Principal Role of Glycoprotein VI in ?2?1 and ?IIb?3
Activation During Collagen-Induced Thrombus Formation
Christelle Lecut, Anne Schoolmeester, Marijke J.E. Kuijpers, Jos L.V. Broers, Marc A.M.J. van Zandvoort,
Karen Vanhoorelbeke, Hans Deckmyn, Martine Jandrot-Perrus, Johan W.M. Heemskerk
Objective—High-shear perfusion of blood over collagen results in rapid platelet adhesion, aggregation, and procoagulant
activity. We studied regulation of ?2?1 and ?IIb?3 integrin activation during thrombus formation on collagen.
Methods and Results—Blockade of glycoprotein (GP) VI by 9O12 antibody or of P2Y purinergic receptors permitted
platelet adhesion but reduced aggregate formation, fibrinogen binding, and activation of ?2?1 and ?IIb?3, as detected
with antibodies IAC-1 and PAC1 directed against activation-dependent epitopes of these integrins. Combined blockade
of GPVI and P2Y receptors and thromboxane formation abolished integrin activation but still allowed adhesion of
morphologically unstimulated, nonprocoagulant platelets. Exogenous ADP partly restored the suppressive effect of
GPVI blockade on integrin ?2?1 and ?IIb?3 activation. Adhesion was fully inhibited only with simultaneous blocking
of GPVI and ?2?1, indicating that the integrin can support platelet–collagen binding in the absence of its activation.
Blockade or absence of GPIb? only moderately influenced integrin activation and adhesion unless GPVI was inhibited.
Conclusions—GPVI- and autocrine-released ADP induce affinity changes of ?2?1 and ?IIb?3 during thrombus formation
on collagen under flow. These integrin changes are dispensable for adhesion but strengthen platelet–collagen
interactions and thereby collagen-induced platelet activation. (Arterioscler Thromb Vasc Biol. 2004;24:1727-1733.)
Key Words: ADP ? collagen ? glycoprotein VI ? integrins ? platelets ? thrombus
platelet interactions with subendothelial matrix components
and for platelet–platelet interactions leading to aggregate and
thrombus formation.1Integrin ?2?1 plays a role in platelet
adhesion to collagen under static2,3and flow conditions.4,5
Integrin ?IIb?3 allows platelets to bind to fibrinogen and von
Willebrand factor (vWF) present on collagen and other
platelets.6This leads to stable platelet adhesion and aggregate
On resting platelets, these integrins are considered to be
present in a low-affinity state. Intracellular signaling or ligand
binding results in conformational changes of the integrins
with a switch to higher-affinity states.6Agonists such as
thrombin, collagen, ADP, and vWF induce ?IIb?3 activation
and platelet aggregation.8,9Full integrin activation with ADP
requires the P2Y1and P2Y12purinergic receptors.10,11Recent
studies show that integrin ?2?1 can also be activated by
inside-out signaling.12,13Thrombin and collagen turn this
integrin into a high-affinity form, whereas ADP changes it to
intermediate affinity.13Although much is known of the
affinity and avidity changes of ?IIb?3 on isolated platelets
latelet integrins are critical in hemostasis. Abundantly
expressed at the platelet surface, integrins are required for
especially,8,14regulation of integrin activation during throm-
bus formation is incompletely understood.
In vivo studies as well as ex vivo experiments, in which
blood was allowed to flow over collagen under arterial shear
conditions, have indicated that glycoprotein (GP) VI is a
principal receptor responsible for collagen-induced activation
of platelets during thrombus formation.5,15–21The ?2?1con-
tribution to platelet–collagen interaction has been debated
extensively.16Current evidence with murine and human
platelets shows that this integrin functions to reinforce the
activating effect of GPVI to produce stable, nonembolizing
thrombi.17,22–24Integrin ?2?1, putatively in its activated
form, synergizes with GPVI to stimulate Ca2?signaling,
granule secretion, and subsequent aggregate forma-
tion.4,15,22,24,26It also assists GPVI in triggering of the
procoagulant platelet response (ie, by stimulating surface
exposure of phosphatidylserine [PS]).3,5,17,22This procoagu-
lant phospholipid strongly potentiates local formation of
thrombin and, hence, coagulation.27In flowing human (but
less clearly so in murine) blood, GPVI blockade still allows
?2?1-dependent platelet adhesion to collagen.5,22This raises
the question of how the activation state of this integrin relates
Received May 6, 2004; accepted May 28, 2004.
From the Departments of Biochemistry (C.L., M.J.E.K., J.W.M.H.), Molecular Cell Biology and Genetics (J.L.V.B.), and Biophysics (M.A.M.J.V.),
CARIM, Maastricht University, The Netherlands; the Laboratory for Thrombosis Research (A.S., K.V., H.D.), KU Leuven, Campus Kortrijk, Belgium;
and E348 Institut National de la Sante ´ et de la Recherche Me ´dicale (C.L., M.J.-P.), Faculte ´ Xavier Bichat, Universite ´ Paris, France.
C.L. and A.S. contributed equally to this work.
Correspondence to J.W.M. Heemskerk, PhD, Department of Biochemistry, Maastricht University, PO Box 616, 6200 MD Maastricht, The Netherlands.
© 2004 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.orgDOI: 10.1161/01.ATV.0000137974.85068.93
to its adhesive and signaling function. Under flow over
collagen, this may involve the GPIb-V-IX complex, which is
another receptor implicated in integrin ?IIb?3 activation on
interaction with vWF.28,29
By using novel antibodies against GPVI and against
activation-dependent epitopes on ?2?1, we investigated
?2?1 and ?IIb?3 activation during human platelet interaction
with collagen and subsequent thrombus formation under
flow. We found that GPVI and P2Y receptor stimulation
caused activation of either integrin, whereas GPIb contributed
to a lesser extent. Surprisingly, we found significant integrin-
dependent adhesion in the absence of its activation.
Materials, blood preparation, and experimental design are available
online at http://atvb.ahajournals.org.
Thrombus Formation Under Flow Conditions
D-phenylalanyl-L-prolyl-L-arginine chloromethylketone (PPACK)-
anticoagulated blood, as described.22Briefly, whole blood was
perfused for 4 minutes over a collagen-coated coverslip through a
parallel-plate transparent flow chamber at a wall-shear rate of 150 to
1000 s?1. PS exposure was detected by postperfusion with rinsing
Hepes buffer, pH 7.45 (in mmol/L: 136 NaCl, 10 glucose, 5 Hepes,
2.7 KCl, 2 MgCl2, and 2 CaCl2, plus 0.1% BSA and 1 U/mL
heparin), containing 1 ?g/mL OG488-annexin-A5. Integrin activa-
tion was monitored by adding fluorescein isothiocyanate (FITC)-
coupled IAC-1 (10 ?g/mL) or PAC1 (1:50) to blood before perfu-
sion. Where indicated, probes were added to the rinsing buffer.
High-resolution phase-contrast and fluorescent images were rec-
orded in real time with intensified cameras as described.17Using
Quanticell software (Visitech), fluorescence images (average 32
camera images) were corrected for background fluorescence by
subtraction of the mean gray level from an adjacent site of the
coverslip. Area coverage by fluorescent platelets was determined
with Quanticell software. Area coverage from phase-contrast images
was analyzed offline using ImagePro software (Media Cybernetics)
by automated threshold settings and application of double masks.
Area coverage data were used to determine platelet deposition on
collagen because they could be collected simultaneously with, and
thus compared with, fluorescence data.
collagen wereperformed with
Confocal and 2-Photon Laser
For confocal microscopy, coverslips with thrombi stained with
FITC-IAC-1, Gi9-FITC, or MOPC-21-FITC were observed with a
Bio-Rad laser-scanning microscope. Where indicated, thrombi were
fixed with 2% formaldehyde and blocked with 15% BSA in PBS, pH
7.5. After permeabilization with 0.005% sodium dodecyl sulfate,
actin cytoskeletal fibers were labeled with Texas-red phalloidin. An
MRC600 laser-scanning microscope system was used, equipped with
argon–krypton and red diode lasers. Optical sections (4 to 8 scans)
were recorded in Kalman filtering mode.
Two-photon laser scanning microscopy (TPLSM) was with a
Bio-Rad 2100 multiphoton system. PPACK blood containing 30%
calcein-labeled platelets17was perfused over collagen, and z-series
of scans were recorded during perfusion. Alternatively, blood was
supplemented with OG488-fibrinogen (0.25 mg/mL). Excitation was
by a Spectra-Physics Tsunami Ti:Sapphire laser, tuned and mode-
locked at 800 nm, producing pulses of ?100 fs wide (repetition rate
82 MHz). Fluorescence was detected at 508 to 523 nm.
Suppressed Aggregation and PS Exposure by
GPVI and ADP Receptor Blockade but Unchanged
Platelet Adhesion to Collagen
The monoclonal antibody (mAb) 9O12, directed against the
GPVI collagen-binding site, acts as an efficient GPVI antag-
onist in vitro.30At 25 ?g/mL, antigen-binding fragments
(Fabs) of 9O12 specifically blocked platelet adhesion to
collagen under static conditions. The fragments also inhibited
collagen-induced platelet aggregation and granule secretion.
These effects were accompanied by suppression of collagen-
induced protein tyrosine phosphorylation (Figure I, available
online at http://atvb.ahajournals.org). Surprisingly, 9O12
stimulated phosphorylation of unknown protein bands at 25
and 36 kDa. The mAb binds to the first Ig domain of GPVI,
distal from the membrane (C. Lecut and M. Jandrot-Perrus,
unpublished data, 2003).
The ability of 9O12 Fab to block GPVI in blood flowing
over type-I collagen fibers was investigated at intermediate
shear stress (1000 s?1). Under control conditions, perfusion
resulted in rapid platelet deposition on collagen, which
increased linearly in time for at least 5 minutes, as recorded
from phase-contrast images.17,22In blood supplemented with
calcein-loaded platelets, the 3D build-up of thrombus forma-
tion was followed by recording of stacks of fluorescence
images using the high-resolution technique of TPLSM, which
provides higher sensitivity and less bleaching than conven-
tional microscopy. After an initial phase of 2 minutes of
adhesion to the collagen, flowing platelets preferentially
incorporated into aggregates (Figure II, available online at
http://atvb.ahajournals.org). In the presence of 9O12 Fab (50
?g/mL), aggregate formation but not initial adhesion was
severely impaired, leaving only 1 to 2 layered platelet groups.
As a result, thrombus volume was reduced greatly with 9O12.
4 minutes of flow (Figure 1), indicated that surface area covered
on average (n?11 subjects), with a variation between donors
Figure 1. Effect of GPVI and P2Y receptor blockade on platelet
aggregation, PS exposure, and integrin activation. Whole blood
was perfused over collagen at 1000 s?1for 4 minutes; preincu-
bation with vehicle (control condition), anti-GPVI 9O12 (50
?g/mL), or P2Y blockers (40 ?mol/L MRS2179 and 20 ?mol/L
AR-C69931MX). A, Representative phase-contrast images
(120?120 ?m) after perfusion. Note the presence of collagen
fibers. Representative fluorescence images (150?150 ?m) after
perfusion with: B, OG488-annexin-A5 to measure PS exposure;
C, FITC-IAC-1 to detect activated ?2?1; or D, FITC-PAC1 to
detect activated ?IIb?3.
1728 Arterioscler Thromb Vasc Biol.
from 9.9% to 25.6%. With 9O12 present, only small aggregates
were observed together with numerous single platelets, whereas
the area covered by platelets remained unchanged (94.9?7.5%
of control condition; Figure 2A). This is an underestimate of the
platelet deposition reduction, bearing in mind the abolishment of
aggregate formation. Higher concentrations of 9O12 up to 200
?g/mL gave similar results.
GPVI-induced PS exposure of the platelets on collagen
was monitored by staining with OG488-annexin-A5.22,31
Under control conditions, many platelets bound annexin-A5,
giving an area coverage with fluorescence of 10.5?1.5%
(n?8). These platelets had a round, blebbing structure, as
described previously.17With 50 ?g/mL 9O12 Fab present,
annexin-A5 staining reduced greatly to 15% of the control
situation (Figures 1B and 2A), along with platelet blebbing.
Thus, 9O12 suppressed collagen-induced aggregate forma-
tion and PS exposure under flow (both effects of GPVI-
induced platelet activation) but not platelet adhesion to
collagen. This was in agreement with earlier results obtained
with the anti-GPVI scFv antibody 10B12.17
Blockers of P2Y1(MRS2179) and P2Y12(AR-C6991MX)
receptors25were used to evaluate the contribution of auto-
crine ADP in platelet deposition under flow. With the P2Y
blockers present, platelets deposited as single cells or as small
2-layered aggregates (Figure 1A). Surface area covered with
platelets was increased slightly to 131.0?19.9% of control
(Figure 2A), likely because of increased contacts of platelets
with collagen fibers. Annexin-A5 fluorescence was increased
similarly to 128.4?29.5% of control (Figures 1B and 2A). In
the presence of P2Y blockers, adhesion and PS exposure were
about halved when the blood was also pretreated with
acetylsalicylic acid (ASA) to block thromboxane formation
(Figure 2A), indicating that released thromboxane is involved
in platelet adhesion.
Platelet deposition was reduced further when 9O12 was
combined with P2Y blockers and ASA. This resulted in
adhesion of merely single platelets (28.9?4.8% of control),
whereas annexin-A5 staining was abolished completely (Fig-
ure 2A). This extends earlier observation14and indicates that
human GPVI, together with the secondary mediators ADP
and thromboxane, is responsible for aggregate formation and
PS exposure but is dispensable for platelet adhesion.
Suppressed ?2?1 and ?IIb?3 Activation by
Blockade of GPVI or ADP Receptors
To study ?2?1 activation, a new antibody IAC-1 was used,
which specifically recognizes high-affinity forms of this
integrin.32IAC-1 does not bind to resting platelets but readily
binds to a neoepitope in the ?2 I-domain that become
exposed during platelet activation with ADP, thromboxane,
or thrombin. Activation uncovers an I-domain region at
amino acids 199 to 201, which is located at the opposite site
of the metal ion-dependent adhesion site domain that is
involved in binding to collagen. IAC-1 is thus of little effect
on collagen binding.32FITC-labeled IAC-1 binds to platelets
on immobilized convulxin (data not shown), indicating that
?2?1is activated after platelet adhesion via GPVI.
When added to blood at 10 ?g/mL, FITC-labeled IAC-1
did not inhibit platelet deposition on collagen under flow
(surface area coverage with all platelets 13.2?1.4% versus
13.6?1.1% in the absence of IAC-1; n?7). Yet, FITC-IAC-1
avidly bound to the platelets adhering to collagen under
control conditions but not in the presence of platelet inhibi-
tors (Figure III, available online at http://atvb.ahajournal-
s.org). Other experiments were performed with FITC-Gi9, a
mAb recognizing all ?2?1 forms, and with FITC-MOPC-21,
a mAb that does not bind to platelets. FITC-Gi9 stained
control and inhibited platelets, but FITC-MOPC-21 failed to
give detectable staining (Figure III).
During perfusions under control condition, fluorescence
staining of FITC-IAC-1 increased in time; it covered
9.5?2.1% (n?8 subjects) of the surface area after 4 minutes.
Addition of 9O12 (50 ?g/mL) greatly reduced staining to
23?1.9% of the control condition, despite the presence of
many adherent platelets (Figures 1C and 2B). Addition of
P2Y blockers also reduced IAC-1 staining to 41.2?22.1%
of control. Blockade of GPVI and P2Y and presence of ASA
resulted in almost complete suppression of IAC-1 fluores-
cence (Figure 2B).
Figure 2. Quantitative effect of GPVI and P2Y receptor blockade
on platelet aggregation, PS exposure, and integrin activation.
Whole blood was perfused over collagen, and platelets were
stained with fluorescent annexin-A5, IAC-1, or PAC-1 (Figure 1).
Blood was untreated (control) or treated with anti-GPVI Fab
9O12 (50 ?g/mL). Alternatively, blood was treated with P2Y
blockers (40 ?mol/L MRS2179 and 20 ?mol/L AR-C69931MX) in
combination with anti-GPVI Fab and ASA; with P2Y blockers
alone; or with P2Y blockers and ASA. A, Surface area coverage
of all platelets and PS-exposing platelets. B, Surface area cov-
erage of FITC-IAC-1 and PAC-1 staining detecting activated
?2?1 and ?IIb?3, respectively. Per parameter, data were
expressed as percentages of respective control condition set at
100% (mean?SEM; n?4 to 5; *P?0.05; **P?0.01 vs control).
Lecut et alThrombus Formation and Integrin Activation
To ensure that the reduced IAC-1 binding was not caused
by a low detection level of the fluorescence camera, platelets
were counterstained for actin with fluorescent phalloidin and
examined by confocal microscopy. Under control conditions,
individual platelets in aggregates were strongly labeled with
FITC-IAC-1, as apparent from overlays of IAC-1 and phal-
loidin images (Figure IV, available online at http://atvb.
ahajournals.org). With 9O12 present, FITC-IAC-1 fluores-
cence of collagen-adherent platelets was reduced greatly, in
contrast to the still bright phalloidin staining.
Because 9O12 inhibited platelet aggregation, its effect was
studied on signaling toward integrin ?IIb?3 using fluores-
cent-labeled PAC1, which is a mAb against activated
?IIb?3.33Under control conditions, addition of FITC-PAC1
to blood or postperfusion with FITC-PAC1 gave fluorescent-
labeled platelet aggregates on collagen (Figure 1D). After 4
minutes of perfusion, staining with FITC-PAC1 was
7.5?2.1% (n?8) of the surface. Blocking of GPVI or P2Y
receptors decreased PAC1 staining (Figure 2B). Similarly, as
observed with IAC-1, the combination of GPVI and P2Y
blockers plus ASA almost completely suppressed PAC-1
To verify that ADP could activate integrins on collagen-
adherent platelets in the absence of GPVI activity, 9O12-
treated platelets were postperfused with 10 ?mol/L ADP.
Perfusion with ADP but not with vehicle gave a substantial
?5-fold increase in IAC-1-labeling or PAC1-labeling of
adherent platelets (Figure 3).
Addition of OG488-labeled human fibrinogen to the blood
provided another means to measure ?IIb?3 activation on
flow. TPLSM with low detection threshold showed that
platelet aggregates on collagen were labeled diffusely with
OG488-fibrinogen (Figure 4A and 4B). In the presence of
9O12, only some of the single, collagen-adherent platelets
showed fluorescence. Total surface area coverage with
OG488-fibrinogen decreased greatly to 13.0?2.8% of control
(Figure 4C). P2Y blockers were less effective in reducing
OG488-fibrinogen binding (ie, to 42.4?5.0% of control);
many individual platelets still bound fibrinogen (Figure 4B).
With 9O12, ASA, and P2Y blockers present, fibrinogen
binding was no longer detected (data not shown).
These results indicate that antagonism of GPVI or the P2Y
receptors severely impaired exposure of activation-dependent
epitopes on ?2?1 and ?IIb?3. Only combined blockade of
GPVI and secondary mediators resulted in full inhibition of
integrins. This confirms that autocrine ADP and thromboxane
play key roles in thrombus build-up24,25and reveals that these
mediators, along with GPVI, mediate ?2?1 and ?IIb?3
GPIb Involved in Collagen Platelet Adhesion but
not in Integrin Activation
GPIb-IX-V mediates platelet adhesion to collagen under
shear. Its role in integrin activation was studied using the
anti-GPIb? mAb 12G1, which specifically hinders shear-
dependent adhesion to vWF.34At maximally effective con-
Figure 3. Effect of ADP addition on integrin activation of GPVI-
inhibited platelets. Blood containing 50 ?g/mL 9O12 was per-
fused over collagen, and FITC-PAC1 or FITC-IAC-1 binding was
measured. Thereafter, vehicle buffer or ADP (10 ?mol/L) with
same fluorescent antibody was superfused at 1000 s?1for 1
minute. Percentages of area coverage with fluorescence are
given at respective control condition (mean?SEM; n?3;
*P?0.05 vs 9O12 alone).
Figure 4. Effect of GPVI and P2Y receptor blockade on platelet–
fibrinogen binding. Blood with OG488-labeled fibrinogen (0.25
mg/mL) was perfused for 4 minutes over collagen in the pres-
ence of vehicle (control), anti-GPVI 9O12, or P2Y blockers (Fig-
ure 1). A, Phase-contrast images (120?120 ?m) and B, merged
stacks of TPLSM images (110?110 ?m) after perfusion. C,
Quantitative effect of receptor blockade on OG488-fibrinogen
fluorescence. Data are percentages of control (mean?SEM;
1730 Arterioscler Thromb Vasc Biol.
centration of 40 ?g/mL, 12G1 F(ab?)2completely blocked
adhesion of platelets to immobilized vWF. However, the
F(ab?)2only partially reduced adhesion to collagen fibers at
1000 s?1(Figure V, available online at http://atvb.ahajournal-
s.org). Although less platelet-collagen contacts were formed,
aggregate formation was not prevented; the area covered by
platelets remained 72.2?10.6% (n?7) of control. GPIb
blockade with 12G1 inhibited staining with OG488-
annexin-A5 to 41.7?3.3% of control (Figure V). This inter-
vention reduced staining with FITC-PAC1 and FITC-IAC-1
only moderately to 70.7?11.4% and 73.8?16.3% of control,
respectively. However, with 9O12 present, 12G1 markedly
inhibited platelet deposition to 12.0?5.3% of control.
The results with blocking antibody were corroborated by
studies with blood from a patient with Bernard-Soulier
syndrome, displaying GPIb-deficient platelets (Figure 5A).
After 4 minutes of perfusion, surface area covered with
patient platelets was 7.6?0.9% (ie, slightly lower than the
averaged value for healthy subjects [16.4?1.3%; n?11]).
With 9O12, adhesion of patient platelets was almost abol-
ished to 8.2?1.8% of control conditions (Figure 5A and 5B).
Thus, at this shear rate, GPIb and GPVI together determine
adhesion to collagen, but GPIb does not have a major role in
Functional Importance of ?2?1 Activation
Under control conditions, most platelets on collagen dis-
played pseudopods, lamellipods, or blebs. When 9O12 was
combined with ASA and P2Y blockers, platelets adhered
individually (coverage 28.9?4.8% of control) and did not
show morphological signs of activation (Figure 6). The
remaining adhesion was integrin-dependent because blocking
anti-?2?1 mAb 6F1 (10 ?g/mL) severely abrogated adhesion
with 9O12 (18.0?3.5% versus control; n?4), and extra
addition of ?IIb?3-blocking arg-gly-asp-ser (400 ?mol/L)
eliminated all platelet deposition (?5%). Thus, integrins
participated in platelet adhesion in the absence of detectable
binding of IAC-1, PAC1, or fibrinogen.
Experiments in which 6F1 was added to the blood in-
formed on the functional importance of (activated) ?2?1.
Blocking of ?2?1 with 6F1 notably reduced pseudopod
formation but still allowed bleb formation (Figure 6). The
6F1 effect was complete with P2Y blockers present as well
(only blebs formed because of GPVI activation). Pseudopod
and lamellipod formation was restored when 9O12-treated
platelets were postperfused with ADP (data not shown). This
suggested that ?2?1-dependent pseudopod formation, which
correlated with IAC-1 binding, was responsible for increased
In this study, we used newly developed tools to determine the
role of human GPVI and ADP in integrin activation during
collagen-induced thrombus formation under flow. The O12
mAb, directed against the collagen-binding site of human
GPVI, was used to block GPVI activity. This inhibited
Figure 5. Abolished adhesion by blockade of GPVI of platelets
from Bernard-Soulier (BSS) patient. Blood from a healthy control
subject or a patient displaying BSS was perfused over collagen
with/without 9O12 (50 ?g/mL). A, Representative phase-contrast
images (120?120 ?m) of deposited platelets; inserts are 3-fold
magnifications showing giant size of BSS platelets. B, Quantita-
tive effect on platelet deposition. Values are percentage of sur-
face area coverage (mean?SEM; n?3; *P?0.05; **P?0.01 vs
absence of 9O12).
Figure 6. Platelet shape and activation of integrin ?2?1. Blood
was perfused over collagen (Figure 2). Blood was treated with
anti-?2?1 6F1 (10 ?g/mL), anti-GPVI 9O12 (50 ?g/mL), or P2Y
blockers (MRS2179?AR-C69931MX) plus ASA, as indicated.
Shown are representative phase-contrast images (bar?10 ?m).
White arrows indicate pseudopods; black arrows, blebbing
Lecut et alThrombus Formation and Integrin Activation
formation of platelet aggregates and staining with
annexin-A5 (detecting PS exposure) and IAC-1 (detecting
activated ?2?1), as well as fibrinogen and PAC1 (detecting
activated ?IIb?3). FITC-IAC-1 is the first described mAb to
specifically recognize high activation states of ?2?1.32
Blockade of the P2Y1and P2Y12receptors partially inhibited
binding of IAC-1, PAC1, and fibrinogen to platelets, but
blockade of GPVI and P2Y receptors in combination with
ASA treatment was needed to abolish all binding completely.
Conversely, postperfusion with ADP resulted in increased
IAC-1 and PAC1 binding to GPVI-inhibited platelets. To-
gether, these results provide direct evidence for a role of
GPVI and autocrine ADP in inside-out integrin signaling. The
inhibitory effects of 9O12 are consistent with studies using
isolated platelets showing that stimulation with GPVI ago-
nists results in integrin activation6,13and exposure of proco-
agulant PS.27In addition, they extend recent evidence that
GPVI and ?2?1 contribute to human thrombus formation.17,23
Although anti-GPVI 9O12 efficiently suppressed aggrega-
tion, procoagulant activity, and integrin activation under
flow, it did not abolish platelet adhesion to collagen, even not
at a high dose of 200 ?g/mL. This is remarkably similar to the
effects of anti-human GPVI scFv, 10B12, which is also
directed against the collagen-binding domain of GPVI.17
Thus, 2 different antibodies against human GPVI appear to
suppress platelet activation under shear but not adhesion to
collagen. However, we find that combined blockade of
human GPVI and ADP/thromboxane effects does lower the
adhesion. For mouse blood, this is less clear because block-
ade or absence of murine GPVI has been found to either
abolish5,18,22or still permit21platelet–collagen adhesion un-
der flow. This discrepancy is probably attributable to differ-
ent experimental conditions.
At the moderately high shear rate of 1000 s?1used, the
anti-GPIb mAb 12G1 only partially reduced platelet adhesion
to collagen/vWF. When given alone, 12G1 inhibited adhesion
slightly and hardly influenced binding of IAC-1 and PAC1.
However, in combination with GPVI blockade, GPIb antag-
onism or absence of GPIb (in a Bernard-Soulier patient)
completely abolished platelet adhesion to collagen. This
indicates that GPIb-V-IX only partially contributes to integrin
activation under conditions in which GPVI and P2Y receptors
are also signaling.
Adhesion of platelets treated with GPVI and P2Y antago-
nists was mostly blocked when anti-?2?1 mAb 6F1 was
added. Furthermore, staining of platelets with IAC-1 or PAC1
correlated with ?2?1-dependent pseudopod formation of the
platelets on collagen. These observations suggest that platelet
adhesion to collagen can occur under conditions in which the
IAC-1/PAC1 epitopes are not or only partially exposed (ie,
with no or local integrin activation), basically in agreement
with earlier suggestions by Inoue et al.35In conclusion, our
results indicate that GPVI is responsible for integrin affinity
regulation on platelet adhesion to collagen under high shear.
Furthermore, autocrine released ADP and subsequent engage-
ment of P2Y receptors play assisting roles. Thus GPVI- and
P2Y-coupled signaling act synergistically to achieve full
integrin activation and thereby stable thrombus formation.
C.L. was supported by a Marie-Curie Fellowship from the European
Community. We acknowledge grant 902-16 to 276 from the Neth-
erlands Organization for Scientific Research. We thank AgroBio for
production of 9O12 ascites.
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Lecut et al Thrombus Formation and Integrin Activation