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INTRODUCTION |
When the integrity of the vascular endothelium is disrupted,
various macromolecular components of the vascular subendothelium become
exposed and accessible to platelets. Although several of these
components, such as laminin, fibronectin, and von Willebrand factor,
all provide a suitable substrate for platelet adhesion, fibrillar
collagen is the most thrombogenic constituent of the vascular
subendothelium since it supports not only adhesion but also causes
platelet activation leading to platelet aggregation (1, 2). Because
platelet-platelet interactions are primarily mediated by the
simultaneous binding of the multivalent adhesive glycoproteins
fibrinogen and/or von Willebrand factor to activated GP1
IIb-IIIa complexes on two different
platelets, collagen must ultimately operate by effecting the activation
of the GP IIb-IIIa complexes on an adhered platelet to either capture
or serve as docking site for a circulating platelet.
Three GPs, namely
2
1 integrin (GPIa-IIa),
CD36 (GPIV, also known as GPIIIb), and GP VI have been implicated in
platelet-collagen adhesive interactions, however, the roles of these
GPs on the adhesion-induced expression of activated GP IIb-IIIa
complexes are not fully understood.
Four patients have been described with mild bleeding diathesis
attributable to deficient expression of
2-integrin and
platelets from these patients had impaired collagen-induced aggregation but aggregated normally to other agonists (3-6). Anti-CD36 antibodies have been shown to partly inhibit platelet adhesion to fibrillar collagen under both static and flow conditions (7-9). However, platelets from Naka-negative donors which constitutively
lack CD36 have been shown to aggregate normally to collagen (10-12).
Several Japanese patients with mild bleeding disorders have been
described whose platelets failed to aggregate in response to collagen
(13-16). Analysis of membrane glycoproteins in these patients revealed
that their platelets either lacked GP VI or had very little of it. Thus
far, lack of either
2
1 integrin or GP VI
in patients has been associated with impaired platelet aggregation in
response to collagen. These results imply that both
2
1 integrin and GP VI have definite roles
in activation of the GP IIb-IIIa complex after platelet binding to
collagen. Clinical studies also suggested that more than a single
partial defect is required for a disorder of collagen platelet
interaction to become clinically important (17). A recent study has
shown that GP VI-deficient platelets bind some fibrinogen in response
to collagen without aggregation, suggesting that collagen may induce
some signaling via
2
1 integrin, leading to the activation of the GP IIb-IIIa complex (18). In addition, another
collagen receptor has recently been identified in human platelets. A
recombinant receptor protein (54 kDa), obtained by using a prokaryotic
expression system, reacted specifically with type I collagen but not
with type III collagen (19).
A large number of investigators have examined the collagen-induced
platelet activation process including the expression of activated GP
IIb-IIIa complex during collagen-induced aggregation of washed
platelets in suspension or during perfusion of whole blood over
collagen-coated coverslips. Three excellent reviews have appeared in
the past year (20-22). However, time-dependent changes in
the expression of activated GP IIb-IIIa complex during platelet
adhesion to immobilized collagen under static conditions have not been
studied in detail. We recently showed the role of GP VI in divalent
cation-independent platelet adhesion to fibrillar collagen under
static conditions and its direct association with the adhesion-induced
thromboxane A2 (TXA2) generating system (9). Using our static adhesion assay, we have now investigated the role of
2
1 integrin, CD36, and GP VI on the
expression of activated GP IIb-IIIa complexes on platelets adhered to
type I fibrillar and monomeric collagens. We simultaneously measured
adhesion rate and the activation of the GP IIb-IIIa complexes by
employing 125I-PAC-1 which binds only to the activated
forms of the GP IIb-IIIa complexes (23). Furthermore, by using confocal
laser scanning microscopy, we were able to visualize adhesion-induced
changes in the morphology of platelets and distribution of activated GP IIb-IIIa complexes as detected by FITC-labeled PAC-1. Our results clearly demonstrate that platelet adhesion to immobilized collagen can
directly induce the activation of the GP IIb-IIIa complex and this
inside-out signaling is mediated by both
2
1 integrin and GP VI.
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EXPERIMENTAL PROCEDURES |
Reagents--
Acid-insoluble equine tendon fibrillar collagen
(Chrono Par) was obtained from Chrono-Log Co. (Broomall, PA).
Acid-soluble rat tail type I collagen was purchased from Collaborative
Biomedical Products Research (Bedford, MA). 51Cr
(Na251CrO4, 250 mCi/mg) and
Na125I (1 mCi/10 µl) were purchased from Amersham Co.
Prostaglandin E1 and SQ29548 were obtained from Cayman
Chemicals (Ann Arbor, MI). Chelex 100 was from Sigma. Unlabeled PAC-1
and fluorescein isothiocyanate (FITC)-labeled PAC-1 were purchased from
Becton Dickinson (San Jose, CA).
Antibodies--
Protein G-affinity purified mouse monoclonal
antibody 6F1, directed against human platelet
2
1 integrin and recognizing the
2-subunit, was generously provided by Dr. Barry S. Coller (School of
Medicine, State University of New York, Stony Brook, NY). An unrelated
mouse monoclonal antibody of the IgG1 subclass (clone-MOPC 21) was purchased from Sigma. A monospecific polyclonal antibody to
human platelet CD36 (number 916) was raised in New Zealand White
rabbits as described previously (24). A monospecific antibody against
GP VI was purified from the plasma of a patient with idiopathic thrombocytopenic purpura who had developed an autoantibody against GP
VI (16). Fab fragments (Fabs) were prepared from the IgG fraction of
the patient's plasma, from normal human plasma and from the rabbit
anti-CD36 serum by digestion with agarose-coupled papain utilizing a
ImmunoPure Fab preparation kit (Pierce Chemical Co., Rockford, IL)
principally according to the manufacturer's instructions but with a
slight modification in temperature and time of incubation (9). The
final product in each case was dialyzed extensively against HEPES
saline, pH 7.4. Fabs thus obtained retained their activity as judged by
their ability to block aggregation of washed platelets induced by the
corresponding intact IgG. In addition, 6F1 and Fabs from control IgG,
rabbit anti-CD36 serum, and anti-GP VI serum did not induce any
platelet aggregation and [14C]serotonin secretion,
suggesting that these antibodies do not activate platelets. For
antibody experiments, platelets were incubated with each antibody for
30 min at room temperature prior to their being allowed to adhere to
collagen-coated wells.
Platelet Preparation--
Human platelet-rich plasma was
prepared as described previously (9). Washed platelets were prepared by
the citrate wash method with minor modifications. Briefly
citrate-washed platelets were suspended in Chelex 100-pretreated
modified Tyrode-HEPES buffer (136.7 mM NaCl, 5.5 mM glucose, 2.6 mM KCl, 13.8 mM,
NaHCO3, 0.36 mM
NaH2PO4·H2O, 0.25% bovine serum
albumin, pH 7.4) at a concentration of 2 × 109
platelets/ml. When required, platelets (1 × 109) were
labeled with Na251CrO4 (50 µCi/ml) for 1 h at room temperature followed by washing twice
with citrate wash buffer containing 0.5% bovine serum albumin. Finally
washed platelets were resuspended in Chelex 100-treated modified
Tyrode-HEPES buffer containing 10 µM Ca2+
with or without further supplementation with 1 mM
Mg2+. To avoid platelet activation during washing and
aggregation during adhesion assays, prostaglandin E1
(250 ng/ml) was included in all buffers used to prepare washed
platelets and in subsequent operations.
Adhesion Assay and Binding of 125I-PAC-1 to Adhered
Platelets--
Monoclonal antibody PAC-1 was radiolabeled with
Na125I using IODO-BEADS as suggested by the manufacturer
(Pierce). Labeled antibody was separated from free Na125I
by gel filtration on a PD-10 column (Pharmacia, Piscataway, NJ) which
was equilibrated with Tyrode-HEPES buffer. The specific activity of
125I-PAC-1 was approximately 200,000 cpm/µg protein.
Microtiter wells were coated with type I acid-insoluble equine tendon
fibrillar collagen or with type I acid-soluble rat tail collagen
maintained under acid conditions to ensure maintenance of monomer
structure. Platelet adhesion assays were performed as described
previously (9), but with a slight modification in preparation of the
suspending medium. Since maximal PAC-1 binding to the activated form of
the GP IIb-IIIa complex is observed at 3-10 µM free
Ca2+ (23), Tyrode-HEPES buffer was first treated with
Chelex 100 to remove any divalent cations and later supplemented with 1 mM Mg2+ and 10 µM
Ca2+ for Mg2+-dependent adhesion
and with 10 µM Ca2+ alone for
Mg2+-independent adhesion. Platelets from a single donor
were aliquoted into two portions. One aliquot was converted to a
51Cr-labeled platelet suspension containing unlabeled PAC-1
(1 µg/ml) while the other aliquot was converted to a nonlabeled
platelet suspension containing 125I-PAC-1 (1 µg/ml). A
portion (50 µl) of the unlabeled platelet preparation was added to
collagen-coated wells. At the desired time adhesion was stopped by
removing nonadhered platelets by washing each well six times by
decantation with 200-µl aliquots of suspending Tyrode-HEPES buffer.
The adhered platelets were solubilized in SDS (2%) for 30 min and
125I-PAC-1 binding was quantitated by counting the lysates
in a
-counter (C). At the end of the incubation, an
aliquot (50 µl) of unused platelet suspension was solubilized with an
equal volume of SDS (2%) and counted for total count (T).
Another aliquot of unused platelet suspension was centrifuged at 13,000 rpm in a microcentrifuge through a 20% sucrose and the pellet was
solubilized in 50 µl of SDS (2%) and counted to obtain nonspecific
binding (NS). Adhesion rate (R = % adhesion × 0.01) was quantitated in parallel experiments using
51Cr-labeled platelets. The adhesion-induced
125I-PAC-1 binding was calculated by the following
equation,
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(Eq. 1)
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Confocal Laser Scanning Microscopy--
Microwells were prepared
by attaching a Flexiperm chamber (Heraeus Instruments, Osterode,
Germany) on a cover glass. Collagen coating and bovine serum albumin
blocking were performed as described above. Washed platelets containing
FITC-labeled PAC-1 (1 µg/ml) were added to the microwells and
incubated for the indicated times. Unbound antibodies and platelets
were removed by washing six times with Tyrode-HEPES buffer and then
adhered platelets were fixed with 1% paraformaldehyde for 30 min at
room temperature. Differential interference contrast images and
fluorescent confocal images of 0.5-µm optical sections were
simultaneously obtained using a Carl Zeiss 510 confocal laser scanning
microscope (Thornwood, NY) with a Zeiss Plan-Apo 100 × 1.40 NA
oil immersion objective. FITC fluorescence was detected at an
excitation wavelength of 488 nm with a barrier filter at 500 nm.
 |
RESULTS |
Platelet Adhesion to Collagen and Adhesion-induced Activation of
the GP IIb-IIIa Complex--
We previously used Mg2+ or
EDTA-containing Tyrode-HEPES buffer to characterize divalent
cation-dependent or -independent platelet adhesion to
fibrillar and monomeric collagens (9). To detect activation of the GP
IIb-IIIa complex in the present work, we employed
125I-PAC-1 which requires 3-10 µM free
Ca2+ to fully bind to the activated form of the GP IIb-IIIa
complex (23). We therefore, modified the suspension medium as described under "Experimental Procedures." Adhesion rate was determined using
51Cr-labeled platelets and the activation of the GP
IIb-IIIa complex was simultaneously determined by measuring
125I-PAC-1 binding to adhered platelets. Typical patterns
of time-dependent platelet adhesion to type I
acid-insoluble fibrillar collagen and acid-soluble monomeric collagen
and concomitant activation of the GP IIb-IIIa complexes both under
Mg2+-dependent and -independent conditions are
shown in Fig. 1, a and
b, respectively. Under these conditions, PAC-1 had a
negligible effect on platelet adhesion (data not shown). Both in the
presence and absence of extracellular Mg2+, platelets
adhered to fibrillar collagen showed time-dependent PAC-1
binding and the Mg2+-independent platelet adhesion was
about half of the adhesion observed in the presence of
Mg2+. As shown in Fig. 1b, platelets adhered to
monomeric collagen also showed PAC-1 binding, however, this adhesion
was exclusively Mg2+-dependent and the extent
of PAC-1 binding was always less than that of platelets adhered to
fibrillar collagen. At the 60-min time point, PAC-1 binding was about
60% of that seen with fibrillar collagen even though the same extent
of adhesion was seen in both cases.

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Fig. 1.
Time-dependent platelet
adhesion to plastic-immobilized type I collagen and
125I-PAC-1 binding. 51Cr-labeled washed
platelets containing 1 µg/ml unlabeled PAC-1 or unlabeled washed
platelets containing 1 µg/ml 125I-PAC-1 were added to
microtiter wells coated with type I acid-insoluble equine tendon
fibrillar collagen (a) or type I acid-soluble rat tail
collagen (b). Mg2+-dependent and
-independent adhesion and corresponding 125I-PAC-1 binding
were measured as described under "Experimental Procedures."
Platelet adhesion and 125I-PAC-1 binding have been
expressed as percentage of total number of platelets and total count of
125I-PAC-1 added. Solid bars represent platelet
adhesion while hatched bars represent 125I-PAC-1
binding. Values given are the mean ± S.D. of at least five
experiments using platelets from different individuals each run in
replicate of three.
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Effects of Anti-
2
1 Integrin,
Anti-CD36, and Anti-GP VI Antibodies and Aspirin on Platelet Adhesion
to Type I Fibrillar Collagen and Activation of the GP IIb-IIIa
Complex--
We next examined the effect of antibodies against three
collagen receptors, namely anti-
2
1
integrin IgG (6F1; 20 µg/ml), anti-CD36 Fabs (916; 300 µg/ml), and
anti-GP VI Fabs (300 µg/ml), on platelet adhesion to fibrillar
collagen and activation of the GP IIb-IIIa complex at the 60-min time
point. These concentrations of antibodies have been shown to be optimal
in previous studies (9). We also examined the effect of aspirin (1 mM) since we previously showed that platelets adhered to
fibrillar collagen generated considerable amounts of TXA2
both in the presence and absence of Mg2+ (9). As shown in
Fig. 2a, in the presence of
Mg2+, anti-
2
1 integrin,
anti-CD36, anti-GP VI antibody, and aspirin showed minimal inhibition
of platelet adhesion to fibrillar collagen (5-15%) at the 60-min time
point. In contrast, anti-
2
1 integrin, anti-GP VI antibody, and aspirin significantly inhibited PAC-1 binding
by about 40, 50, and 30%, respectively: the TXA2 receptor antagonist SQ29548 (10 µM) showed similar effects to
aspirin (data not shown). Anti-CD36 antibody had no effect on adhesion
or on PAC-1 binding. The combination of
anti-
2
1 integrin and anti-GP VI antibody
inhibited platelet adhesion completely as reported earlier (9) and no
activation of the GP IIb-IIIa complex was detected. We then examined
the combination of each antibody and aspirin. No apparent additive or
synergistic inhibitory effects were observed. As shown in Fig.
2b, in the absence of Mg2+ anti-GP VI antibody
inhibited completely both adhesion and PAC-1 binding whereas no effect
was seen with anti-
2
1 integrin antibody which had shown significant inhibition of PAC-1 binding in the presence
of Mg2+. Anti-CD36 antibody inhibited platelet adhesion to
collagen about 30% which correlated with the inhibition of PAC-1
binding. Aspirin showed minor inhibitory effects on both platelet
adhesion and PAC-1 binding. A combination of
anti-
2
1 integrin or anti-CD36 antibody
with aspirin did not show significantly additive or synergistic effects
on either adhesion or PAC-1 binding. Under each set of conditions,
unrelated mouse IgG1 and control Fabs prepared from rabbit
and human normal IgG used as negative controls had negligible effects
on adhesion and PAC-1 binding (data not shown). Platelets adhered to
fibrillar collagen secreted their
and dense granule contents even
in the absence of Mg2+ (9). Therefore we examined the
possible role of released ADP and serotonin on activation of the GP
IIb-IIIa complex. Neither ADP removal by creatine phosphate/creatine
phosphokinase nor the presence of serotonin receptor antagonist,
ketanserin, had any significant effect on the activation of the GP
IIb-IIIa complex (data not shown).

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Fig. 2.
Effects of
anti- 2 1
integrin, anti-CD36, anti-GP VI antibodies, and aspirin (ASA) on
platelet adhesion to type I acid-insoluble fibrillar collagen and
125I-PAC-1 binding. 51Cr-labeled platelets
were incubated with anti- 2 1 integrin
(6F1; 20 µg/ml), anti-CD36 (number 916 Fabs; 300 µg/ml), anti-GP VI
(anti-p62 Fabs; 300 µg/ml) antibodies, and aspirin (1 mM)
for 30 min at room temperature prior to their addition to the
microtiter wells. Adhesion and 125I-PAC-1 binding
were measured as described under "Experimental Procedures."
Nonimmune IgG subclass-matched antibodies, used as a negative
control, had no effect on platelet adhesion and 125I-PAC-1
binding. Platelet adhesion and 125I-PAC-1 binding are
expressed as percentage of the corresponding control value. Adhesion
and 125I-PAC-1 binding were studied in the presence of
Mg2+ (a) or in its absence (b).
Values shown are the mean ± S.D. of four experiments each run in
replicate of three.
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Effects of Anti-
2
1 Integrin,
Anti-CD36, and Anti-GP VI Antibodies on Platelet Adhesion to Type I
Monomeric Collagen and Activation of the GP IIb-IIIa Complex--
As
reported earlier, platelet adhesion to type I monomeric collagen is
exclusively Mg2+-dependent and solely mediated
by
2
1 integrin (9). This interaction does
not cause any release reaction and generation of TXA2. As shown in Fig. 3,
anti-
2
1 integrin antibody inhibited both
platelet adhesion and PAC-1 binding completely at the 60-min time
point. However, anti-CD36 and anti-GP VI antibody alone or in
combination had minimal effects on both platelet adhesion and PAC-1
binding.

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Fig. 3.
Effects of
anti- 2 1
integrin, anti-CD36, and anti-GP VI antibodies on
Mg2+-dependent platelet adhesion to monomeric
type I rat tail collagen and 125I-PAC-1 binding.
Conditions were similar to those used in Fig. 2a except that
acid-soluble rat tail collagen was used. Platelet adhesion and
125I-PAC-1 binding are expressed as percentages relative to
the corresponding control values. Values shown are the mean ± S.D. of four experiments each run in replicate of three.
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Visualization of Adhesion-induced Morphological Changes and the
Expression of the Activated GP IIb-IIIa Complex--
To visualize
time-dependent platelet adhesion to collagen and changes in
PAC-1 binding, FITC-labeled PAC-1 (1 µg/ml) was added to washed
platelet suspensions and adhesion assays were performed as described
under "Experimental Procedures." Fig.
4, a-i, shows the time course
of differential interference contrast images of platelets adhered to
type I fibrillar and monomeric collagen and the fluorescent confocal
images of the surface activation of the GP IIb-IIIa complexes as
detected by FITC-labeled PAC-1. FITC-labeled PAC-1 binding was specific
since FITC-labeled control antibody showed no binding to adherent
platelets and prostaglandin E1-treated resting platelet
showed negligible FITC-labeled PAC-1 binding (data not shown). These
series of photomicrographs depict direct induction of the activated GP
IIb-IIIa complexes and their distribution after platelet adhesion to
fibrillar and monomeric collagen. At the 15-min time point, in the
presence of Mg2+, platelets in contact with fibrillar
collagen showed pseudopodia followed by broad lamellae formation (Fig.
4a). PAC-1 binding was evident on partially spread
platelets. It should be noted that some platelets showed blebbing (Fig.
4a, arrow). This formation is further evident at the 30-min
time point (Fig. 4b). Platelets adhered to fibrillar
collagen spread out with time and the surface was almost covered with
fully spread out platelets at the 60-min time point, however, no
apparent aggregates were seen (Fig. 4c). To evaluate
possible platelet aggregate formation, adhered platelets at the 60-min
time point were fixed with 1% paraformaldehyde and incubated with
mouse monoclonal anti-fibrinogen antibody and further incubated with
FITC-labeled anti-mouse antibody. Anti-fibrinogen antibody binding was
found to be localized at the boundary of adjacent platelets that had
already spread onto collagen. However, no anti-fibrinogen antibody
binding was observed on the platelet surface, confirming that no
aggregates were formed in this assay (data not shown). In the absence
of Mg2+, platelets adhered to fibrillar collagen and also
showed PAC-1 binding, however, spreading was minimal, suggesting that
Mg2+ is required for platelet spreading (Fig. 4,
d-f). Fig. 4, g-i, shows the time course of
platelet adhesion to type I monomeric collagen in the presence of
Mg2+. Platelets adhered to monomeric collagen also showed
some spreading and a lesser extent of activation of the GP IIb-IIIa
complex as compared with fibrillar collagen. Fig. 4, j-l,
shows the effects of anti-
2
1 integrin,
anti-GP VI antibody, and aspirin on platelet adhesion to fibrillar
collagen and PAC-1 binding in the presence of Mg2+ at the
60-min time point. In the presence of
anti-
2
1 integrin antibody, platelet
spreading was strongly reduced but not completely inhibited, suggesting
that some integrin other than
2
1
integrin, possibly GP IIb-IIIa, may be involved in platelet spreading.
As for PAC-1, anti-
2
1 integrin antibody
slightly inhibited its binding and redistribution was minimal as
compared with control (Fig. 4c). In the presence of anti-GP
VI antibody, PAC-1 binding was remarkably reduced and platelet
spreading was slightly inhibited. Aspirin had minimal effects on
platelet adhesion and spreading, however, a significant decrease of
PAC-1 binding was observed confirming our 125I-PAC-1
binding data.

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Fig. 4.
Confocal microscopic images showing
fluorescent PAC-1 binding and differential interference contrast images
showing morphology of platelets adhered to type I collagen in the
presence of Mg2+. Washed platelets containing 1 µg/ml FITC-labeled PAC-1 were added to microwells coated with type I
acid-insoluble fibrillar collagen or acid-soluble monomeric collagen.
At the desired time points, unbound antibodies were washed and adhered
platelets were fixed with 1% paraformaldehyde for 30 min at room
temperature. Samples were analyzed by using confocal laser scanning
microscopy (LSM 510) as described under "Experimental Procedures."
Typical patterns of FITC-PAC-1 binding (left panel),
differential interference contrast imaging (middle panel),
and overlapped imaging (right panel) are shown. Platelet
adhesion to fibrillar collagen in the presence of Mg2+ at
15- (a), 30- (b), and 60-min (c) time
points and in the absence of Mg2+ at 15- (d),
30- (e), and 60-min (f) time points are shown.
Arrows indicate bleb formation. Platelet adhesion to
monomeric collagen at 15- (g), 30- (h), and
60-min (i) time points are shown. Platelets were incubated
with anti- 2 1 integrin (6F1; 20 µg/ml)
(j), anti-GP VI (anti-p62 Fabs; 300 µg/ml) (k),
antibodies and aspirin (1 mM) (l) for 30 min at
room temperature and platelet adhesion at 60-min time points are shown.
Scale bar = 5 µm.
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|
 |
DISCUSSION |
The accessibility of activated GP IIb-IIIa complexes on the
surface of platelets adhered to a damaged vessel plays a crucial role
in subsequent platelet recruitment to a thrombus. In this study, we
have investigated the role of collagen receptors, namely
2
1 integrin, CD36, and GP VI, on the
expression of activated GP IIb-IIIa complexes induced by platelet
adhesion to type I fibrillar and monomeric collagen under static conditions.
Platelet adhesion to monomeric collagen requires Mg2+ and
is exclusively mediated by
2
1 integrin
and there is no significant role of CD36 or GP VI in this interaction.
Platelets adhered to monomeric collagen neither secrete their granule
content nor generate TXA2 (9). However, these platelets
show significant spreading and induce activation of the GP IIb-IIIa
complex although this expression is about 40% less than adhesion to
fibrillar collagen. This is consistent with the earlier report which
showed the binding of thromboerythrocytes to platelets adhered to type
I rat skin collagen (25). Taken together, these results suggest that
2
1 integrin-mediated inside-out signaling
is able to induce partial platelet spreading and partial activation of
the GP IIb-IIIa complex. Monomeric collagen effectively supports
platelet adhesion, whereas polymerization of monomeric collagen is
required to induce platelet aggregation and secretion (26-30). These
observations may be explained by the fact that the number of activated
GP IIb-IIIa complexes and their localization induced by monomeric
collagen is not sufficient to elicit a full aggregatory response.
Platelet adhesion to fibrillar collagen is composed of
Mg2+-dependent and -independent adhesion (9,
31). In the presence of Mg2+, platelets adhered to
fibrillar collagen showed full spreading and increased activation of
the GP IIb-IIIa complex than that of platelets adhered to monomeric
collagen. Resting platelets have been shown to have approximately
50,000 molecules of GP IIb-IIIa complex on their surface. Upon
activation with strong agonist such as thrombin, this number increases
to 100,000 sites per platelet, probably reflecting the recruitment from
the internal pool of GP IIb-IIIa complex (23, 32). However, the maximum
number of PAC-1-binding sites on activated platelets under saturating conditions has been reported to be only 25,000 sites per platelet (23).
In the presence of Mg2+, platelets adherent to fibrillar
collagen at the 60-min time point bound about 12,000 125I-PAC-1 molecules per platelet. This number is about
half of the total PAC-1-binding sites as determined by thrombin
stimulation (23). In our confocal images, PAC-1 binding was observed
only on the nonadhered surface of the platelets; no apparent PAC-1 binding was observed by Z-sectioning or vertical images (X-Z
sectioning) on the side of the cell adhering to collagen fibers (data
not shown). Thus our PAC-1 binding data may reflect only one side surface expression of the activated GP IIb-IIIa complex. In our previous report (9), we measured both
- and dense granule secretion
during platelet adhesion and found that only fibrillar collagen can
induce these granules secretion (50% in the presence of
Mg2+). Therefore, the increased number of PAC-1-binding
sites on platelets adherent to fibrillar collagen may partly reflect
the recruitment of the GP IIb-IIIa complex originating from internal pools.
Anti-
2
1 integrin antibody inhibited
activation of the GP IIb-IIIa complex about 40% and significantly
inhibited but did not eliminate platelet spreading. These results
confirm that
2
1 integrin-mediated
signaling is partly responsible for the activation of the GP IIb-IIIa
complex and platelet spreading once platelets have adhered to fibrillar
collagen. The mechanism of platelet spreading which is not attributable
to
2
1 integrin remains to be elucidated.
It has been reported that
2
1
integrin-deficient platelets were not able to spread on collagen and
collagen failed to induce platelet aggregation (3, 4, 6). In the
presence of anti-
2
1 integrin antibody,
collagen-induced platelet aggregation was also abolished (5, 33).
Accordingly, it could be concluded that platelet spreading on collagen
fibers is mainly mediated by
2
1 integrin
and that nearly full activation of the GP IIb-IIIa is required for
platelet aggregation induced by collagen. It is also possible that
2
1 integrin may be necessary for an
initial tethering of floating platelets to the collagen fiber (22,
34-37).
In our previous study, Mg2+-independent adhesion was
studied in the presence of EDTA to chelate any Mg2+ arising
from the impurities of the reagents used to make Tyrode-HEPES buffer
(9). In order to remove Mg2+ and other divalent cations
from the buffer, we used Chelex 100. Divalent cations were added as
needed. The results of our previous study indicated that divalent
cation-independent adhesion amounts to about one-fourth of the adhesion
observed in the presence of Mg2+. Using the chelation
method we found that Mg2+-independent adhesion was about
half that seen in the presence of Mg2+ suggesting that
EDTA, even at only 50 µM concentration, can affect platelet adhesion. The results of this study, as well as our earlier study, clearly show that in the absence of Mg2+ platelets
adhered to fibrillar collagen and this interaction is mainly dependent
on GP VI and there seems to be no role of
2
1 integrin. Platelets adhered to
fibrillar collagen in the absence of Mg2+ do secrete and
generate TXA2 (9). Although this interaction does not
induce platelet spreading, apparent activation of the GP IIb-IIIa
complex was observed. In the presence of Mg2+, anti-GP VI
Fabs also inhibited the activation of the GP IIb-IIIa complex by about
50% and platelet spreading was slightly inhibited. These results
suggest that GP VI-mediated signaling also plays an important role in
adhesion-induced activation of the GP IIb-IIIa complex. GP VI-deficient
platelets failed to aggregate in response to collagen (13-16) and
anti-GP VI Fabs also abolished collagen-induced platelet aggregation
(16). In addition, a recent study under flow conditions has shown that
GP VI-deficient platelets exhibit almost normal primary adhesion to
collagen fibers but that platelet aggregates formation is abrogated
(38). These observations are consistent with the role of GP VI and the
activation of the GP IIb-IIIa complex.
Although G proteins, intracellular calcium, protein kinases, and many
other proteins are thought to be involved in the expression of
activated GP IIb-IIIa complex, the exact mechanisms that control the
expression of activated GP IIb-IIIa complex are still obscure (39, 40).
Several lines of evidence suggest that activation of platelets
stimulated by collagen is mediated through a tyrosine kinase pathway
that involves the Fc receptor
chain, Syk, and phospholipase C
2
(41-43). Phosphorylated phospholipase C
2 becomes activated leading
to the generation of inositol triphosphate and elevation of
intracellular Ca2+. These phosphorylation events seem to be
mediated by both
2
1 integrin and GP VI
(44-47). In other words,
2
1 integrin-
and GP VI-mediated signaling converge on intracellular Ca2+
elevation. In fact, it has been shown that collagen directly induces a
rise in cytosolic calcium in single human platelets (48). The
intracellular Ca2+ chelator, BAPTA, inhibited
125PAC-1 binding in a dose-dependent manner
although it does not itself affect platelet
adhesion,2 suggesting that
intracellular Ca2+ seems to be a key second messenger for
the inside-out signaling leading to the activation of the GP IIb-IIIa
complex. Further experiments are currently underway to elucidate the
relationship between intracellular calcium ion and the expression of
activated GP IIb-IIIa complexes.
We have previously reported that platelets adhered to fibrillar
collagen are able to secrete dense and
granule contents and
generate TXA2: in particular TXA2 generation
was found to be directly associated with GP VI (9). TXA2,
ADP, and serotonin are considered as positive feedback agonists in that
they bind back to platelets and synergistically potentiate platelet
activation processes leading to large aggregate formation. Therefore,
we estimated the contribution of TXA2, ADP, and serotonin
to the activation of the GP IIb-IIIa complex. In the presence of
Mg2+, aspirin, and the TXA2 receptor
antagonist, SQ29548, showed about 30% inhibition of activation of the
GP IIb-IIIa complex induced by platelet adhesion to fibrillar collagen,
but only minimal effects on adhesion and spreading of platelets were
observed. These results suggest that generated TXA2 binds
to adhered platelets and induces activation of the GP IIb-IIIa complex.
In the presence of Mg2+, a combination of aspirin with
anti-
2
1 integrin did not show any
additive inhibitory effects on the activation of the GP IIb-IIIa complex. Furthermore, in the absence of Mg2+, aspirin did
not inhibit activation of the GP IIb-IIIa complex. These results
suggest that TXA2-mediated activation of the GP IIb-IIIa
complex may require platelet spreading. In contrast, removal of
secreted ADP by the creatine phosphate/creatine phosphokinase system or
the presence of serotonin receptor antagonist, ketanserin, did not show
any significant inhibition of activation of the GP IIb-IIIa complex,
suggesting that ADP and serotonin do not have a significant effect on
platelets already adhered to collagen. Platelets deficient in GP VI
failed to aggregate and bind fibrinogen upon challenge with
collagen-related peptide (CRP-X) suggesting a role of GP VI in the
activation process and subsequent fibrinogen binding and aggregation.
Although a reduced but significant amount of fibrinogen bound to GP
VI-deficient platelets when they were challenged with fibrillar
collagen (18). Convulxin, a minor component of tropical rattle snake
venom, has been shown to activate platelets by interacting exclusively
with GP VI (49). However, in a related study, an antibody to
2
1 integrin inhibited convulxin-induced platelet aggregation by about 60%. The authors have proposed that during later stages of convulxin-induced platelet activation
2
1 integrin may be partially responsible
for the expression of activated GP IIb-IIIa complex (50). These
observations and our own results further support the view that both GP
VI and
2
1 integrin play important roles
in the expression of the activated GP IIb-IIIa complex induced by
platelet-collagen interactions.
In summary, platelet adhesion to collagen can directly induce
activation of the GP IIb-IIIa complex. Fibrillar collagen induced increased expression than monomeric collagen. This activation process
is mediated by both
2
1 integrin and GP VI
and enhanced by TXA2. Although our static adhesion assay
system was able to dissect the individual role of collagen receptors on
activation of the GP IIb-IIIa complex, it is imperative to elucidate
their exact roles under more physiological conditions such as whole blood under flow conditions. Finally, considering the role of GP VI in
adhesion-induced TXA2 generation and the activation of the
GP IIb-IIIa complex, blocking collagen-GP VI interaction may prove to
be an alternative therapeutic approach to the treatment of thrombotic disease.