From the Department of Protein Biochemistry, Institute of Life
Science, Kurume University, Kurume-shi,
Fukuoka-ken 839-8016, Japan
Platelet interaction with soluble and insoluble
collagens was characterized through binding studies. In contrast
to resting platelets, cells reacted with activators, TS2/16 (integrin
2
1-activating antibody), thrombin,
collagen-related peptide, or ADP, exhibited specific soluble collagen
binding that is Mg2+-dependent, but inhibited
by prostaglandin I2, Ca2+, and Gi9
(anti-integrin
2
1 antibody). Each
platelet has 1500-3500 soluble collagen binding sites, with a
dissociation constant of 3.5-9 × 10
8
M. This is the first study to show the specific binding of
soluble collagen to platelets; our data strongly suggest that the
receptor is integrin
2
1 after it becomes
activated upon platelet activation. These results suggest that
activation of platelets transforms integrin
2
1 to a state with higher affinity
binding sites for soluble collagen. The soluble collagen-platelet
interaction was compared with the platelet interaction with fibrillar
collagen, which has until now not been demonstrated to bind
specifically to platelets. Here, we demonstrated specific, biphasic
fibrillar collagen binding. One phase is rapid and metal
ion-independent, and accounts for most of the binding. The other phase
is slow and Mg2+-dependent. The characteristic
differences in the specific bindings of soluble and fibrous collagens
demonstrate the different contributions of two different collagen
receptors.
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INTRODUCTION |
When platelets react with collagen, they form aggregates after
activation of their signal transduction systems through processes involving many reactions including calcium release, formation of
phosphoinositides, and phosphorylation of proteins. Platelets can also
adhere to an immobilized collagen surface. Collagen is one of the main
components of the vascular subendothelium and becomes exposed to the
blood stream when endothelial cells are damaged. These observations
suggest that the interaction of platelets with collagen is one of the
first, requisite reactions in thrombus formation under physiological
conditions. The interaction of platelets with collagen has been studied
under two conditions. One approach is to analyze platelet adhesion and
aggregation onto a collagen-coated surface under flow conditions using
whole blood (1-5). This method observes platelet adhesion under flow
conditions that closely simulate physiological conditions, and the fact
that platelets adhere and aggregate under these conditions shows that
the interaction with collagen is involved in physiological thrombus
formation. Alternatively, platelet adhesion on a collagen-coated
surface can be analyzed under static conditions; this type of assay
uses washed platelets instead of whole blood (6, 7). In both systems,
two platelet glycoproteins, glycoprotein
(GP)1 Ia/IIa, also known as
integrin
2
1, and GP VI were found to participate in platelet adhesion to the collagen surface.
However, the direct binding of collagen to platelets has not been
studied in detail because soluble collagen did not bind to platelets.
Resting platelets do not react with soluble collagen. They only react
with insoluble collagen fibers or an immobilized collagen surface, both
substrates having physical properties that made it very difficult to
analyze the binding reaction of platelets and collagen. Gordon and
Dingle (8) reported that collagen did not bind to platelets until it
became fibrillar. Binding of platelets to radiolabeled insoluble
collagen particles was indicated to be a very fast reaction and
Mg2+-independent (9), but they could not find measurable
specific binding of labeled collagen fibrils to platelets under their
conditions. Thus, their binding data were not suitable for kinetic
analysis of the binding reaction.
Our present study is the first to demonstrate the binding of soluble
collagen to platelets and the kinetic analyses of this reaction. The
soluble collagen binding is Mg2+-dependent, and
the activation of integrin
2
1 is involved
in this reaction. In addition, we were also able to demonstrate the specific binding of fibrous collagen particles by platelets, finding that the major portion of this binding is Mg ion-independent. These
results suggest that the two collagen receptors, integrin
2
1 and a Mg2+-independent
collagen receptor, probably GP VI, would be involved in the
collagen-platelet interaction with different specificities.
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MATERIALS AND METHODS |
Preparation of Soluble and Insoluble (Fibrillar)
Collagen--
Bovine type III collagen and human type III collagen
were obtained from Koken Co., Ltd. (Tokyo, Japan) and Fuji Chem. Ind. Co., Ltd. (Takaoka, Toyama, Japan), respectively. Both collagens were
dialyzed against 136 mM NaCl, 2.7 mM KCl, 0.42 mM NaH2PO4, 12 mM
NaHCO3, 5.5 mM glucose, and 5 mM
HEPES, pH 7.4 (buffer A) at 4 °C. After dialysis, the collagen
solution was incubated at 37 °C for 1 h. The resultant collagen
gel was centrifuged at 8000 × g for 20 min to obtain
the supernatant and the pellet. The supernatant was further centrifuged
at 100,000 × g for 1 h at 25 °C, and the obtained supernatant solution was used as soluble collagen. This soluble collagen was stored at 4 °C until used in the experiments. The collagen pellet obtained after centrifugation was washed once with
buffer A and then suspended with buffer A at the concentration of about
1 mg/ml. The suspended collagen was sonicated for 1 min three times
with a sonicator attached with a cup horn (Heat Systems-Ultrasonics Inc., Farmingdale, NY), which was cooled by ice. The obtained microparticles of collagen suspensions were used as insoluble collagen.
Soluble collagen was radiolabeled with Na125I by using
IODO-BEADS (Pierce) at room temperature according to the
manufacturer's instructions. For the preparation of
125I-labeled insoluble collagen, collagen dialyzed against
buffer A was labeled on ice by using IODO-BEADS and dialyzed against buffer A at 4 °C. 125I-Labeled collagen was polymerized
at 37 °C and centrifuged, and the pellet of labeled collagen
obtained was processed as described above to make labeled insoluble
collagen microparticles (fibrillar collagen). The specific
radioactivities ranged between 3 and 9 × 104
cpm/µg. The data presented in this paper are those obtained with bovine collagen. Similar data were obtained with human collagen, but
its limited availability made it more feasible to employ bovine collagen for extensive studies.
Determination of Collagen Concentrations--
The concentrations
of soluble and fibrillar collagen were determined by assaying the
protein by the bicinchoninic protein assay (10), with collagen as the
standard protein. The molar concentration was calculated with 3 × 105 as the molecular weight of collagen.
Platelet Preparation--
Whole blood was drawn from the cubital
vein of healthy volunteers into 0.1 volume of 3.8% sodium citrate.
After platelet-rich plasma (PRP) was obtained, platelets were
sedimented from PRP by centrifugation at 900 × g for
12 min after the addition of sodium prostaglandin I2
(PGI2) (Funakoshi, Tokyo, Japan) at the final concentration
of 0.1 µg/ml. The centrifuged platelets were washed once with 6.85 mM citrate, 130 mM NaCl, 4 mM KCl,
and 5.5 mM glucose, pH 6.5; and the washed platelets were
finally suspended with buffer A containing 2% bovine serum albumin
(Sigma) at concentrations of 1-3 × 109 cells/ml.
Binding of Soluble Collagen--
To ensure that the
125I-labeled soluble collagen mixture consisted of only
soluble collagen, just prior to use in each experiment, an aliquot of
the preparation was warmed at 37 °C for 30 min, followed by
centrifugation at 56,000 × g for 60 min at 25 °C
(Beckman TL-100 Ultracentrifuge with a TLA 100.3 rotor; Beckman, Inc., Palo Alto, CA). The supernatant was removed and used as the soluble collagen preparation for the experiments. Routinely, an aliquot of the
supernatant was retained for subsequent determination of protein
concentration.
The selection of the optimum reaction conditions and separation
procedures was based on the data described under "Results." For the
soluble collagen binding experiments, non-siliconized tubes were used.
Washed platelet suspensions were used for all the experiments. The
complete mixture (final volume of 50-70 µl) for determination of
total binding consisted of 2 mM MgCl2,
125I-labeled soluble collagen, any desired reagents such as
inhibitors or antibodies, 0.2% BSA, and 4 × 108
platelets/ml (unless otherwise stated) in buffer A. 125I-Labeled soluble collagen was used at a concentration
of about 75-100 nM in most experiments, except for the
kinetic studies for determination of binding parameters where the
stated range of concentrations was employed. Nonspecific binding was
determined by adding an approximately 100-fold excess of unlabeled
soluble bovine collagen or 5 mM EDTA (further described
under "Results") to reaction mixtures containing the other
components of the total binding mixture. Platelets were usually added
last to the reaction tubes containing the remaining reagents to
initiate the binding reaction. Four to eight replicates for each
binding condition were used. Binding was allowed to occur for 80 min at
room temperature, without agitating the reaction mixtures. Then, each
reaction mixture was processed by a procedure based on the method
described by Mazurov et al. (9), which was modified to
optimize the separation of platelet-bound soluble collagen from the
unbound soluble collagen; each reaction mixture was layered over a
250-µl pad of 20% sucrose in buffer A containing 0.2% BSA in a
needle-tipped 0.3-ml narrow-tipped microcentrifuge tube (Assist, Tokyo,
Japan) and centrifuged at 10,000 × g in a
microcentrifuge (model MRX-150, MRX-150 rotor; Tomy, Tokyo, Japan) for
5 min at 20 °C. The tubes were placed in a
80 °C freezer for at
least 30 min. The tips of the frozen tubes containing the
platelets/platelet-bound soluble collagen were then cut off by
scissors, individually placed in counting tubes, and the radioactivity
determined in an Aloka Auto Well
system (model ARC-1000
M; Aloka, Tokyo, Japan).
Binding of Insoluble Collagen--
Just prior to use in each
experiment, the 125I-labeled collagen microparticles
(prepared as described above) were processed by the following procedure
to sort out collagen microparticles of an appropriate size range,
i.e. would bind well to resting platelets, but also not
large enough to be pelleted under the conditions of the centrifugation
assay (see "Results" for description of how the optimum conditions
were chosen). An aliquot of the labeled microparticular collagen was
sonicated for 1 min at 60% power, three times with a 1-min interval
between each, by a sonicator attached with a cup horn under cooling
with ice. The sonicated suspension was then centrifuged at 2000 rpm
(250 × g) for 3 min in the MRX-150 high speed
micro-refrigerated centrifuge; the supernatant obtained was used for
the binding studies. For the binding experiments of insoluble collagen,
siliconized tubes and tips were used. The total binding mixture (total
volume of 50 µl) contained 5 mM EDTA or 2 mM
Mg2+, any agents to be tested, microparticular suspension
of 125I-labeled fibrillar collagen at the desired
concentrations, and 0.4-4 × 108 platelets/ml in
buffer A containing 0.2% BSA. In the experiments where
Mg2+ was employed, the washed platelet suspensions were
added with 0.1 µg/ml PGI2 and 10 µM
indomethacin, to prevent aggregation, and incubated at room temperature
for 10 min before aliquots of them were added to the binding mixtures.
When binding was performed in the presence of EDTA, PGI2
and indomethacin were not added unless the EDTA-containing binding
experiment was performed to compare the results with those in the
Mg2+-containing one. Nonspecific binding was determined in
the presence of an 80-100-fold excess, as estimated by protein
concentration, of unlabeled bovine fibrillar collagen, microparticle
suspension prepared in a manner similar to the labeled material.
Platelets were added last to tubes containing the remaining binding
mixture components to initiate the binding reaction, and incubation was carried out for 60 min at room temperature with gentle shaking on a
shaking table, to prevent the settling of the microparticles of
collagen. Each sample was then layered over a 250-µl pad of 20%
sucrose and 2% BSA, centrifuged at 12,000 rpm for 10 min at room
temperature, and processed for counting as described above for soluble
collagen binding.
Data Analyses--
In the soluble collagen binding experiments,
specific binding was calculated by subtracting the nonspecific binding
obtained in the presence of excess unlabeled collagen or 5 mM EDTA.
In the fibrillar collagen binding experiments, total binding and
nonspecific binding were first corrected by subtracting the experimentally determined value for pelleted labeled fibrillar collagen
in the absence and presence of excess unlabeled fibrillar collagen,
respectively (see "Results" for further description). Specific
binding was then calculated by subtracting the corrected nonspecific
binding from the corrected total binding.
Kinetic analyses of the data to obtain the binding parameters were
performed by non-linear regression analyses by the software Prism
(Version 2.01; GraphPad Software, Inc., La Jolla, CA). Other data
analyses were also performed by the same program.
Other Materials--
Collagen-related peptide (CRP) was
synthesized by the method of Morton et al.(11). The peptide
Tyr-Gly-Lys-Hyp-(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly, was
synthesized by Tana Laboratories, L.C. (Houston, TX) using standard
Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid-phase chemistry. The peptide was dissolved in phosphate buffer, pH 7.6, and then it was
cross-linked with 0.25% glutaraldehyde for 1 h on ice (11). The
obtained CRP was dialyzed against buffer A and used for the experiments. The obtained CRP aggregated platelets in PRP at the concentrations of 0.2 µg/ml or higher. Monoclonal antibodies, P2
(anti-GP IIb/IIIa), SZ2 (anti-GP Ib), and Gi-9 (anti-integrin
2
1), were purchased from Immunotech
(Luminy, France). The anti-GP Ib monoclonal antibody NNKY5-5 (12) and
the integrin
2
1-activating antibody,
TS2/16 (13) were kindly given to us by Dr. Shosaku Nomura, Kansai
Medical University, Osaka, Japan and by Drs. Yuji Saito and Junichi
Takagi, Tokyo Institute of Technology, Tokyo, Japan, respectively.
Ristocetin and ADP were obtained from Chrono-Log Corp., Havertown, PA.
Peptides Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Arg-Gly-Glu-Ser (GRGES)
were synthesized in our laboratory with a model 431A peptide
synthesizer (Applied Biosystems, Foster City, CA) and the FastMoc
method according to the company's protocol (5). Thrombin and the
thrombin receptor agonist TRAP (SFLLRNPNDKYEPF) were obtained from
Sigma and Calbiochem (San Diego, CA), respectively.
 |
RESULTS |
Conditions for Optimum Separation of Samples--
The
centrifugation conditions for maximizing platelet sedimentation with
the minimum of labeled collagen sedimentation was established by using
51Cr-labeled platelets and 125I-labeled soluble
or microparticular fibrillar collagen. As shown in Fig.
1, platelet sedimentation was not
affected by the presence of an excess of unlabeled soluble collagen in
either the presence or absence of 2 mM Mg2+;
greater than 96% platelet pelleting was obtained in about 4-5 min at
12,000 rpm as judged by the amount of sedimented 51Cr.
Addition of 5 mM EDTA to the platelet mixtures containing excess collagen and 2 mM MgCl2 did not affect
the sedimentation properties (data not shown). There was very little
pelleting of the precentrifuged labeled soluble collagen at
centrifugation times as high as 10 min; the amount sedimented varied
among different soluble collagen preparations, but usually was less
than 1% of the added 25I counts, and the sedimented amount
plateaued before 5 min of centrifugation.

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Fig. 1.
Platelet sedimentation time in the presence
of an excess of soluble collagen. The following mixtures were
incubated for 80 min at room temperature without agitation:
51Cr-labeled platelets (4 × 108 cells/ml) + 2 mM Mg + 300 µg/ml unlabeled soluble collagen ( ),
51Cr-labeled platelets (4 × 108 cells/ml)
only ( ), 125I-labeled soluble collagen + 2 mM Mg2+ ( ), and 125I-labeled
soluble collagen only ( ). Then 50-µl aliquots of each mixture were
individually centrifuged over a pad of 250-µl 20% sucrose (in
HEPES-Tyrode's buffer, pH 7.4) in a needle-tipped centrifuge tube for
various times at 12,000 rpm, and the amount of sedimented radioactivity
(51Cr or 125I) was determined as described
under "Materials and Methods." The pelleted radioactivity is
reported as a percent of the total label (51Cr or
125I) in the sample. Each data point and error bar is the
mean ± S.D. of quadruplicate determinations; where error bars are
not visible, the S.D. is smaller or equal to the size of the symbol.
The recovery of counts (pellet counts + supernatant counts) was greater
than 98% in all cases.
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In contrast, the situation with microparticles of labeled fibrillar
collagen was quite different. The presence of the fibrillar collagen
substantially slowed the rate of platelet sedimentation (Fig.
2, left graph). Furthermore,
the presence of excess unlabeled fibrillar collagen affected the
sedimentation of labeled collagen fibrils (Fig. 2, right
graph). Although some labeled collagen was sedimented in the
absence of excess unlabeled collagen, its inclusion resulted in
1.5-3-fold higher levels of labeled collagen sedimentation at all
centrifugation times and speeds. The conditions of 10-min
centrifugation time at 12,000 rpm were chosen to optimize the pelleting
of platelets (>95%), and the effect of high concentrations of
unlabeled collagen fibrils, which were required for the establishment of nonspecific binding, was corrected for by determining how much 125I-labeled fibrillar collagen is sedimented in the
presence or absence of excess unlabeled fibrillar collagen at each
concentration of labeled fibrillar collagen employed in an experiment.
This had to be done for each experimental session because the
preparation of collagen fibrils would be expected to be different on
each occasion, even though the size of the fibrils may lie within a similar range.

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Fig. 2.
Effects of a high concentration of unlabeled
fibrillar collagen on the sedimentation of platelets and
125I-labeled fibrillar collagen. Left graph, the
following mixtures (all in HEPES-Tyrode's buffer, pH 7.4 containing
2% BSA) were incubated in siliconized tubes for about 40 min at room
temperature, with agitation of the tubes: 51Cr-labeled
platelets + 5 mM EDTA ( ), 51Cr-labeled
platelets + unlabeled fibrillar collagen (350 µg/ml) + 5 mM EDTA ( ), 125I-labeled fibrillar collagen + 5 mM EDTA ( ), and 125I-labeled fibrillar
collagen + excess of unlabeled fibrillar collagen (350 µg/ml) + 5 mM EDTA ( ). After incubation, 50-µl aliquots of each
mixture were individually centrifuged over 250 µl of 20% sucrose for
various times at 12,000 rpm for various times, and the amounts of
radioactivities of the obtained pellets and supernatants were counted.
The ordinate shows the percentage of counts relative to the
total counts added of either 51Cr (radiolabeled platelets)
or 125I (radiolabeled fibrillar collagen). Right
graph, mixtures containing a fixed concentration of
125I-labeled fibrillar collagen (about 2 µg/ml), 5 mM EDTA, and varying amounts of unlabeled fibrillar
collagen were centrifuged over 250-µl of 20% sucrose for 10 min at
12,000 rpm, and the radioactivities of the pellet and supernatant were
measured. The ordinate shows the percentage of counts
relative to the total counts of 125I-labeled fibrillar
collagen added. In both graphs, each data point is the mean ± S.D. of 10 determinations.
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Preincubation Time with Activation Agents and the Specific Binding
of Soluble Collagen--
Platelets were preincubated with TS2/16 (5 µg/ml), thrombin (0.5 units/ml), or CRP (0.5 µg/ml) for 0-30 min
to determine if preincubation of platelets with the agents affected the
activation of their soluble collagen binding. As shown in Fig.
3, the bindings of the platelets
preincubated with TS2/16, thrombin, or CRP for 5, 10, 20, and 30 min
were similar to that observed when the respective agent was added
simultaneously (0 min) with the 125I-labeled collagen
(about 100 nM) to initiate the binding reaction. Platelets
not activated by the addition of one of the agents showed little or no
binding. Therefore, we performed the following binding experiments
without preincubation.

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Fig. 3.
Effect on soluble collagen binding of
preincubating platelets with activating agents for varying times.
Platelets were incubated with TS2/16 (5 µg/ml) ( ), thrombin (0.5 units/ml) ( ), or CRP (0.5 µg/ml) ( ) for various times, and then
the binding was initiated by adding 125I-labeled soluble
collagen and the reaction allowed to occur for 80 min at room
temperature. For the zero time point, the activating agent was added
simultaneously with the ligand. All samples were processed by the
centrifugation method, and the radioactivities in the platelet pellet
were determined and reported as femtomoles/106 platelets.
Each data point is the mean ± S.D. of four determinations.
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Time Course of Soluble Collagen Binding to Activated
Platelets--
Soluble collagen binding to agonist-activated platelets
reached equilibrium within about 70-80 min. Typical of the time
courses observed are those for TS2/16- and thrombin-activated platelets shown in Fig. 4. Control mixtures that
contained no activating agents showed a very low level of counts
sedimenting along with the platelet pellet; this might be due to a
small amount of sedimentable soluble collagen, the amount which varied
with the collagen preparation, or platelets may have become slightly
activated during the platelet preparation procedures or during the
incubation period.

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Fig. 4.
Time course of soluble collagen binding to
activated platelets. A platelet suspension (4 × 108 cells/ml, Hepes-Tyrode's buffer plus 2% BSA) was
incubated with TS2/16 (5 µg/ml, ), thrombin (0.5 units/ml, ),
or buffer (control unactivated platelets, ×); 2 mM
Mg2+; and 125I-labeled soluble collagen for
varying times and the binding assessed by the centrifugation procedure.
Mixtures for determining nonspecific binding contained 5 mM
EDTA in addition to the other components. Each data point is the
mean ± S.D. of four determinations.
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In this figure, TS2/16-activated platelets bound more soluble collagen
than the thrombin-activated platelets. Although we observed a similar
phenomena numerous times in the course of our experiments, in other
instances, thrombin induced soluble collagen binding to levels
comparable to those by TS2/16. This could be explained by the
instability of platelet activation with thrombin. Platelets apparently
have an optimum concentration of thrombin for their maximum activation,
which varied from individual to individual. In contrast,
TS2/16-activated platelets showed stable binding activity with high
capacity.
Effects of Metal Ions on Soluble Collagen Binding to Platelets
Activated by Various Agonists--
The effects of metal ions on
soluble collagen binding to activated platelets activated by ADP,
thrombin, TS2/16, or CRP are shown in Table
I. The actual amounts of binding and the
extent of metal ion effect differed among individuals. For platelets activated by any of the agents, soluble collagen binding was
Mg2+-dependent. More specific binding was
observed in the presence of 2 mM Mg2+ than 1 mM; and in each experiment, specific binding induced by the
agonists in the presence of either concentration of Mg2+
was higher than that observed in the control platelets to which no
metal ions had been added. The control platelets in the 2 mM Mg2+ experiment showed a significant amount
of soluble collagen binding in the absence of added activator,
suggesting that in some cases, there may have been some activation
during the platelet isolation procedures. Because of this, the percent
increase in soluble collagen binding relative to the control was less
prominent, especially in the 2 mM Mg2+
experiments (see explanation in the next paragraph). Compared with the
binding in the presence of 2 mM Mg2+, that in
the presence of Mn2+ was higher.
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Table I
Effect of metal ions on the specific binding of soluble collagen to
activated platelets
Each experiment was performed on a different day with platelets from a
different individual. Because platelets from different individuals
showed variable sensitivities to the activating agents, only values
from the same person can be validly compared with each other.
Nonspecific binding was determined in the presence of a 100-fold excess
of unlabeled soluble collagen in all experiments except for experiment
4, in which 5 mM EDTA was used. Specific binding is
reported as femtomoles/106 platelets.
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As illustrated in Fig. 5, a more detailed
examination of the Mg2+ concentration dependence in the
cases of two activators, TS2/16 (5 µg/ml) and thrombin (0.1 units/ml), indicates that saturation with Mg2+ occurs
around 2 mM for the bindings induced by both activators, since the specific binding of soluble collagen plateaus after this
level of metal ion. As seen from the control curve (no added activators), there is indeed some increase in soluble collagen binding
as higher concentrations of this divalent ion are included in the
reaction mixture; because the saturation level for this low level of
binding (a maximum of about 10%) occurs at about the same
concentration as the TS2/16 and thrombin curves, it is likely due to
some activation of the platelets during the isolation procedures or
incubation, instead of an independent activation by Mg2+
itself which would likely show a different Mg2+ ion
dependence.

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Fig. 5.
Mg 2+ dependence of soluble
collagen binding to TS2/16- and thrombin-activated platelets. The
binding of soluble collagen to platelets activated with TS2/16 (5 µg/ml) ( ) or thrombin (0.1 unit/ml) ( ) was determined in the
presence of various concentrations of Mg2+. Control
mixtures ( ) contained no activating agent. Nonspecific binding was
determined in the presence of 5 mM EDTA (0, 0.5, 1, and 2 mM Mg2+) or 10 mM EDTA (5 and 10 mM Mg2+). Samples were processed by the
centrifugation procedure, and the pellets were counted. The specific
binding is reported as femtomoles/106 platelets, and each
value is the mean ± S.D. of quadruplicate determinations.
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For all the activators, little binding occurred in the presence of 5 mM EDTA. The level observed in the presence of 5 mM EDTA was similar to that observed in the presence of an
excess of unlabeled soluble collagen (about 350 µg/ml), which we
added to assess nonspecific binding in the preliminary experiments.
Because of this, we employed 5 mM EDTA to assess
nonspecific binding; this allowed us to determine nonspecific binding
at higher concentrations of labeled soluble collagen where it would
otherwise not have been possible to obtain the 100-fold excess of
unlabeled ligand required because of the limitation of how high a
concentration of soluble collagen in solution can be achieved.
For the ADP-, thrombin-, TS2/16-, or CRP-activated platelets, little
binding was seen in the presence of 2 mM Ca2+;
when both 1 mM Ca2+ and 1 mM
Mg2+ were present, specific binding was lower than that
seen in the presence of 1 mM Mg2+ alone. These
observations indicate the inhibitory effect of calcium ion as reported
by Santoro (6).
A similar study performed with thrombin receptor agonist peptide (TRAP)
(20-95 µM) indicated that it could also activate
platelets to bind soluble collagen, although it was a weaker activator
than thrombin because the maximum amount of binding induced by this agent was less than that obtained with thrombin (data not shown). The
specific binding of soluble collagen to TRAP-activated platelets showed
the same ion dependence as the bindings induced by the above agents
(data not shown).
Effect of PGI2 on Soluble Collagen Binding to Activated
Platelets--
As shown in Fig. 6, the
presence of PGI2 markedly decreased the specific binding in
the presence of 2 mM Mg2+ in thrombin- or
CRP-activated platelets. On the other hand, it did not inhibit the
specific binding of TS2/16-activated platelets. In the ADP-induced
platelets, soluble collagen binding was low, so it was difficult to
assess the effect of PGI2 accurately.

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Fig. 6.
Effect of PGI2 on soluble
collagen binding to activated platelets. The binding of soluble
collagen to platelets were determined in the presence of one of the
following activating agents: ADP (20 µM), TS2/16 (5 µg/ml), thrombin (0.1 units/ml), and CRP (0.5 µg/ml), with 2 mM Mg2+ (empty bars) or 2 mM Mg2+ + PGI2 (solid
bars) in the binding mixture. To determine the effect on
non-activated platelets, platelets were incubated with PGI2
(0.1 µg/ml) (dotted bars) or without PGI2 in
the absence of Mg2+ (diagonally shaded bars).
The samples were processed by the centrifugation procedure and the
pellets were counted. Each bar represents the mean ± S.D. from
eight determinations.
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Effect of Inhibitors on Soluble Collagen Binding to Activated
Platelets--
The results illustrated in Fig.
7 show typical data observed in four
separate experiments with different platelets and soluble bovine
collagen preparations, with 0.1 units/ml thrombin as the platelet
activating agent. Compared with the specific binding in the absence of
any inhibitor, taken as 100% in the figure, the specific binding of
soluble collagen was inhibited by the anti-GP Ia/IIa antibody Gi9 (10 µg/ml) to a level similar to that observed with PGI2 (0.1 µg/ml), but antibodies P2 (anti-GP IIb/IIIa, 10 µg/ml), NNKY5-5
(anti-GP Ib, 10 µg/ml), GRGDS (1 mM), and GRGES (negative
control, data not shown) were not inhibitory.

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Fig. 7.
Effect of inhibitors on soluble collagen
binding to activated platelets. Soluble collagen binding to
platelets activated by thrombin (0.1 units/ml) was determined with the
presence of no added inhibitor or one of the following agents:
PGI2 (0.1 µg/ml), P2 (10 µg/ml), NNKY5-5 (10 µg/ml),
Gi9 (10 µg/ml), and GRGDS (1 mM). Each bar shows the
mean ± S.D. of six determinations. The data shown here is
representative of four experiments performed with different platelets
and different soluble collagen preparations.
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Activating Agent Concentration Dependence of Soluble Collagen
Binding to Platelets--
Fig. 8 shows
the concentration dependence curve for CRP, which shows an apparent
optimum concentration for activation of soluble collagen binding. To
determine if this is due to CRP competing with soluble collagen for
binding to activated platelets, the effect of high CRP concentrations
on the binding of soluble collagen to TS2/16-activated platelets was
assessed. The data shown in Fig. 9 shows
that this is the case; CRP inhibited the specific binding of soluble
collagen to TS2/16-activated platelets in a concentration-dependent manner, with the inhibitory
concentrations of CRP corresponding to those at which soluble collagen
binding to CRP-activated platelets were decreased in Fig. 8.

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Fig. 8.
CRP concentration dependence of soluble
collagen binding to platelets. Binding of soluble collagen to
platelets was determined in the presence of various concentrations of
CRP and 2 mM Mg2. Each data point is the
mean ± S.D. of six determinations.
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Fig. 9.
CRP competes with binding of soluble collagen
to TS2/16-activated platelets. Platelets (4 × 108 cells/ml) were activated by incubation with 5 µg/ml
TS2/16 in the presence of 2 mM Mg2+ for 20 min.
Then binding was initiated by adding a mixture containing various
concentrations of CRP and 125I-labeled soluble collagen in
HEPES-Tyrode's buffer plus 2% BSA and after 80 min at room
temperature, the samples were processed by the centrifugation method.
The data are expressed relative to the specific soluble collagen
binding in TS2/16-activated platelets in the absence of CRP (100%).
Each data point is the mean ± S.D. of six determinations.
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Kinetic Analyses of Soluble Collagen Binding by Activated
Platelets--
The binding parameters for soluble collagen binding to
platelets activated with TS2/16 (5 µg/ml) and thrombin (0.1 units/ml) were determined by measuring the specific binding of soluble collagen as a function of soluble collagen in the range of 4-456 nM
soluble collagen. Fig. 10 shows the
binding curve obtained for TS2/16. Data were analyzed by non-linear
regression of the binding curve, fitting the data to either a one-site
or two-site model. Both the TS2/16 and thrombin data showed a better
fit to the one-site model, although it should be noted that the
Scatchard plots in all experiments (see Fig. 10, inset, for
the Scatchard plot in one experiment with TS2/16) appear to fit better
to a curve than a straight line. The binding parameters
(Km and Vmax) obtained are
summarized in Table II; within the
experimental error for the parameters, TS2/16-activated platelets show
binding constants similar to those of thrombin-activated ones.

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Fig. 10.
Binding curve of soluble collagen to
TS2/16-activated platelets. The binding of soluble collagen to
platelets activated with TS2/16 (5 µg/ml) in the presence of 2 mM Mg2+ was determined at various
concentrations of 125I-labeled soluble collagen.
Nonspecific binding was measured in the presence of 5 mM
EDTA. The binding data were analyzed by non-linear regression analysis;
the data best fit to a one-site binding equation. The inset
shows the Scatchard plot of the data. These data correspond to
experiment 1 in Table II, which summarizes the binding
parameters.
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Time Course of Fibrillar Collagen Binding to Platelets--
In
contrast to soluble collagen binding by activated platelets, the
collagen fibrils bound to platelets even in the presence of 5 mM EDTA. Furthermore, the time courses of fibrillar
collagen binding in either the presence of Mg2+ or 5 mM EDTA was very rapid (Fig.
11). The specific binding in the
presence of Mg2+ reached a level that was about 30% higher
than the binding in the presence of EDTA, which would suggest that the
fibrillar collagen binding consists of a
Mg2+-dependent component and a
Mg2+-independent component, which accounts for most of the
binding.

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Fig. 11.
Time course of fibrillar collagen binding to
platelets. Fibrillar collagen binding to platelets (7.5 × 107 cells/ml, HEPES-Tyrode's buffer plus 2% BSA;
pretreated by the addition of PGI2 and indomethacin as
indicated under "Materials and Methods") was assessed in the
presence of 5 mM EDTA ( ) or 2 mM
Mg2+ ( ). Nonspecific binding was determined in the
presence of about a 100-fold excess of unlabeled fibrillar collagen
(about 350 µg/ml). The reaction mixtures were incubated for various
times at room temperature, with slow agitation on a shaking table.
Processing of the samples by the centrifugation assay and calculation
of specific binding were performed as described in the text. Each data
point is the mean ± S.D. of five determinations.
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Other Characteristics of Microparticular Fibrillar Collagen
Binding--
Platelets were able to specifically bind fibrillar
collagen in either the presence of Mg2+ (2 mM)
or EDTA (5 mM), as described above. The specific binding was concentration-dependent; a typical example is shown in
Fig. 12. However, it was not possible
to obtain concentration data in a sufficient range to permit an
analyses of the binding curve because collagen is a macromolecule whose
solution properties (e.g. viscosity and stickiness) limit
its workable concentration to a maximum of about 500 µg/ml; it was
therefore impossible to determine nonspecific binding at the higher
concentrations of ligand because a 80-100-fold excess (from
preliminary experiments, data not shown) of unlabeled fibrous collagen
would be necessary. Furthermore, the heterogeneity of fibrillar
collagen makes it far from an ideal defined ligand, so binding analyses
techniques are not applicable. In this figure, the concentration of
collagen was calculated using 300,000 as the molecular mass of
collagen. However, we measured the binding of fibrous collagen
aggregates, so the actual molar concentration of collagen should be
much less. Thus, this figure shows the concentration dependence at a
very low concentration range of fibrous collagen (compare this with Fig. 10).

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Fig. 12.
Binding of fibrillar collagen to
platelets. The binding of fibrillar collagen to platelets
(7.5 × 107 cells/ml) in the presence of 5 mM EDTA was determined at various concentrations of
125I-labeled fibrillar collagen. Nonspecific binding was
measured in the presence of an excess of unlabeled fibrillar collagen.
Details about the method and calculation of specific binding are given
in the text. The inset shows the data for total binding
( --- ) and nonspecific binding (× - - ×) from which the
specific binding was calculated. Each point is a single determination
(150 in total for each curve). Each curve was obtained by fitting the
data by non-linear regression analysis using a second degree
polynomial.
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The effect of several anti-platelet glycoprotein antibodies on the
specific binding of fibrous collagen was tested. The anti-GP Ia/IIa
antibody Gi-9 inhibited the specific binding of fibrous collagen by
25% relative to the control (100%) that did not contain any antibody,
whereas antibodies P2 (anti-GP IIb/IIIa) and SZ-2 (anti-GP Ib) had
little effect (Fig. 13).

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Fig. 13.
Effect of antibodies against specific
platelet glycoproteins on fibrillar collagen binding to platelets.
The binding mixtures contained platelets (7 × 107
cells/ml, pretreated with PGI2 and indomethacin as
indicated under "Materials and Methods"), HEPES-Tyrode's buffer,
2% BSA, 1 mM Mg2+, 125I-labeled
fibrillar collagen and one of the following antibodies: Gi9 (10 µg/ml), P2 (10 µg/ml), and SZ-2 (10 µg/ml), or contained an equal
volume aliquot of buffer in the control experiment (no added antibody).
Nonspecific binding was determined in the presence of 350 µg/ml
unlabeled fibrillar collagen. The mixtures were incubated for 60 min,
with agitation, at room temperature and then processed by the
centrifugation procedure. The graph is a plot of the specific binding
in the presence of each of the antibodies relative to the control (no
added antibody, 100%). Each bar is the mean ± S.D. of 10 determinations.
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DISCUSSION |
Platelet-collagen binding has yet to be thoroughly investigated,
even though an analysis of this interaction would be prerequisite to
understanding the mechanism of platelet adhesion to collagen and
subsequent aggregation, the important initial steps of thrombus formation in vivo. This is primarily due to the properties
of fibrous collagen that make it a far from an ideal, homogeneous ligand. Several reports indicated that only fibrous collagen can bind
to platelets, and no binding of soluble collagen could be demonstrated
(8, 9). Two collagen receptors have been identified on the platelet
surface, integrin
2
1 (also known as GP
Ia/IIa) (6, 14) and GP VI (15, 16). However, the interrelationship of
these two collagen receptors in the adhesion and aggregation of
platelets with collagen has not been elucidated. In this paper, we
showed the specific binding of soluble and fibrous collagens to
platelets and obtained data demonstrating the different contributions of these collagen receptors to the bindings of these two types of
collagens.
As indicated by other researchers, we could not detect any specific
binding of soluble collagen to resting platelets. However, we found
that platelets reacted with a monoclonal anti-integrin
2
1 antibody, TS2/16, exhibited
significant specific binding of soluble collagen. TS2/16 is an
activating antibody that binds to the
1-subunit of
integrin
2
1 (17) and indicated to
increase the binding of fibronectin and collagen to the corresponding
integrin receptors (17, 18). Therefore, in our experiments, TS2/16 would induce the activation of integrin
2
1 molecules, enabling them to bind to
soluble collagen; this indicates that the activated form of integrin
2
1 would have high affinity to bind
soluble collagen. We could also detect the specific soluble collagen
binding to platelets that were treated with other platelet stimulants such as thrombin, ADP, and CRP (11). The effect of ADP was weak compared with that of thrombin and also the amount of specific binding
was variable from experiment to experiment. Soluble collagen binding to
platelets in response to any of these stimulants, with the exception of
TS2/16, was inhibited by PGI2 (Fig. 6). These results
strongly suggest that integrin
2
1 became
activated when platelets were reacted with these stimulants. A similar
mechanism was found for the binding of fibrinogen to GP IIb/IIIa
(integrin
IIb
3) (19, 20). Thus, both GP
IIb/IIIa and integrin
2
1 would become
activated when platelets are reacted with various types of stimulants,
gaining the ability to bind fibrinogen and soluble collagen,
respectively.
The binding of soluble collagen to platelets required the presence of
Mg2+ ions, but Ca2+ ions were inhibitory. This
metal ion dependence profile is similar to the one reported for the
interaction of integrin
2
1 and collagen (6), supporting the above hypothesis that integrin
2
1 mainly contributes to the binding of
soluble collagen. We also found that manganese ions had a stimulatory
effect. They not only induced the activations of integrin
2
1 in the absence of other activators, but also stimulated the collagen binding of platelets activated by
thrombin or TS2/16. This result suggests that Mn2+ ion
would activate integrin
2
1 through a
mechanism different from that responsible for the TS2/16- or
thrombin-induced activation Other
1- and
2-integrins were shown to have a similar metal ion
dependence: stimulation by Mn2+ and inhibition by
Ca2+ (21-23). In addition, Mn2+-induced
activation of
1-integrin, was suggested to be different from the activation induced by TS2/16 in the presence of
Mg2+ (24). In contrast to Mn2+, the stimulatory
effect of Mg2+ would be attributed to the partial
activation of platelets that had occurred during the preparation of
washed platelets, because the extent of the platelet activation was low
even in the presence of a high concentration of Mg2+ (Fig.
5).
The kinetic analysis of soluble collagen binding showed that the
binding is saturable and fit to a one-site binding model. The
dissociation constant and the number of binding sites per cell were
calculated to be 3.5-9 × 10
8 M and
1500-3500 sites per cell, respectively (Table II). Platelets activated
with thrombin and those activated by TS2/16 showed similar binding
parameters, indicating that the integrin activated by TS2/16 through
direct binding and that activated by thrombin through inside-out
signaling would have a similar active conformation. By using monoclonal
antibodies, the number of integrin
2
1
molecules on platelets was calculated to be 1000-2800 per platelet
(25, 26). The number of soluble collagen binding sites determined in
the present experiments is well consistent with those reported numbers
of integrin
2
1. We used 300 kDa as the
molecular mass of collagen to calculate the number of binding sites,
which assumes that the soluble collagen is present in the monomeric
form. Thus these binding data suggest that the collagen bound to
integrin
2
1 is mainly the monomeric form.
Our present study is the first to determine the dissociation constant
for the interaction of integrin
2
1 with
collagen.
The finding that soluble collagen can bind to CRP-activated platelets
should provide some clues about the relationship between GP VI and
integrin
2
1 because CRP was reported to
bind to GP VI and aggregate platelets (11, 27). Soluble collagen
binding was inhibited by PGI2, which suggests that
platelets were first activated by CRP and then integrin
2
1 becomes activated through the
inside-out signaling induced by CRP. These results suggest the
relationship between the GP VI and integrin
2
1.
In contrast to the binding of soluble collagen, which behaves more like
a classical ligand, it is very difficult to measure the specific
binding of fibrous collagen to platelets. Previous studies could not
demonstrate the specific binding of fibrous collagen to platelets and
reported only the total binding of the fibrous collagen (8, 9). Our
binding studies indicated that one of the reasons for this is the fact
that a higher amount of labeled collagen sedimented in the presence of
excess cold collagen (Fig. 2), the condition usually employed to
determine nonspecific binding; this would be due to the formation of
bigger collagen fibrils in the presence of a high concentration of
unlabeled collagen. Furthermore, a small portion of the labeled
collagen precipitated even in the absence of excess cold collagen.
Because of this, we performed control experiments that measured the
sedimentation of labeled fibrous collagen in the presence and absence
of excess unlabeled fibrous collagen; these values were subtracted from those of total binding and those of nonspecific binding, respectively, to obtain the actual value for the specific binding. Without these corrections, the specific binding appears to be very low; this would be
the reason that the specific binding of fibrous collagen could not be
detected by previous workers. Other precautions were also necessary to
optimize the fibrous collagen binding assay and increase
reproducibility. Since fibrous collagen readily sticks to laboratory
ware, we used siliconized pipette tips and tubes, ran at least six
replicates for each data point, and measured both the pellet and
supernatant (in the binding tube) after removing the tips to obtain the
total radioactivity we actually added to the tube. The specific fibrous
collagen binding increased almost linearly as a function of added
labeled fibrous collagen (Fig. 12), consistent with specific binding
observed in the very low ligand concentration range, much lower than
the dissociation constant. We could not perform a kinetic analysis of
the fibrous collagen binding because fibrous collagen is not
homogeneous and we could not measure the specific binding of fibrous
collagen at the higher concentrations, which was precluded by the
inability to add unlabeled collagen at the necessary 100-fold excess at
high concentrations of labeled ligand.
We found that the binding of fibrous collagen had characteristics very
different from those of soluble collagen binding. The initial binding
of fibrous collagen is very rapid, accounts for most of the observed
specific binding, and most notably is not dependent on the presence of
metal ions. In the presence of Mg2+, there is a slow
gradual increase in fibrous collagen binding after the initial rapid
binding (Fig. 11). The amount of the
Mg2+-dependent portion of the specific binding
is similar to the percent decrease (about 30%) in specific binding
observed in the presence of the anti-integrin
2
1 antibody Gi9 (Fig. 13). These results suggest that the slow and Mg2+-dependent
binding of fibrous collagen would be due to the contribution of
integrin
2
1. Considering the previously
reported metal ion independence of the bindings of GP VI with collagen
and CRP (11, 15), the main portion of the fibrous collagen binding
observed in our present study, fast and also metal ion-independent, may be attributable to GP VI.
Soluble collagen binding was decreased when platelets were activated
with high concentrations of CRP (Fig. 8). This is because soluble
collagen binding is inhibited by CRP, as seen in Fig. 9, which
illustrates the CRP concentration-dependent inhibition of
soluble collagen binding to TS2/16 activated platelets. Since CRP is a
synthetic polypeptide composed mainly of repeating tripeptide (Gly-Pro-Hyp) units, these results suggest that the basic triple helical structure of collagen would also contribute to the binding of
soluble collagen to integrin
2
1. The same
conclusion was also deduced by Morton et al. (28) from their
study of a synthetic polypeptide specifically recognized by integrin
2
1. They determined the specific peptide
sequence recognized by integrin
2
1 by
using overlapping peptide sequences of collagen type III covalently linked to a homotrimeric triple-helical peptide. Residues 522-528 of
the collagen
1(III) chain were found to be the minimum structure required for the recognition of integrin
2
1. Although a linear DGEA sequence of
collagen
1(I) was assigned to the binding site of collagen (29), the
effect of DGEA is questionable and attempts to synthesize a linear
peptide that can react with integrin
2
1 have not been successful (30).
Recent work from a number of laboratories have indicated the
participation of two different signaling pathways in the platelet activation induced by collagen through experiments using GP
VI-deficient platelets and GP VI-activating reagents, CRP and
convulxin. CRP and convulxin induced tyrosine phosphorylation of
platelet proteins, especially, tyrosine kinase Syk, Fc receptor
chain, and phospholipase (PL)C
2 independently of integrin
2
1 (27, 31-34). In the GP VI-deficient
platelets, the tyrosine phosphorylation of Syk, Fc receptor
chain
and PLC
2 was decreased and the phosphorylation of c-Src of the
patient's platelets was inhibited by an anti-integrin
2
1 antibody (33), suggesting that
integrin
2
1 would activate platelets
through the phosphorylation of c-Src. Our data indicate that platelets
have at least two different collagen receptors: integrin
2
1, which binds with high affinity to
soluble collagen, and another receptor, which may be GP VI, that
rapidly binds to fibrous collagen. Since the signaling by the binding
of fibrous collagen to GP VI was indicated by the above mentioned
reports, it would be meaningful to analyze the effect of the binding of soluble collagen to integrin
2
1 on the
signaling system in platelets. The binding of soluble collagen to
platelets should be a useful tool for analyzing the reaction of
platelets with collagen.
We thank Drs. Yuji Saito and Junichi Takagi,
Tokyo Institute of Technology, Tokyo, Japan for giving us the antibody
TS2/16 and Dr. Shosaku Nomura, Kansai Medical University, Osaka, Japan, for the antibody NNKY5-5. We also thank Dr. Yoshiki Miura of our department for the preparation of CRP.