki (P.S.), Helsinki, Finland.
Correspondence to Dr Riitta Lassila, Wihuri Research Institute, Kalliolinnantie 4, SF-00140 Helsinki 14, Finland. E-mail riitta.lassila{at}wri.fimnet.fi
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Abstract—The molecular differences between native-type collagen type I fibrils (NC) and their pepsinated monomers (PC) were used to uncover receptors involved in platelet-collagen interaction along the adhesion-activation axis. The platelet-depositing capacity of NC and PC under blood flow and their adhesive properties and respective morphologies, aggregation, procoagulant capacity, and tyrosine phosphorylation were compared under different cationic milieus, including or excluding the glycoprotein (GP) Ia/IIa. NC was consistently a more preferable and activating substrate than PC during flow (5 minutes) and in platelet aggregation. In PPACK-treated blood, both NC (3.3-fold) and PC (2.7-fold) increased platelet attachment on elevation of the shear rate from 500 to 1640 s-1, whereas in citrated blood, adhesion and thrombus growth on PC were negligible under the high shear rate, unlike on NC (1.9-fold increase). The complete lack of platelet deposition on PC in citrated blood could be overcome by restoring physiological Mg2+ concentration, and in contrast to NC, platelets interacting with PC were highly dependent on Mg2+ during adhesion, aggregation, and procoagulant response. Monoclonal antibody (mAb 131.7) against GP IV inhibited platelet deposition to NC in citrated blood (2 minutes) by 49%, which was not further increased by coincubation with mAb against GP Ia (6F1). These results stress the importance of GP Ia/IIa in shear-resistant platelet deposition on collagen monomers. In native fibers, however, the preserved quaternary structure with telopeptides activates additional platelet receptors capable of substituting GP Ia/IIa and GP IV.
Key Words: hemostasis • platelet • collagen • receptor • divalent cations
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Introduction |
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On exposure of subendothelial collagen to flowing blood, adhering platelets, with their subsequent activation and procoagulant capacity, initiate hemostasis and thrombosis.1 2 3 4 Pathophysiological platelet-collagen interactions include bleeding disorders due to either defective receptors or autoantibodies2 3 5 or to impaired collagen synthesis,6 7 8 indicating several functional determinants on both the platelets and collagen.
Thus far, glycoprotein (GP) Ia/IIa, integrin
2ß1, has been
recognized as the major platelet receptor for collagen in primary
adhesion, and accordingly, dysfunctional GP Ia/IIa associates with
prolonged bleeding tendencies in patients.9 10 11 Although
GP IV has been implicated in the very early phases of adhesion, the
impact of GP IV,12 13 14 as well as the other receptor
candidates,15 16 17 on hemostasis has remained unsettled. GP
VI is involved in collagen-induced platelet activation, evidenced
both by a poor aggregation response in patients lacking this
receptor5 and by collagen-related peptides that likely
induce strong platelet aggregation in normal platelets via this
receptor.18 19 However, patients lacking GP VI have only
mild bleeding tendencies,5 and under blood flow, the
collagen-related peptides are unable to retain firm
adhesion.20 Qualitative differences in collagen
preparations complicate the identification of the determinants in
platelet-collagen interaction. The term "native collagen
fibers" has been used to cover a spectrum of preparations, from
suspensions of native fibrils (eg, Horm) to dialysed fibrils consisting
of salt-soluble tropocollagens, but also "fibrils" formed after
dialysis of protease-extracted collagen monomers. However, the removal
of telopeptides, eg, by pepsin, prevents the formation of fibrils,
resulting instead in small nonbanded fibrous aggregates,21
and the severity of the impaired fibril formation depends on the extent
of the pepsin digestion.22 Thus, the structural
differences in collagen preparations are at least partly responsible
for the inconclusive data on the determinants of the
platelet-collagen interaction. Moreover, studies focusing on these
effects under blood-flow conditions have been limited,23
most of the studies having used static adhesion and traditional
platelet aggregation techniques.24 25
To obtain more information about the platelet-recruiting capacity and activating potential of type I collagen, the collagen monomers derived from fibrils by extensive pepsin treatment (eliminating telopeptides and quaternary structure) were used as a tool to investigate the responsible determinants. These fibrils and monomers were compared as substrates in experiments that dissociated the hemostatic events evolving from the platelet-collagen interaction under flowing blood: adhesion, aggregate formation, tyrosine phosphorylation, and procoagulant activity.
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Methods |
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5-Hydroxy(G-3H)tryptamine creatinine sulfate (5-HT) was obtained from Amersham International plc. Aurin tricarboxylic acid (ATA, MW 473.4) and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) peptide were from Sigma. Horseradish peroxidase (HRP)–conjugated mouse anti-human von Willebrand factor (vWF), HRP-conjugated goat anti-mouse IgG antibodies, and monoclonal antibody (mAb) AN51 (against GP Ib) were from Dakopatts A/S; mAbs Gi9 (against GP Ia), SZ2 (against GP Ib), and SZ22 (against GP IIb) were from Immunotech SA; and 4G10 (against phosphotyrosine) was from Upstate Biotechnology, Inc. S-2238 was from Chromogenix AB and Thromborel S from Behringwerke AG. Bovine
-thrombin was from Dade
(Baxter Healthcare Co). mAbs 131.7 (against GP IV) and 6F1 (against GP
Ia, 2) were kind gifts from Dr Narendra Tandon
(Otsuka America Pharmaceutical, Inc, Rockville, Md) and Dr Barry Coller
(Mount Sinai Medical Center, New York, NY), respectively, and vWF was
obtained from Dr Thierry Burnouf (Center Regional de Transfusion
Sanguine, Lille, France).
Preparation of Collagen
Collagen type I fibrils from bovine Achilles’ tendon were
extracted with 0.5 mol/L acetic acid and salt-precipitated with 1.7
mol/L NaCl, essentially as previously described.26 To
obtain collagen monomers (PC) devoid of quaternary structure and
telopeptides, native-type collagen fibrils (NC) were extensively
treated with pepsin [3 additions of pepsin (Sigma Chemical Co), 1
mg/mL, rotation for 3 weeks at 4°C] and salt-precipitated. After the
collagen stock solutions were centrifuged at
100 000g for 1 hour at 4°C, only 2% of NC
(acid-insoluble collagen) but >90% of PC (acid-soluble collagen)
remained in the supernatant. The collagen concentrations were
determined by a modified hydroxyproline assay27 or
with Sircoll dye from Biocolor Ltd. The structural properties of the
collagen preparations were analyzed by transmission electron
microscopy (TEM) (JEOL 1200EX). Collagen stocks were
immobilized onto gold grids supported with
carbon-coated polyvinyl formal plastic film, and negatively stained
with 1% potassium phosphotungstic acid, pH 7.3, for 1 minute at
22°C.
Blood Collection
The study was approved by an institutional review board. Blood
was obtained from healthy volunteers who had not used any medication
during the previous 10 days. Nine volumes of free-flowing blood were
collected into 1 volume of either (1) 30 µmol/L (final
concentration)
D-phenylalanyl-L-prolyl-L-arginine
chloromethyl ketone (PPACK) (Calbiochem-Novabiochem Corp); (2) 90
mmol/L sodium citrate, additionally adjusted depending on the donor’s
hematocrit28 ; or (3) acidic citrated dextrose, pH 4.5, for
gel-filtered platelets (GFPs). The anticoagulants were chosen to
yield various concentrations of divalent cations that affect the
function of GP Ia/IIa.10 29 The concentration of ionized
magnesium in blood anticoagulated with citrate was 8 µmol/L, and
with PPACK, it was 0.62 mmol/L, as measured with a Microlyte 6
electrolyte analyzer (Kone Instruments Corp). The
concentrations of ionized magnesium in platelet-rich plasma (PRP)
were 4 µmol/L in citrate and 0.61 mmol/L in PPACK. The
concentrations of ionized calcium were 86 µmol/L in citrated and
1.37 mmol/L in PPACK-treated blood, and 79 µmol/L and
1.37 mmol/L in PRP, respectively. Platelet counts and
hematocrit were determined by a Thrombocounter Coulter T-540 (Coulter
Electronics, Inc).
Platelet Preparation
GFPs for adhesion studies were prepared from PRP after a single
washing step in the presence of prostaglandin
E1 (25 ng/mL) and apyrase (1 U/mL) (Sigma) by
elution of the platelet suspension through a Sepharose 2B column
(Pharmacia LKB Biotechnology Inc) in HEPES buffer without divalent
cations.30 Platelets were labeled with
[3H]5-HT (specific activity, 13 Ci/mmol), 12
nmol/mL of PRP, or GFPs for 15 minutes at 37°C. The
[3H]5-HT labeling method has been validated
previously.31
Coverslips With Immobilized Collagen
Thermanox coverslips (NUNC), to be used during the same day,
were coated by spraying 10 times with NC or PC (0.36 mg/mL)
alternating with neutralizing buffer and then incubated (90
minutes, 35°C) to induce in situ fibril formation.32 For
observation under light microscope, the coverslips were stained with
Coomassie brilliant blue R250.
Platelet Deposition on Immobilized Collagen Under
Flow Conditions in Whole Blood
To study platelet deposition (adhesion-dependent
platelet aggregate formation), whole blood was perfused in
parallel-plate perfusion chambers with defined rheological
characteristics.31 33 34 After the PRP was labeled, the
blood was reconstituted without adjustment of the platelet counts.
When indicated, the PRP was incubated with mAb 6F135
(final concentration, 10 µg/mL) and/or mAb 131.736 (5
µg/mL) for 20 minutes at 22°C before the blood was reconstituted
and then allowed to stabilize for 30 minutes. The prewarmed (37°C)
blood aliquots were recirculated over coverslips for 5 minutes at shear
rates of 500 or 1640 s-1. Then the samples were
briefly perfused and rinsed with PBS, and the deposited
3H activity was measured in a liquid
scintillation counter (1414 Rackbeta, Wallac), as described
previously.31 The perfusions were completed within 3.5
hours of blood collection.
Scanning electron microscopy (SEM; JEOL SEM-820) was used to analyze the morphology of platelet deposition and adhesion. The samples were fixed with 2.5% phosphate-buffered glutaraldehyde at 22°C for 2 hours. After rinsing with PBS, the samples were dehydrated, critical point–dried under CO2, and sputter-coated with gold or platinum.
Platelet Adhesion on Immobilized Collagen
GFPs (100x106/mL) were labeled as
described. Collagen-coated coverslips were placed in 24-well
flat-bottom multidishes (NUNC) precoated with 2% human serum
albumin, and GFPs (0.5 mL) were incubated stationary for 30
minutes at 22°C in the presence of 2 mmol/L of either
Ca2+ or
Mg2+.10 Afterwards, the coverslips
were rinsed in PBS and subjected to scintillation counting. This
platelet density and stationary conditions were chosen to promote
adhesion without inducing platelet-platelet interaction. When
indicated, GFPs were preincubated for 30 minutes35 with
mAb Gi9 (20 µg/mL) or 6F1 (10 µg/mL), which had been dialyzed to
remove NaN3.
Platelet Aggregation
Aggregation of platelets (300x106 mL
in suspension or in PRP anticoagulated with either acidic citrated
dextrose, pH 6.5, or PPACK) was measured turbidimetrically in a
dual-channel aggregometer (Payton Associates Inc). The samples were
stirred at 700 rpm and preincubated for 1 minute at 37°C before the
addition of collagen. The rates of primary aggregation
(min-1) and maximal aggregation (%) at 5
minutes were assessed.
Tyrosine Phosphorylation
GFP suspensions (500x106/mL),
preincubated with 300 µmol/L of KQAGDV, were stimulated in an
aggregometer with 1.5 µg/mL of NC or preformed PC "fibrils"
(HEPES buffer, 1.5 hours, 35°C) in the presence of 2 mmol/L of
either Ca2+ or Mg2+. At
selected time points, an equal volume of 2x SDS Laemmli sample buffer
with 3 mmol/L sodium orthovanadate was added. The samples (20
µL) were electrophoresed on a 7.5% SDS-polyacrylamide gel
and transferred onto nitrocellulose membrane (Probind 45, Pharmacia).
After blocking, the membranes were incubated with 0.2 µg/mL of 4G10
and HRP-conjugated goat anti-mouse antibody. The tyrosine
phosphorylation patterns were visualized with the
enhanced chemiluminescence technique (ECL kit, Amersham). The blots
were stripped and then reprobed with SZ22 to control the platelet
amounts on different lanes.
Assessment of Procoagulant Activity of Collagen-Adherent
GFPs
After adhesion of GFPs on either NC or PC in the presence of
2 mmol/L of either Ca2+ or
Mg2+, the coverslips were washed and incubated
for 10 minutes with PBS containing 0.9 mmol/L
CaCl2. Then a 1:20 dilution of platelet-poor
plasma (PPP) was added, and the generation of thrombin was enhanced by
trace amounts of tissue thromboplastin (1:5000 final dilution of
Thromborel S) under agitation for 10 minutes at 37°C.37
From each well, 180 µL of supernatant was reacted with a
chromogenic substrate, S-2238 (200 µmol/L), for 6
minutes. Absorbance was measured at 405 nm (Labsystems Multiscan MCC).
A control was included by incubating PPP and thromboplastin over
collagen-coated coverslips without adhered platelets. This control
value was deducted from the values obtained with platelet-adhered
coverslips. The thrombin amounts formed were calculated by use of known
concentrations of bovine -thrombin (Dade) in a standard curve.
Statistics
The data are presented as mean±SD and were
analyzed with a paired Student’s t test. Regression
analysis was applied to study the association between the
adhesion results. All assays were done in duplicate, and n refers to
the number of donors. Because of the great interindividual differences
in platelet responses to collagen,38 all
comparisons between NC and PC in a given experiment were performed in
samples from the same donor.
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Results |
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Collagen Structure
The Coomassie blue–stained collagen-coated coverslips were viewed by light microscopy after the treatment to induce fibrils. These were thick bundles of collagen fibrils that clearly differed from segmented strands of collagen monomers (Figure 1
, A
and B). The collagen preparations were negatively stained and
analyzed for periodicity in TEM to verify that the native type
I collagen was in the form of true fibrils. Indeed, this preparation
displayed periodic fibrils, whereas the PC monomers failed to do so
(Figure 1, C and D), even after the treatment to induce
"fibrils."
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Whole-Blood Perfusion Studies
To dissociate the involvement of different receptors during
platelet-collagen interaction, the platelet-recruiting capacity
of NC and PC was compared under various shear rates and anticoagulants:
a low to intermediate shear rate of 500 s-1 and
a high shear rate of 1640 s-1 and blood
anticoagulated with either citrate (86 µmol/L
Ca2+, 8 µmol/L Mg2+)
or 30 µmol/L PPACK (normal cations; 1.37 mmol/L
Ca2+, 0.62 mmol/L
Mg2+), respectively. Under the perfusion
conditions tested, platelets predominantly preferred NC over PC
(Figure 2A).
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In PPACK-anticoagulated blood, platelet deposition was not
significantly larger on NC than on PC at the low shear rate of 500
s-1 (5.4±2.7 versus
3.7±1.7x106/cm2), but at
the high shear rate of 1640 s-1, almost 2 times
more platelets deposited on NC than on PC (17.8±10.3 versus
10.0±6.8x106/cm2) (Figure 2A
). The shear-dependent increase in platelet deposition was
of similar magnitude on both substrates: 3.3-fold on NC and 2.7-fold on
PC, suggesting that the vWF-mediated platelet deposition was not
markedly affected by the differences between NC and PC. This result was
further confirmed by use of ATA as an inhibitor for
vWF-mediated platelet deposition on collagen.39 At the
high shear rate, ATA inhibited platelet deposition in a similar
manner for both PC and NC, by 
40% (data not shown).
In citrated blood devoid of Mg2+, which is
important for GP Ia/IIa activity, the total platelet deposition
diminished on both substrates (Figure 2A), and the
platelet-recruiting differences between NC and PC were accentuated.
Over 3-fold more platelets deposited on NC than on PC at the shear
rate of 500 s-1 (3.9±0.7 versus
1.2±0.4x106/cm2). The
difference between the collagens was even more profound when the shear
rate was elevated to 1640 s-1, which resulted in
15 times larger platelet deposition on NC than on PC (7.6±3.1
versus 0.5±0.2x106/cm2).
NC showed a shear-induced increase in platelet deposition, in
contrast to PC, on which platelet adhesion and subsequent
aggregation were severely impaired at the high shear rate. This effect
was also clear in the SEMs of the perfusion channels subjected to the
low versus high shear rate: whereas full aggregates were able to form
on PC under the low shear force (Figure 2B), platelets and
platelet aggregates were absent under the high-shear-rate
conditions (Figure 2C).
Because of the lack of platelet deposition on PC under the high shear rate, we assessed the binding of vWF from citrated plasma to NC and PC under both stationary conditions and flow. Detected with HRP-conjugated mAb, NC bound more vWF than PC under stationary conditions. However, in flow under high-shear-rate conditions, vWF binding from citrated PPP with washed and glutaraldehyde-fixed red blood cells28 did not differ (data not shown). Furthermore, incubation of the PC surface with vWF (10 µg/mL) before the perfusion did not improve the defective platelet deposition (data not shown). However, when the physiological level of Mg2+ cations was restored in citrated blood (by addition of 7 mmol/L MgCl2, Microlyte 6 analyzer), platelet deposition on PC was corrected, reaching up to 84% (66±20%, n=4) of that displayed in PPACK-treated blood under the high-shear-rate conditions. The restoration of physiological magnesium concentration allowed platelet deposition on PC to reach 61% and 83% of the NC-induced platelet deposition at the high and the low shear rates, respectively (n=2). In PPACK blood at 3 minutes of perfusion, 6F1 dropped the deposition to PC by 92% (n=1). Interestingly, additional determinants of NC could circumvent the functionally restricted GP Ia/IIa, as evidenced by the moderate platelet deposition in citrated blood at both shear rates. Furthermore, at the low shear rate, the platelet deposition on NC in citrated blood did not essentially differ from platelet deposition in PPACK blood (P=0.16). Finally, the inhibitory effects of mAbs against GP IV and GP Ia were tested on platelet deposition to NC after 2 minutes of perfusion. mAb 131.7 inhibited 49±16% (n=5) of platelet deposition on NC at high shear rate in citrated blood. Under these conditions, the addition of 6F1 did not further inhibit platelet deposition (data not shown).
SEM supported the results of [3H]5-HT–labeled
perfusions, illustrating both the surface coverage and the morphology
of deposited platelets (Figure 3). At
the low shear rate, irrespective of the collagen surface, and in the
same anticoagulant, platelets and aggregates were fairly evenly
distributed, but the size of the aggregates varied (Figure 3, A
through D). The high shear rate increased the morphological
differences, which depended on both the anticoagulant and the substrate
(Figure 3, E and F). The relatively low PPACK concentration was
chosen to allow adhesion-dependent activation events and possible
formation of local fibrin. In PPACK-anticoagulated blood, platelet
deposition on NC ranged from single platelets to multilayered
aggregates (Figure 3E), whereas platelet deposition on PC
seemed more uniform (Figure 3F). Strand-like structures, most
likely representing locally formed fibrin, were found
between the aggregates. However, it has been shown previously that at
this PPACK concentration, circulating thrombin–antithrombin III
complexes were not elevated after the perfusion.31 In
citrated blood, platelet recruitment on NC resembled that on PC in
PPACK blood (Figure 3G). Contrasting the evenly covered
perfusion channel with PC at 500 s-1 (Figure 3, B and D) or with NC under all conditions (Figure 3, A,
C, E, and G), platelets were virtually absent on PC in citrated
blood under the high shear rate (Figure 3H). Figure 3H
shows the few platelets deposited only close to the channel edges
(Figure 2C), where the shear rate decreases.
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Altogether, the necessity of functional GP Ia/IIa was obvious in platelets adhering to PC under high-shear-rate conditions: The addition of Mg2+ to blood was able to correct the lacking platelet deposition in citrated blood, and a mAb against GP Ia significantly inhibited platelet deposition in PPACK blood. The structural determinants of NC, however, were able to attract platelets via receptors other than GP Ia/IIa and GP IV and to enhance platelet activation.
Adhesion Studies
Because platelet deposition depended on the
shear-resistant buildup of the thrombus on adhesion, we next
studied the primary adhesion of platelets on PC and NC in the
presence of 2 mmol/L Mg2+ or
Ca2+ and specifically the role of GP Ia/IIa
therein.10 In the presence of Mg2+,
PC was consistently a significantly preferable substrate for
platelet adhesion than NC (Figure 4).
The adhesion to PC correlated well with the adhesion to NC
(r=0.83, P<0.005), indicating the involvement of
the same platelet receptor, ie, GP Ia/IIa, in adhesion to both PC
and NC. On the contrary, switching the cations from
Mg2+ to Ca2+ strongly
decreased platelet adhesion to PC, resulting in 2.4 times larger
adhesion to NC than to PC, probably because of the closure of GP Ia/IIa
for collagen.40 In sharp contrast to PC, switching
the cations from Mg2+ to
Ca2+ enhanced platelet adhesion on NC (Figure 4). Ca2+-dependent platelet adhesion
to NC did not correlate with Mg2+-dependent
adhesion to PC (r=0.41), but it did correlate with the
adhesion to NC in the presence of Mg2+
(r=0.76, P<0.01), suggesting distinct receptors
for NC under the differing divalent cation milieus as well as an
interplay between GP Ia/IIa and another receptor(s).
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Blocking GP Ia with mAbs (6F1 or Gi9, both IgG1 subclasses) decreased
Mg2+-dependent platelet adhesion to PC (63%
and 52%, respectively) (Figure 5A). The extent of this inhibition
was similar to that caused by the switching of
Mg2+ to Ca2+, and in the
presence of Ca2+, 6F1 did not further inhibit
adhesion to PC. The mAbs did not inhibit platelet adhesion on NC
when either Mg2+ or Ca2+
was provided (Figure 5B). In contrast, in the presence of
Mg2+, 6F1, but not Gi9, rather increased adhesion
on NC. As a control, mAbs against GP Ib did not affect adhesion on
either PC or NC (data not shown).
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SEM revealed interesting cation-dependent aspects of platelet
morphology and confirmed that the selected assay conditions favored
adhesion of single platelets (Figure 6). In the presence of
Mg2+, PC- and NC-adherent platelets appeared
similar, showing many pseudopods and full spreading of the cytoplasm
(Figure 6, A and C). In the presence of
Ca2+, only a few platelets adhered on PC, and
they retained a round shape with a few projections (Figure 6B). In contrast, platelets on NC projected pseudopods
and were still able to spread (Figure 6D). In addition, a
notable proportion of "spongy"-appearing platelet ghosts was
detected, which had swollen and no longer remained spread or had
pseudopods.
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The adhesion results confirmed the blood flow studies in that GP Ia/IIa was essential for primary platelet adhesion to PC, whereas it was not mandatory for NC. Unlike PC, NC possessed the ability for adhesion and activation-related morphological changes in the presence of Ca2+.
Aggregation Studies
Because of the better aggregate-forming capacity of NC during
blood flow, platelet aggregation was studied with PPACK-treated and
citrated PRP and GFPs in the presence of divalent cations. Here, the
platelet preference for NC over PC was also more obvious in
citrated PRP, in which a 4-fold higher concentration of PC was
needed to induce maximal aggregation of an extent similar to that with
NC, but the lag time still remained twice as long (Table 1). In PPACK PRP, the 4-fold
higher concentration of PC resulted in a compatible lag time, rate, and
maximal aggregation. Although during the NC-induced aggregation the lag
time did not vary with the anticoagulation, the rate was enhanced,
benefiting from the presence of cations. The same
cation-dependent enhancement was observed with PC. To exclude the role
of fibril formation in the extended lag time, PC was preincubated at
35°C for 2 hours in a neutralizing buffer. Although this treatment
improved the aggregating capacity of PC, the lag time, rate, and
maximal aggregation all failed to match aggregation induced by the
corresponding concentrations of NC (data not shown). 6F1 affected PC
significantly more than NC by prolonging lag time and reducing the rate
and maximal aggregation in citrated PRP, and these effects were even
more pronounced in PPACK PRP (data not shown). The aggregation data of
GFPs were complementary to those of PRP (Table 2). The aggregating capacity of PC was
severely impaired when Mg2+ was omitted and
Ca2+ was present. This also prolonged the lag
time of NC-triggered aggregation, but once the aggregation started, its
rate and maximum were the same irrespective of the ionic environment.
In contrast to plasma milieu, fibril-forming treatment improved the
PC-induced aggregation response of GFPs, especially in the presence of
Mg2+ (data not shown).
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Studies on Tyrosine Phosphorylation as a Marker
of Activation
Time-dependent tyrosine phosphorylation was also
assessed to capture the differential NC- and PC-induced activation of
platelets. To discard the phosphorylation events
dependent on GP IIb/IIIa activation, an inhibitory peptide
(KQAGDV) was used. In the presence of Mg2+, both
NC and prefibrillated PC induced similar patterns of
tyrosine-phosphorylated proteins, but with NC, the
kinetics of phosphorylation was slightly faster than
with PC (Figure 7). The differences in
phosphorylation patterns were accentuated in the
presence of Ca2+, when several bands were not
visible during PC induction or appeared only at the latest time points.
The switch of cations also changed the NC-induced
phosphorylation: in the presence of
Ca2+, several bands appeared slowly or their
intensity decreased.
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Studies of Procoagulant Activity on Collagen-Adherent
Platelets
Finally, the procoagulant activity of NC- and PC-adherent
platelets was assessed by measurement of thrombin formation.
Platelets adhering on NC had consistently increased
procoagulant capacity compared with platelets adhering on PC, and
platelets, which originally adhered to NC in the presence of
Ca2+, generated the most thrombin (Figure 8). However, if the GFPs were first
allowed to adhere in the presence of Mg2+ and the
procoagulant activity was then developed by the addition of external
Ca2+, significant thrombin formation was observed
on PC, albeit still less than on NC when related to the number of bound
platelets. Thrombin formation (µU ·
106 GFPs-1 ·
min-1) was 11±4 (PC,
Mg2+) <18±11 (NC,
Mg2+) < 30±15 (NC,
Ca2+), n=4.
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Structural differences resulting from the removal of collagen telopeptides and native quaternary structure (Figure 1
) lead to
reduced activation potential of the collagen, whereas the adhesive
capacity remained, as illustrated by relatively similar surface
coverages on both the collagen fibrils and monomers during perfusion
(Figures 2 and 3) (except at the high shear rate in
citrated blood) and adhesion experiments with GFPs in the presence of
Mg2+ cations (Figure 4). As previously
reported, adhesion to monomeric collagen (1) was
Mg2+-dependent and mediated by GP
Ia/IIa,10 (2) was inhibited by Ca2+
cations,10 29 and (3) resulted in complete spreading of
platelets41 (Figures 4, 5, and 6). Our most interesting finding was the complete cation-dependence of the shear force–resistant thrombus formation on PC, in contrast to NC, which were capable of inducing thrombus formation cation-independently. Compatible with our other findings, this result manifests the importance of GP Ia/IIa as a mandatory primary receptor of platelet interaction with the collagen triple helix, as also reported by others.42 In support of our results, it was recently highlighted that GP Ia/IIa is irreplaceable in resisting shear forces.20 43 Being a poor adhesive substrate for GFPs in the presence of Ca2+ or in the absence of divalent cations in blood flowing at the high shear rate, monomers revealed the importance of the first collagen-adherent platelets in thrombus formation.37 Collagen monomers may offer a way to study the role and donor variability of GP Ia/IIa, which are likely to be associated with certain receptor polymorphisms and even thrombotic events.44 45
The differences between native-type collagen fibrils and monomers cannot be explained by differential vWF-binding capacity, because (1) vWF binding to collagen has been reported to be cation-independent,46 (2) the vWF-dependent increase in platelet deposition was observed on both substrates in PPACK-anticoagulated blood, (3) it was similarly inhibited by ATA,39 and (4) incubation of the monomer surface with vWF before perfusion (citrated blood, high shear rate) was unable to correct for the lacking platelet deposition, in contrast to restoration of the physiological Mg2+ concentration.
Adhesion to monomers correlated strongly with adhesion to fibrils in
the presence of Mg2+, whereas
Mg2+-dependent adhesion on monomers did not
correlate with adhesion on fibrils in the presence of
Ca2+, suggesting that at least 2 different
receptors mediate platelet adhesion to collagen, depending on the
experimental conditions and the substrate. Existence of additional
collagen receptor(s) capable of replacing GP Ia/IIa was further
supported by the ability of collagen fibrils to withhold thrombus
formation under the high shear force (Figures 2 and 3)
and the unchanged adhesion under the conditions in which GP
Ia/IIa–inhibiting antibodies and Ca2+ cations
were present (Figures 4 and 5). In the presence of GP
Ia– and GP IV–blocking antibodies, 50% of the platelet
deposition remained in citrated blood at high shear rate, compatible
with an unknown platelet adhesion receptor for native-type collagen
fibrils.
In addition to containing the molecular determinants for GP Ia/IIa–
and GP IV–independent adhesion, collagen fibrils induced platelet
activation more efficiently than monomers, as also recently suggested
by increased thromboxane A2
production triggered with acid-insoluble
collagen.14 The better platelet-binding and
aggregate-forming capacity and independence of divalent cations of
fibrils observed during perfusions were supported by the aggregation
studies, in which 
4 times fewer native-type fibrils than collagen
monomers were needed, as reported previously.47 The
morphology of adherent GFPs suggested that collagen fibrils
activated platelets, in contrast to monomers (Figure 6). The receptor(s) using fibrils benefited from the presence of
Ca2+ and the conditions that favored
platelet-platelet interaction and activation (PPACK, high
shear, 37°C). In the presence of Ca2+,
augmented platelet activation by fibrils was also evidenced by the
tyrosine-phosphorylation events (Figure 7) and
enhanced procoagulant capacity (Figure 8). Conversely, the
similarities in tyrosine phosphorylation induced by NC
and PC in the presence of Mg2+ and the distinct
NC-induced tyrosine phosphorylation patterns in
different cationic milieus (Figure 7), as well as the equal
procoagulant responses of NC and PC if the platelets were first
allowed to bind in the presence of Mg2+ (Figure 8), all suggest that GP Ia/IIa also participates in platelet
activation.48 49 Recently, the interplay in the GP
Ia/IIa–GP IIb/IIIa axis was disturbed by mast cell–derived heparin
proteoglycans, which strongly inhibited aggregate formation subsequent
to adhesion on collagen.50
The identity of the hypothetical collagen receptor(s) acting on
determinants on native-type fibrils was not elucidated by these
experiments. The activating receptor is likely to be GP VI, which is
reported to induce potent signal transduction by
collagen51 52 or by agents acting solely through
it.53 54 In this study, GP VI may participate, because NC
induced faster tyrosine phosphorylation patterns, as
well as a procoagulant response,55 which could accord with
the morphological changes of platelet deposition on NC in the
presence of Ca2+ (Figure 6). However, it
has been reported that under blood flow, GP VI alone is incapable of
retaining adhesion, so it is unlikely that it acts as a primary
adhesive receptor.3 20 56 We found that in citrated blood,
an mAb against GP IV (131.7) was able to inhibit platelet
deposition on NC in the early adhesive phase by 49%, as reported
previously.36 Thus, it is possible that when GP Ia/IIa and
GP IV are eliminated in our perfusion experiments and when vWF is
unable to firmly stop platelets, a third, as yet unidentified
receptor plays a necessary part in the early platelet interaction
with native collagen fibrils.
In the light of the present understanding of platelet-collagen interaction,3 5 57 our results suggest that (1) GP Ia/IIa is an absolute requirement for the collagen triple helix to support shear-resistant thrombus growth, and thus monomers provide a tool to study GP Ia/IIa activity in individual donors; (2) the native fibril structure of the collagen molecule is involved in platelet-activating events such as aggregate formation, tyrosine phosphorylation signaling, and development of procoagulant activity; (3) an unknown receptor acting via quaternary structure/telopeptides is capable of replacing GP IV–, GP Ia/IIa–, and cation-dependent functions in primary adhesion; and (4) when functional, GP Ia/IIa also participates in the events leading to platelet activation.
|
Acknowledgments |
|---|
The authors acknowledge the excellent technical assistance of Marja Lemponen and Tuula Järvenpää. We thank Dr Matti Vauhkonen for assistance in the collagen preparations and Dr Michael Hess (Electron Microscopy Unit, Institute of Biotechnology, University of Helsinki) for the TEM micrographs. While this work was in press, related findings were published: Savage B, Glusberg MH, Ruggeri ZM. Influence of fibrillar collagen structure on the mechanisms of platelet thrombus formation under flow. Blood. 1999;94:2704–2715.
Received February 3, 1999; accepted March 19, 1999.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
1. Baumgartner HR. Platelet interaction with collagen fibrils in flowing blood: reaction of human platelets with
-chymotrypsin-digested subendothelium.
Thromb Haemost. 1977;37:1–16.[Medline]
[Order article via Infotrieve]2. Kehler B. Platelet receptors for collagen. Platelets. 1995;6:11–16.
3. Sixma JJ, van Zanten H, Huizinga EG, van der Plas RM, Verkleij M, Wu Y-P, Gros P, de Groot PG. Platelet adhesion to collagen: an update. Thromb Haemost. 1997;78:434–438.[Medline] [Order article via Infotrieve]
4.
Heemskerk JWM, Vuist WMJ, Feijge MAH, Reutelingsperger
CPM, Lindhout T. Collagen but not fibrinogen surfaces induce bleb
formation, exposure of phosphatidylserine, and
procoagulant activity of adherent platelets: evidence for
regulation by protein tyrosine kinase-dependent
Ca2+ responses. Blood. 1997;90:2615–2625.
5. Moroi M, Jung SM. Platelet receptors for collagen. Thromb Haemost. 1997;78:439–444.[Medline] [Order article via Infotrieve]
6. Siegel BM, Friedman IA, Schwartz SO. Hemorrhagic disease in osteogenesis imperfecta: study on platelet functional defect. Am J Med. 1957;22:315–322.[Medline] [Order article via Infotrieve]
7. Johnson GJ, Holloway DE, Hutton SW, Duane WC. Platelet function in scurvy and experimental human vitamin C deficiency. Thromb Res. 1981;24:85–96.[Medline] [Order article via Infotrieve]
8. Hindriks GA, Sixma JJ, de Groot PG. Ascorbic acid increases thrombogenicity of cellular matrices. Thromb Haemost. 1991;66:505–509.[Medline] [Order article via Infotrieve]
9. Nieuwenhuis HK, Akkerman JWN, Houdijk WPM, Sixma JJ. Human blood platelets showing no response to collagen fail to express surface glycoprotein Ia. Nature. 1985;318:470–472.[Medline] [Order article via Infotrieve]
10. Santoro SA. Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen. Cell. 1986;46:913–920.[Medline] [Order article via Infotrieve]
11.
Kunicki TJ, Nugent DJ, Staats SJ, Orchekowski RP,
Wayner EA, Carter WG. The human fibroblast class II extracellular
matrix receptor mediates platelet adhesion to collagen and is
identical to the platelet glycoprotein Ia-IIa complex.
J Biol Chem. 1988;263:4516–4519.
12.
Saelman EUM, Kehler B, Hese KM, de Groot PG, Sixma JJ,
Nieuwenhuis HK. Platelet adhesion to collagen and
endothelial cell matrix under flow conditions is not
dependent on platelet glycoprotein IV.
Blood. 1994;83:3240–3244.
13.
Diaz-Ricart M, Tandon NN, Gomez-Ortiz G, Carretero M,
Escolar G, Ordinas A, Jamieson GA. Antibodies to CD36 (GPIV) inhibit
platelet adhesion to subendothelial surfaces under
flow conditions. Arterioscler Thromb Vasc Biol. 1996;16:883–888.
14.
Nakamura T, Jamieson GA, Okuma M, Kambayashi J, Tandon
NN. Platelet adhesion to native type I collagen fibrils: role of
GPVI in divalent cation-dependent and -independent adhesion and
thromboxane A2 generation.
J Biol Chem. 1998;273:4338–4344.
15.
Kotite NJ, Cunningham LW. Specific adsorption of a
platelet membrane glycoprotein by human insoluble
collagen. J Biol Chem. 1986;261:8342–8347.
16.
Deckmyn E, van Houtte E, Vermylen J. Disturbed
platelet aggregation to collagen associated with an antibody
against an 85/90 kD platelet glycoprotein in a patient
with prolonged bleeding time. Blood. 1992;79:1466–1471.
17. Chiang TM, Rinaldy A, Kang AH. Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen. J Clin Invest. 1997;100:514–521.[Medline] [Order article via Infotrieve]
18.
Morton LF, Hargreaves PG, Farndale RW, Young RD, Barnes
MJ. Integrin
2ß1-independent
activation of platelets by simple collagen-like peptides: collagen
tertiary (triple-helical) and quaternary (polymeric) structures are
sufficient alone for
2ß1-independent
platelet reactivity. Biochem J. 1995;306:337–344.
19. Barnes MJ, Knight CG, Farndale RW. Collagens and atherosclerosis: cell-collagen interaction. In: Jacotot B, Mathé D, Fruchart J-C, eds. Atherosclerosis XI. Singapore: Elsevier Science; 1998:299–306.
20.
Verkleij MW, Morton LF, Knight CG, de Groot PG, Barnes
MJ, Sixma JJ. Simple collagen-like peptides support platelet
adhesion under static but not under flow conditions: interaction
via 2ß1 and von
Willebrand factor with specific sequences in native collagen is
a requirement to resist shear forces. Blood. 1998;91:3808–3816.
21. Kadler KE, Holmes DF, Trotter JA, Chapman JA. Collagen fibril formation. Biochem J. 1996;316:1–11.
22. Leibovich SJ, Weiss JB. Electron microscope studies of the effects of endo- and exopeptidase digestion on tropocollagen: a novel concept of the role of terminal regions in fibrillogenesis. Biochim Biophys Acta. 1970;214:445–454.[Medline] [Order article via Infotrieve]
23.
Saelman UM, Morton LF, Barnes MJ, Gralnick HR, Hese KM,
Nieuwenhuis HK, de Groot PG, Sixma JJ. Platelet adhesion to
cyanogen-bromide fragments of collagen
1(I) under flow conditions. Blood. 1993;82:3029–3033.
24. Morton LF, Peachey AR, Barnes MJ. Platelet-reactive sites in collagens type I and III: evidence for separate adhesion and aggregatory sites. Biochem J. 1989;258:157–163.[Medline] [Order article via Infotrieve]
25.
Santoro SA, Walsh JJ, Staatz WD, Baranski KJ. Distinct
determinants on collagen support
2ß1 integrin-mediated
platelet adhesion and platelet activation. Cell
Regul. 1991;2:905–913.[Medline]
[Order article via Infotrieve]
26. Miller EJ, Rhodes RK. Preparation and characterization of the different types of collagen. Methods Enzymol. 1982;82:36–64.
27. Kivirikko KI, Laitinen O, Prockop DJ. Modifications of a specific assay for hydroxyproline in urine. Anal Biochem. 1967;19:249–255.[Medline] [Order article via Infotrieve]
28. Sakariassen KS, Muggli R, Baumgartner HR. Measurement of platelet interaction with components of the vessel wall in flowing blood. Methods Enzymol. 1989;169:37–70.[Medline] [Order article via Infotrieve]
29.
Grzesiak JJ, Davis GE, Kirchhofer D, Pierschbacher MD.
Regulation of
2ß1-mediated
fibroblast migration on type I collagen by shifts in the concentrations
of extracellular Mg2+ and
Ca2+. J Cell Biol. 1992;117:1109–1117.
30. Timmons S, Hawiger J. The isolation of platelets by albumin gradient and gel filtration. Methods Enzymol. 1989;169:11–22.[Medline] [Order article via Infotrieve]
31. Mustonen P, Lassila R. Epinephrine augments platelet recruitment to immobilized collagen in flowing blood: evidence for a von Willebrand factor-mediated mechanism. Thromb Haemost. 1996;75:175–181.[Medline] [Order article via Infotrieve]
32. Holmes DF, Capaldi MJ, Chapman JA. Reconstitution of collagen fibrils in vitro: the assembly process depends on the initiating procedure. Int J Biol Macromol. 1986;8:161–166.
33.
Badimon L, Badimon JJ, Galvez A, Fuster V. Influence of
arterial damage and wall shear rate on platelet
deposition. Arteriosclerosis. 1986;6:312–320.
34. Lassila R, Badimon JJ, Vallabhajosula S, Badimon L. Dynamic monitoring of platelet deposition on severely damaged vessel wall in flowing blood. Arteriosclerosis. 1990;16:306–315.
35.
Coller BS, Beer JH, Scudder LE, Steinberg MH.
Collagen-platelet interactions: evidence for a direct interaction
of collagen with platelet GPIa/IIa and an indirect interaction with
platelet GPIIb/IIIa mediated by adhesive proteins.
Blood. 1989;74:182–192.
36. Matsuno K, Diaz-Ricart M, Montgomery RR, Ashter RH, Jamieson GA, Tandon NN. Inhibition of platelet adhesion to collagen by monoclonal anti-CD36 antibodies. Br J Haematol. 1996;92:960–967.[Medline] [Order article via Infotrieve]
37.
Siljander P, Carpén O, Lassila R.
Platelet-derived microparticles associate with fibrin during
thrombosis. Blood. 1996;87:4651–4663.
38.
Kunicki TJ, Orchekowski R, Annis D, Honda Y.
Variability of integrin
2ß1 activity on human
platelets. Blood. 1993;82:2693–2703.
39.
Phillips MD, Moake JL, Nolasco L, Turner N. Aurin
tricarboxylic acid: a novel inhibitor of the association of
von Willebrand factor and platelets. Blood. 1988;72:1898–1903.
40. Staatz WD, Peters KJ, Santoro SA. Divalent cation-dependent structure in the platelet membrane glycoprotein Ia-IIa (VLA-2) complex. Biochem Biophys Res Commun. 1990;168:107–113.[Medline] [Order article via Infotrieve]
41.
Nieuwenhuis HK, Sakariassen KS, Houdijk WPM,
Nievelstein PFEM, Sixma JJ. Deficiency of platelet membrane
glycoprotein Ia associated with a decreased platelet
adhesion to subendothelium: a defect in platelet
spreading. Blood. 1986;68:692–695.
42.
Saelman EUM, Nieuwenhuis HK, Hese KM, de Groot PG,
Heijnen HFG, Sage EH, Williams S, McKeown L, Gralnick HR, Sixma JJ.
Platelet adhesion to collagen types I through VIII under conditions
of stasis and flow is mediated by GPIa/IIa
(2ß1-integrin).
Blood. 1994;83:1244–1250.
43.
Tanaka M, Handa M, Kawakami K, Kokubum T, Oda A, Ohta
Y, Kudoh S, Ikeda Y.
2ß1 integrin and
GPIb/IX complex engagements in platelet adhesion to type I collagen
under flow conditions. Blood. 1997;90:27a. Abstract.
44. Kritzik M, Savage B, Nugent DJ, Santoso S, Ruggeri ZM, Kunicki TJ. Nucleotide polymorphism in the alpha 2 gene define multiple alleles that are associated with differences in platelet alpha 2 beta 1 density. Blood. 1998;92:382–388.
45. Moshfegh K, Wuillemin WA, Redondo M, Lämmle B, Beer JH, Liechti-Gallati S, Meyer BJ. Association of two silent polymorphisms of platelet glycoprotein Ia/IIa receptor with risk of myocardial infarction: a case-control study. Lancet. 1999;353:351–354.[Medline] [Order article via Infotrieve]
46. Usui T, Fujisawa T, Takagi J, Saito Y. Propolypeptide and mature portions of von Willebrand factor of bovine origin recognize different sites on type-I collagen obtained from bovine tendon. Eur J Biochem. 1992;205:363–367.[Medline] [Order article via Infotrieve]
47. Csako G, Suba EA, Herp A. Monomeric and polymeric collagen-induced platelet aggregation in citrated and heparinized platelet-rich plasma. Connect Tissue Res. 1985;13:299–311.[Medline] [Order article via Infotrieve]
48.
Keely PJ, Parise LV. The
2ß1 integrin is a
necessary co-receptor for collagen-induced activation of Syk and the
subsequent phosphorylation of phospholipase C
2 in
platelets. J Biol Chem. 1996;271:26668–26676.
49.
Kamiguti AS, Markland FS, Zhou Q, Laing GD, Theakston
RGD, Zuzel M. Proteolytic cleavage of the
ß1 subunit of platelet
2ß1 integrin by the
metalloproteinase jararhagin compromises collagen-stimulated
phosphorylation of pp72syk. J Biol
Chem. 1997;272:32599–32605.
50.
Lassila R, Lindstedt K, Kovanen PT. Native
macromolecular heparin proteoglycans exocytosed from stimulated rat
serosal mast cells strongly inhibit platelet-collagen interactions.
Arterioscler Thromb Vasc Biol. 1997;17:3578–3587.
51.
Ichinohe T, Takayama H, Ezumi Y, Arai M, Yamamoto N,
Takahashi H, Okuma M. Collagen-stimulated activation of Syk but not
c-Src is severely compromised in human platelets lacking membrane
glycoprotein VI. J Biol Chem. 1997;272:63–68.
52.
Kehrel B, Wierwille S, Clemetson KJ, Anders O, Steiner
M, Knight CG, Farndale RW, Okuma M, Barnes MJ. Glycoprotein
VI is a major collagen receptor for platelet activation: it
recognizes the platelet-activating quaternary structure of
collagen, whereas CD36, glycoprotein IIb/IIIa, and von
Willebrand factor do not. Blood. 1998;91:491–499.
53. Achison M, Joel C, Hargreaves PG, Sage SO, Barnes MJ, Farndale RW. Signal elicited from human platelets by synthetic, triple helical, collagen-like peptides. Blood Coagul Fibrinolysis. 1996;7:149–152.[Medline] [Order article via Infotrieve]
54.
Polgar J, Clemetson JM, Kehrel BE, Wiedemann M,
Magnenat EM, Wells TNC, Clemetson KJ. Platelet activation and
signal transduction by convulxin, a C-type lectin from Crotalus
durissus terrificus (tropical rattlesnake) venom via the p62/GPVI
collagen receptor. J Biol Chem. 1997;272:13576–13583.
55.
Heemskerk JWM, Siljander P, Vuist WMJ, Breikers G,
Reutelingsperger CPM, Barnes MJ, Knight CG, Lassila R, Farndale RW.
Function of glycoprotein VI and integrin
2ß1 in the
procoagulant response of single, collagen-adherent platelets.
Thromb Haemost.. 1999;81:782–792.[Medline]
[Order article via Infotrieve]
56.
Moroi M, Jung SM, Shinmyozu K, Tomiyama Y, Ordinas A,
Diaz-Ricart M. Analysis of platelet adhesion to a
collagen-coated surface under flow conditions: the involvement of
glycoprotein VI in the platelet adhesion.
Blood. 1996;88:2081–2092.
57. Barnes MJ, Knight CG, Farndale RW. The collagen-platelet interaction. Curr Opin Hematol. 1998;5:314–320.[Medline] [Order article via Infotrieve]
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