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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 491-499
Glycoprotein VI Is a Major Collagen Receptor for Platelet
Activation: It Recognizes the Platelet-Activating Quaternary
Structure of Collagen, Whereas CD36, Glycoprotein IIb/IIIa, and von
Willebrand Factor Do Not
By
Beate Kehrel,
Sonja Wierwille,
Kenneth J. Clemetson,
Olaf Anders,
Michael Steiner,
C. Graham Knight,
Richard W. Farndale,
Minoru Okuma, and
Michael J. Barnes
From the Department of Internal Medicine A, University of
Münster, Münster, Germany; the Theodor Kocher Institute,
University of Berne, Berne, Switzerland; the University Department of
Internal Medicine and Pathobiochemistry, University of Rostock,
Rostock, Germany; the Strangeways Research Laboratory, Cambridge, UK;
the Department of Biochemistry, University of Cambridge, Cambridge, UK;
and the Department of Hematology and Oncology, Graduate School of
Medicine, Kyoto University, Kyoto, Japan.
 |
ABSTRACT |
Simple collagen-related peptides (CRPs) containing a repeat
Gly-Pro-Hyp sequence are highly potent platelet agonists. Like collagen, they must exhibit tertiary (triple-helical) and quaternary (polymeric) structure to activate platelets. Platelet signaling events
induced by the peptides are the same as most of those induced by
collagen. The peptides do not recognize the
 2 1 integrin. To identify the signaling
receptor involved, we have evaluated the response to the CRP,
Gly-Lys-Hyp(Gly-Pro-Hyp)10-Gly-Lys-Hyp-Gly of platelets
with defined functional deficiencies. These studies exclude a primary
recognition role for CD36, von Willebrand factor (vWF), or glycoprotein
(GP) IIb/IIIa. Thus, both CD36 and vWF-deficient platelets exhibited
normal aggregation, normal fibrinogen binding, and normal expression of
CD62 and CD63, measured by flow cytometry, in response to the peptide,
and there was normal expression of CD62 and CD63 on thrombasthenic
platelets. In contrast, GPVI-deficient platelets were totally
unresponsive to the peptide, indicating that this receptor recognizes
the Gly-Pro-Hyp sequence in collagen. GPVI-deficient platelets showed
some fibrinogen binding in response to collagen but failed to aggregate
and to express CD62 and CD63. Collagen, but not CRP-XL, contains
binding sites for 21. Therefore, it is
possible that collagen still induces some signaling via 21, leading to activation of GPIIb/IIIa.
Our findings are consistent with a two-site, two-step model of collagen
interaction with platelets involving recognition of specific sequences
in collagen by an adhesive receptor such as
21 to arrest platelets under flow and
subsequent recognition of another specific collagen sequence by an
activatory receptor, namely GPVI.
| |
INTRODUCTION |
COLLAGENS OF THE subendothelium are major
determinants of the thrombogenicity of the blood vessel
wall.1 In particular, the fibrous collagens I and III,
exposed as a consequence of injury to the vessel wall, are strong
platelet agonists that induce shape change, release reaction, and
aggregation. The mechanism of collagen-platelet interaction involves
both the direct recognition of collagen receptors and indirect binding
of collagen to the platelet surface via intermediary proteins.2,3 As an example of the latter, von Willebrand factor (vWF) plays an essential role in hemostasis by complexing to
collagen(s) in the vessel wall and concomitantly binding to specific
receptors on the platelet surface,4,5 the glycoprotein (GP)
Ib/V/IX complex, and the activated GPIIb/IIIa complex,6 thus forming a bridge between collagen(s) and platelets.
A number of platelet membrane proteins have been proposed as collagen
receptors, including integrin 21
(GPIa/IIa; as reviewed by Santoro and Zutter7), CD36
(GPIIIb, GPIV), and GPVI (p62). Their precise role in the overall
process of interaction and the relationship between them remains
unclear.
Nieuwenhuis et al8 and Kehrel et al9 have
described patients with mild bleeding disorders attributable to
deficient expression of platelet 21. The
platelets from these patients had impaired collagen-induced aggregation
but responded normally to all other platelet agonists. Further evidence
for the involvement of 21 is that
monoclonal antibodies (MoAbs) directed against the receptor are able to
inhibit adhesion of platelets to collagen under either static or flow
conditions.7,10-12
CD36 binds to collagen type I fibers,13 and Fab fragments
of polyclonal antibodies against CD36 have been reported to inhibit collagen-induced platelet aggregation.13,14 However, the
role of CD36 as a platelet collagen receptor is not clear, because a
significant proportion (1% to 4%) of Japanese and other East Asian
populations lack this receptor on platelets but have no obvious
bleeding disorder. Platelet adhesion to collagens I and III15,16 and platelet aggregation and secretion induced by
these collagens is normal in CD36-deficient platelets.17,18
Other Japanese patients with mild bleeding disorders have been
described whose platelets show reduced responses to collagen associated
with functional deficiency in GPVI.19-21 One of these patients developed antibodies to GPVI, which, when added to normal platelets, caused their aggregation and whose Fab fragments were able
to reduce platelet aggregation induced by collagen.21,22 GPVI-deficient platelets showed defective second phase adhesion in
flowing blood, a reaction that is attributable mainly to the platelet-platelet interaction.23
The interaction of platelets with collagen involves firstly adhesion
and, subsequently, activation leading to second phase adhesion,
secretion, and ultimately aggregation. Different mechanisms and
receptor populations may be involved in these two
processes.24,25 Collagen-induced platelet activation
requires the expression by collagen of both tertiary (triple-helical)
and quaternary (polymeric) structure.26-28 Recently, Morton
et al29 have described simple collagen-related peptides
(CRPs), comprising a repeating Gly-Pro-Hyp motif, that mimic the
collagen tertiary (triple-helical) structure. They have shown that,
when cross-linked via either lysyl or cysteine residues to impart
quaternary (polymeric) structure, these peptides (CRP-XL) are extremely
active platelet agonists, being more reactive than collagen fibers. In
contrast, non-cross-linked (nonpolymeric) CRP antagonizes platelet
activation stimulated by either native collagen or
CRP-XL.29 CRPs possess the same tertiary structure as
collagen and, like collagen, this conformation is essential for
activity. Like collagen, CRP molecules spontaneously assemble into an
organized quaternary structure and, again like collagen, the polymeric
structure is essential for activity. It is therefore most likely that
the potent platelet reactivity of CRP-XL reflects the platelet
reactivity to collagen.
The interaction of CRP-XL with platelets does not involve the integrin
21; thus, CRP supports static platelet
adhesion that is not blocked by the
21-directed MoAbs, 6F1 and
MoAb13.29 These same antibodies fully block
21-mediated adhesion to immobilized monomeric collagen. The MoAbs are also without effect on aggregation stimulated by CRP-XL.29 The recognition of
21 by collagen is essential for adhesion
of platelets under flow. However, CRP-XL is unable to support adhesion
under flow conditions.30 Furthermore, the purified I-domain
of the 2 subunit, which mediates the adhesion of native collagens to
21, does not recognize
CRP-XL,31 and affinity chromatography using CRP-XL does not
extract 21 from platelet lysates (Barnes
et al, unpublished data). All of this establishes CRP as
an agonist that operates entirely independently from
21.
Despite the absence of recognition of the integrin
21, CRPs signal in platelets in precisely
the same way as native collagen fibers.32,33 Specifically,
they stimulate tyrosine phosphorylation of the Fc receptor  chain,34 of p72syk, and of phospholipase C2
(PLC2)35 all responses characteristic of platelet
activation by collagen. CRPs are therefore valuable tools for the
identification of the collagen receptor that recognizes the quaternary
structure of collagen and is responsible for signaling leading to
activation. To this end, we have evaluated the response to CRP-XL of
platelets deficient in GPIIb/IIIa, CD36, vWF, and GPVI.
| |
MATERIALS AND METHODS |
Materials.
Ristocetin was obtained from Paesel (Frankfurt, Germany).
Human vWF was purified from Haemate HS (a kind gift of Centeon,
Marburg, Germany), as previously described.36
The vWF-directed MoAb, 4F9 (Immunotech, Marseilles, France), was
conjugated to fluorescein isothiocyanate (FITC) using the antibody
conjugation kit from Sigma (Deisenhofen, Germany) according to the
manufacturer's instructions. The F/P ratio, calculated from the FITC
absorbance at 495 nm and the IgG content, was 1.4. FITC-coupled MoAbs
recognizing CD62 (P-selectin, GMP140, and PADGEM) or CD63 (GP53 and
granulophysin), clone CLB-thromb/6 and CLB-gran/12, respectively, were
obtained from Immunotech (Marseilles, France).
Highly purified human fibrinogen (Enzyme Research Labs, South Bend, IN)
was conjugated with FITC using the FITC-celite (Calbiochem, Bad Soden,
Germany) method according to Xia et al37 but using pH 7.8 buffer for 48 hours at 4°C, resulting in an F/P ratio of 5.0 to
5.2.
Collagen type I for use as a reference reagent has been described
previously.18
The triple-helical peptide,
Gly-Lys-Hyp-[Gly-Pro-Hyp]10-Gly-Lys-Hyp-Gly (CRP), was
synthesized and cross-linked via its lysyl residues to yield CRP-XL as
described in detail by Morton et al29 and Barnes et
al.32
Patients.
All studies were performed with the patients or other blood donors
giving informed consent. Samples from healthy controls were processed
in parallel with all patient samples. None of the subjects had taken
any medication affecting platelet functions for at least 2 weeks before
the study.
The CD36-deficient platelets of a Japanese male blood donor Y.A. have
been described before.18,38 Platelets from patient A.M.
with Glanzmann's thrombasthenia type I were shown to contain less than
1% of normal amounts of GPIIb/IIIa.39 Patient P.R. had von
Willebrand disease type 3 (according to the Sadler and Gralnick
classification40). In addition, the patient suffered from
an intestinal angiodysplasia, which is associated with life-threatening bleeding episodes, requiring frequent replacement therapy with vWF. The
blood for the experiments was taken 2 weeks after the last treatment
and directly before new medication. At that time the vWF:RCO was less
than 1% of normal and vWF antigen was undetectable (Asserachrom vWF;
Stago-Diagnostica, Asniers, France) in both the plasma and the
platelets. The GPVI-deficient platelets of the female Japanese patient
Y.A. have been described in detail.21,22,41
Platelet aggregation.
Platelet aggregation studies were performed in an aggregometer
(Chrono-Log, Haverton, PA) within 3 hours of venipuncture using platelet-rich plasma (PRP) containing 2 × 108
platelets/mL, as described by Born and Cross.42 Blood was
anticoagulated with 0.1 vol of 3.8% Na-citrate and PRP was prepared by
centrifugation at 200g for 10 minutes at room temperature.
Flow cytometric analysis of ristocetin-induced vWF binding to
platelets.
PRP was diluted to 5 × 107 platelets/mL with
Tyrode's buffer, pH 7.4, to minimize the formation of platelet
aggregates. Platelet suspension (200 µL) was added to 20 µL of
ristocetin (at a range of concentrations in Tyrode's buffer, pH 7.4)
without stirring to avoid aggregate formation. After 180 seconds, the
platelets were fixed for 30 minutes with 1% formaldehyde (final
concentration) in phosphate-buffered saline (PBS). Platelets were
washed and resuspended in 200 µL of PBS, and FITC-conjugated anti-vWF
MoAb was added at a saturating concentration (determined beforehand). After 1 hour of incubation at room temperature, 104 single
platelets were analyzed in a flow cytometer (FACScan; Becton Dickinson,
Heidelberg, Germany). Background binding obtained from parallel samples
with FITC-conjugated isotype-specific mouse IgG was subtracted from
each test sample. Excitation was at a wavelength of 488 nm. The FACScan
was used in a standard configuration with a 530 nm bandpass filter.
Data were obtained from fluorescence channels in a logarithmic mode.
Flow cytometric analysis of fibrinogen-FITC binding to CRP-activated
platelets.
Diluted PRP (5 × 107 platelets/mL) was preincubated
for 3 minutes at room temperature with 150 µg/mL fibrinogen-FITC
(saturating concentration). Two hundred microliters of platelet
suspension was added to 20 µL of a solution containing a range of
concentrations of CRP-XL in 0.01 mol/L acetic acid at room temperature.
The reaction was stopped after 120 seconds by fixation with 1%
formaldehyde as described above. Platelets were washed, resuspended in
200 µL of PBS, and analyzed in a flow cytometer as described above. The nonspecific background labeling was determined using platelets from
different patients with thrombasthenia type I and control platelets
treated with 10 mmol/L of the inhibitory peptide GRGDSP (Novabiochem,
Bad Soden, Germany) to prevent specific fibrinogen binding.
Flow cytometric analysis of CD62 and CD63 expression on
CRP-activated platelets.
Two hundred microliters of diluted PRP (5 × 107
platelets/mL) was added at room temperature to 20 µL 0.01 mol/L
acetic acid containing CRP-XL at a range of concentrations. The
reaction was stopped after 120 seconds with 1% formaldehyde, as
described above. Platelets were washed and resuspended in PBS, and
FITC-coupled MoAbs recognizing CD62 or CD63 were added and incubated
for 1 hour at room temperature. The concentrations required for
saturated binding of both antibodies were determined beforehand. After
incubation, platelets were washed, resuspended in 200 µL of PBS, and
analyzed as described above.
| |
RESULTS |
The interaction of CD36-deficient platelets with CRP.
Platelets from 8 control donors were aggregated by CRP-XL at
concentrations from 20 to 100 ng/mL. Aggregation curves from 2 such
donors are shown in Fig 1A. The
CD36-deficient platelets of donor Y.A. reacted as normal control
platelets when treated with CRP-XL, which induced full aggregation at a
concentration of 37 ng/mL (Fig 1B). Fibrinogen binding to
CRP-XL-activated control and CD36-deficient platelets did not differ
significantly (Fig 2). CD36-deficient
platelets activated by CRP-XL expressed CD62 and CD63 to the same
extent as control platelets (data not shown).
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| Fig 1.
Normal platelet aggregation induced by CRP-XL in
platelet-rich plasma (PRP). (A) Control platelets; (B) CD36-deficient
platelets from donor Y.A. The platelet counts were adjusted to 2 × 108/mL. The experiment shown is representative of three
similar experiments.
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| Fig 2.
Normal binding of fibrinogen to CD36-deficient platelets
and control platelets activated with CRP-XL measured by flow cytometry using FITC-labeled fibrinogen. Fluorescence of unstimulated control platelets is defined as 1. Data shown are representative of two similar
experiments.
|
|
The reaction of CRP with thrombasthenic platelets.
Because, as expected, specific fibrinogen binding after exposure to
agonist was not observed in these GPIIb/IIIa-deficient platelets,
activation was determined on the basis of the surface expression of
CD62 (Fig 3A) and of CD63 (Fig 3B).
Expression of both in GPIIb/IIIa-deficient platelets treated with
CRP-XL was not lower than in controls.
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| Fig 3.
Normal expression of CD62 (A) and of CD63 (B) on
GPIIb/IIIa-deficient platelets and control platelets induced by CRP-XL,
measured by flow cytometry after labeling with anti-CD62-FITC or
anti-CD63-FITC, respectively. Fluorescence of unstimulated control
platelets is defined as 1. Data shown are representative of two similar
experiments.
|
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The reaction of CRP with platelets in von Willebrand disease.
Ristocetin, at concentrations up to 4 mg/mL, did not induce
vWF-mediated platelet agglutination in PRP from patient P.R. (data not
shown). The addition of human vWF (10 µg/mL) corrected the defect. No
vWF binding to the surface of the patient's platelets was observed
when these platelets were treated with ristocetin at concentrations
ranging from 0.2 to 2 mg/mL, whereas 1.0 mg/mL induced maximum vWF
binding to control platelets (data not shown). No vWF was detectable in
samples of the patient's platelets and plasma taken at the time these
platelets were activated with CRP-XL. CRP-XL induced normal aggregation
of vWF-deficient platelets in vWF-deficient plasma
(Fig 4A) and induced fibrinogen binding to a somewhat greater extent than in controls (Fig 4B). The surface expression of CD62 (Fig 5A) and of CD63
(Fig 5B) induced by CRP-XL in PRP of patient P.R. were identical in
terms of both extent and concentration dependence to that occurring in
control platelets.
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| Fig 4.
Normal platelet aggregation induced by CRP-XL in PRP from
a von Willebrand disease type 3 patient and from a control (A). Normal
binding of fibrinogen to vWF-deficient platelets and to control
platelets in vWF-deficient plasma activated with CRP-XL, measured by
flow cytometry using FITC-labeled fibrinogen (B). Fluorescence of
unstimulated control platelets is defined as 1. Data shown are
representative of two similar experiments.
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| Fig 5.
Normal expression of (A) CD62 and (B) CD63 on
vWF-deficient platelets in vWF-deficient plasma and control platelets
in control plasma, induced by CRP-XL, measured by flow cytometry using
FITC-labeled anti-CD62 antibody or anti-CD63 antibody, respectively.
Fluorescence of unstimulated control platelets is defined as 1. Data
shown are representative of two similar experiments.
|
|
The reaction of CRP with GPVI-deficient platelets.
Aggregation of GPVI-deficient platelets was totally absent even in the
presence of a concentration of either collagen or CRP-XL 20-fold higher
than was needed to aggregate normal platelets
(Fig 6A and B). In contrast, aggregation of
these platelets by ADP was normal (Fig 6C). Neither collagen nor CRP-XL
induced any surface expression of CD62 (Fig
7A and B) or CD63 (Fig 8A and B). There was
no expression of fibrinogen binding in response to CRP-XL (Fig 9B), although collagen-induced
fibrinogen binding was not absent but expressed to a decreased extent
compared with normal platelets, especially at low concentrations of
collagen (Fig 9A).
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| Fig 6.
Aggregation of GPVI-deficient and control platelets
induced by (A) collagen type I, (B) CRP-XL, and (C) ADP. The platelet count had to be adjusted to 1.8 × 108/mL because the
patient was slightly thrombocytopenic. Collagen and CRP-XL did not
induce platelet aggregation in GPVI-deficient platelets, although these
platelets were able to aggregate in response to ADP. Data shown are
representative of three similar experiments.
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| Fig 7.
Expression of P-selectin (CD62) on GPVI-deficient
platelets and control platelets induced by (A) collagen type I and (B)
CRP-XL, measured by flow cytometry using FITC-labeled anti-CD62
antibody. Neither collagen nor CRP-XL induced CD62 expression on
GPVI-deficient platelets. Fluorescence of unstimulated control
platelets is defined as 1. Data shown are representative of two similar
experiments.
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| Fig 8.
Expression of CD63 on GPVI-deficient platelets and
control platelets induced by (A) collagen type I and (B) CRP-XL,
measured by flow cytometry using FITC-labeled anti-CD63 antibody.
Neither collagen nor CRP-XL induced CD63 expression on GPVI-deficient platelets. Fluorescence of unstimulated control platelets is defined as
1. Data shown are representative of two similar experiments.
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| Fig 9.
Binding of fibrinogen to GPVI-deficient platelets and
control platelets activated by (A) collagen type I and (B) CRP-XL,
measured by flow cytometry using FITC-labeled fibrinogen. In
GPVI-deficient platelets, there was no expression of fibrinogen binding
in response to CRP-XL; collagen-induced fibrinogen binding was
expressed to a decreased extent, especially at low concentrations of
collagen. Fluorescence of unstimulated control platelets is defined as
1. Data shown are representative of two similar experiments.
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| |
DISCUSSION |
All collagens share a common tertiary structure, the collagen triple
helix. The collagen molecule contains three chains, each with a
repeating Gly-X-Y sequence in which X and Y are frequently the
amino acids, Pro and Hyp, respectively. The Gly-Pro-Hyp
triplet represents approximately 12% of the primary sequence of type I collagen. Within the molecule each of these chains forms a left-handed polyproline II helix and the three helices are intertwined to form a
right-handed superhelix, the collagen triple helix. Molecules then
associate very specifically to yield the highly ordered quaternary structure of the collagen fiber.43
The native triple helical structure of collagen is required for
platelet secretion and aggregation. Furthermore, collagen molecules
must also assemble into fibrous form for collagen to be an effective
agonist.26-28 In contrast, monomeric collagen immobilized on plastic can serve as a very effective substrate for adhesion without
any activation.44 This adhesion is mediated by the integrin 21 and can be blocked by antibodies to
21.7,10-12
Although evidence has been presented suggesting that signaling in
platelets by collagen is subsequent to recognition of
21,35,45-47 it is not clear
how far 21 is directly responsible for
signaling leading to platelet activation. Although collagen-induced
phosphorylation events may under some circumstances be attenuated by
blockade of 21 by antibodies, persistence
of signaling has been reported in response to collagen or
CRP-XL.29,34,35,47 Indeed, in platelets lacking GPVI, but
in which 21 is expressed normally, collagen is able to activate the tyrosine kinase c-src, but not p72syk, PLC2, or p125fak.41 All
of these are regarded as important signaling molecules in the
activation of normal platelets by collagen. This indicates the
possibility that GPVI (rather than 21)
may be crucial as an activatory receptor for collagen in platelets.
A triple-helical synthetic analogue of collagen, composed simply of a
repeating Gly-Pro-Hyp sequence, has been shown in polymeric form
(CRP-XL) to elicit a full aggregatory response from human platelets
without the involvement of the integrin
21.29 The triple-helical
non-cross-linked peptide (CRP) is not activatory but inhibits platelet
activation by collagen and by CRP-XL.29 This corroborates
the existence of a signaling receptor on platelets other than, or in
addition to, 21, which requires the
tertiary and quaternary structures of collagen for platelet activation.
As shown here, CD36-deficient platelets bind fibrinogen and exhibit
normal secretion and aggregation in response to CRP-XL (as is true for
collagen15-18). CD36 cannot, therefore, be this collagen
receptor.
Platelets bind indirectly to collagen, via vWF, through GPIb and
GPIIb/IIIa complexes.48 This binding confers resistance to
the shear forces generated by blood flowing along the vessel wall.49,50 However, platelets from a Glanzmann's
thrombasthenia type I patient, totally deficient in GPIIb/IIIa,
responded normally to CRP-XL, which induced normal secretion, measured
as expression on the plasma membrane of CD62 (from the -granules)
and CD63 (from the dense bodies). These data exclude GPIIb/IIIa as the primary signaling receptor that recognizes the quaternary structure of
collagen.
The same can be concluded for vWF. Despite the absence of vWF, both in
the platelets and plasma of patient P.R., CRP-XL induced normal
fibrinogen binding, normal secretion (CD62 and CD63 expression), and
normal aggregation.
These results confirm the view that CD36, GPIIb/IIIa, and vWF are not
essential for platelet activation by collagen.
In contrast, GPVI-deficient platelets were unresponsive to CRP-XL,
showing that GPVI is a signaling receptor for the polymeric cross-linked triple-helical Gly-Pro-Hyp motif. Our findings are in line
with the observation that antibody-induced cross-linking of GPVI in
normal platelets results in activation of src and p72syk in
parallel with activation of PLC222 and that CRP-XL
stimulates tyrosine phosphorylation of PLC2 and p72syk
in normal platelets independent of the integrin
21.35
In GPVI-deficient platelets CRP-XL did not induce the
activation-dependent conformational changes in GPIIb/IIIa that are
necessary for fibrinogen binding. However, it is interesting that
fibrinogen binding was induced in these platelets by increasing
collagen levels, although to a lower degree than in equivalent controls (Fig 9A). Collagen (but not CRP-XL) contains binding sites for 21, and GPVI-deficient platelets express
21, which may possibly be involved in
signal transduction by collagen, sufficient for partial activation of
GPIIb/IIIa. Nevertheless, collagen fibers provided insufficient signals
to cause platelet aggregation in GPVI-deficient platelets, even at
collagen levels 20-fold higher than are needed to aggregate normal
platelets. Granule release was completely absent, as with CRP-XL (Figs
7 and 8). Recently, the activation of c-src, but not of
p72syk or PLC2, has been shown in GPVI-deficient
platelets stimulated with collagen,41 providing firm
evidence for signaling, but not sufficient to cause aggregation,
arising from a second population of collagen receptors as well as GPVI.
It is plausible that GPVI may be linked more to positive feedback
events, such as thromboxane production, a process essential for the
aggregation of platelets by collagen, or granule release, providing
further activation mediated by secreted ADP, so leading to full
activation of GPIIb/IIIa. Results from these and other studies suggest
that, in platelets, 21 has a major role
in adhesion to collagen and a less-well defined role in platelet
activation in support of GPVI. These ideas are consistent with the
two-site two-step model of platelet activation by
collagen.24,25,51 The major role for
21 may be to provide a stable
interaction, especially under nonstatic conditions, to allow subsequent
interaction of the Gly-Pro-Hyp motif with other, activatory receptors.
This study establishes GPVI as an activatory receptor crucial for the
full activation of platelets by collagen.
Un-cross-linked CRP, although not causing platelet activation, does
inhibit aggregation by both CRP-XL and collagen fibers, which may
suggest that GPVI can recognize the monomeric molecule. Lack of
activation by monomeric CRP suggests, in turn, that clustering of GPVI
molecules may be necessary for activation.
In view of the potency of the repeating Gly-Pro-Hyp sequence in
triple-helical, polymeric form, we believe that Gly-Pro-Hyp in collagen
may be a highly specific recognition sequence for GPVI. However, we
cannot rule out the possibility, as we have recently
conjectured,29 that GPVI recognizes the triple helix per se
and that any assembly of Gly-X-Y amino acid triplets generating a
triple-helical structure may be active. Further studies are in progress
to affirm the crucial importance of the Gly-Pro-Hyp motif in the
recognition of collagen by GPVI.
| |
FOOTNOTES |
Submitted April 28, 1997;
accepted September 8, 1997.
Supported in part by the Deutsche Forschungsgemeinschaft (Grant No.
Ke397/1-3) and the Gesellschaft für Thrombose- und
Hämostaseforschung (travel support). K.J.C. was supported by
Grant No. 31-42336.94 from the Swiss National Science Foundation.
R.W.F., C.G.K., and M.J.B. were supported by the Medical Research
Council, UK, to whose External Scientific Staff M.J.B. belongs.
Address reprint requests to Beate Kehrel, PhD, Experimentelle
Haemostaseforschung, Medizinische Klinik und Poliklinik, Innere Medizin A, Universität Münster, Domagkstrasse 3, D-48129 Münster, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank all blood donors and patients involved in this study
for their kind cooperation and Joachim Kardoeus for preparation of the figures and editorial assistance.
| |
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22(1):
14 - 20.
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A. Navdaev, D. Dormann, J. M. Clemetson, and K. J. Clemetson
Echicetin, a GPIb-binding snake C-type lectin from Echis carinatus, also contains a binding site for IgM{kappa} responsible for platelet agglutination in plasma and inducing signal transduction
Blood,
April 15, 2001;
97(8):
2333 - 2341.
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B. Nieswandt, V. Schulte, W. Bergmeier, R. Mokhtari-Nejad, K. Rackebrandt, J.-P. Cazenave, P. Ohlmann, C. Gachet, and H. Zirngibl
Long-Term Antithrombotic Protection by in Vivo Depletion of Platelet Glycoprotein VI in Mice
J. Exp. Med.,
February 19, 2001;
193(4):
459 - 470.
[Abstract]
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D. Dormann, K. J. Clemetson, and B. E. Kehrel
The GPIb thrombin-binding site is essential for thrombin-induced platelet procoagulant activity
Blood,
October 1, 2000;
96(7):
2469 - 2478.
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J. Hartleib, N. Kohler, R. B. Dickinson, G. S. Chhatwal, J. J. Sixma, O. M. Hartford, T. J. Foster, G. Peters, B. E. Kehrel, and M. Herrmann
Protein A is the von Willebrand factor binding protein on Staphylococcus aureus
Blood,
September 15, 2000;
96(6):
2149 - 2156.
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E. Monnet and F. Fauvel-Lafeve
A New Platelet Receptor Specific to Type III Collagen. TYPE III COLLAGEN-BINDING PROTEIN
J. Biol. Chem.,
April 6, 2000;
275(15):
10912 - 10917.
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P. Siljander and R. Lassila
Studies of Adhesion-Dependent Platelet Activation : Distinct Roles for Different Participating Receptors Can Be Dissociated by Proteolysis of Collagen
Arterioscler Thromb Vasc Biol,
December 1, 1999;
19(12):
3033 - 3043.
[Abstract]
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B. Savage, M. H. Ginsberg, and Z. M. Ruggeri
Influence of Fibrillar Collagen Structure on the Mechanisms of Platelet Thrombus Formation Under Flow
Blood,
October 15, 1999;
94(8):
2704 - 2715.
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M. Bauer, M. Retzer, J. I. Wilde, P. Maschberger, M. Essler, M. Aepfelbacher, S. P. Watson, and W. Siess
Dichotomous Regulation of Myosin Phosphorylation and Shape Change by Rho-Kinase and Calcium in Intact Human Platelets
Blood,
September 1, 1999;
94(5):
1665 - 1672.
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S. A. Santoro
Platelet Surface Collagen Receptor Polymorphisms: Variable Receptor Expression and Thrombotic/Hemorrhagic Risk
Blood,
June 1, 1999;
93(11):
3575 - 3577.
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S. J. Briddon, S. K. Melford, M. Turner, V. Tybulewicz, and S. P. Watson
Collagen Mediates Changes in Intracellular Calcium in Primary Mouse Megakaryocytes Through syk-Dependent and -Independent Pathways
Blood,
June 1, 1999;
93(11):
3847 - 3855.
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T. Nakamura, J.-i. Kambayashi, M. Okuma, and N. N. Tandon
Activation of the GP IIb-IIIa Complex Induced by Platelet Adhesion to Collagen Is Mediated by Both {alpha}2{beta}1 Integrin and GP VI
J. Biol. Chem.,
April 23, 1999;
274(17):
11897 - 11903.
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B. S. Gross, J. R. Lee, J. L. Clements, M. Turner, V. L. J. Tybulewicz, P. R. Findell, G. A. Koretzky, and S. P. Watson
Tyrosine Phosphorylation of SLP-76 Is Downstream of Syk following Stimulation of the Collagen Receptor in Platelets
J. Biol. Chem.,
February 26, 1999;
274(9):
5963 - 5971.
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C.G. Knight, L. F Morton, D. J Onley, A. R Peachey, T. Ichinohe, M. Okuma, R. W Farndale, and M. J Barnes
Collagen-platelet interaction: Gly-Pro-Hyp is uniquely specific for platelet Gp VI and mediates platelet activation by collagen
Cardiovasc Res,
February 1, 1999;
41(2):
450 - 457.
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C. G. Knight, L. F. Morton, D. J. Onley, A. R. Peachey, A. J. Messent, P. A. Smethurst, D. S. Tuckwell, R. W. Farndale, and M. J. Barnes
Identification in Collagen Type I of an Integrin alpha 2beta 1-binding Site Containing an Essential GER Sequence
J. Biol. Chem.,
December 11, 1998;
273(50):
33287 - 33294.
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Y. Ezumi, K. Shindoh, M. Tsuji, and H. Takayama
Physical and Functional Association of the Src Family Kinases Fyn and Lyn with the Collagen Receptor Glycoprotein VI-Fc Receptor {gamma} Chain Complex on Human Platelets
J. Exp. Med.,
July 20, 1998;
188(2):
267 - 276.
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M. W. Verkleij, L. F. Morton, C. G. Knight, P. G. de Groot, M. J. Barnes, and J. J. Sixma
Simple Collagen-Like Peptides Support Platelet Adhesion Under Static But Not Under Flow Conditions: Interaction Via alpha 2beta 1 and von Willebrand Factor With Specific Sequences in Native Collagen Is a Requirement to Resist Shear Forces
Blood,
May 15, 1998;
91(10):
3808 - 3816.
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B. Nieswandt, W. Bergmeier, V. Schulte, K. Rackebrandt, J. E. Gessner, and H. Zirngibl
Expression and Function of the Mouse Collagen Receptor Glycoprotein VI Is Strictly Dependent on Its Association with the FcRgamma Chain
J. Biol. Chem.,
July 28, 2000;
275(31):
23998 - 24002.
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D. J. Onley, C. G. Knight, D. S. Tuckwell, M. J. Barnes, and R. W. Farndale
Micromolar Ca2+ Concentrations Are Essential for Mg2+-dependent Binding of Collagen by the Integrin alpha 2beta 1 in Human Platelets
J. Biol. Chem.,
August 4, 2000;
275(32):
24560 - 24564.
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K. Suzuki-Inoue, Y. Ozaki, M. Kainoh, Y. Shin, Y. Wu, Y. Yatomi, T. Ohmori, T. Tanaka, K. Satoh, and T. Morita
Rhodocytin Induces Platelet Aggregation by Interacting with Glycoprotein Ia/IIa (GPIa/IIa, Integrin alpha 2beta 1). INVOLVEMENT OF GPIa/IIa-ASSOCIATED Src AND PROTEIN TYROSINE PHOSPHORYLATION
J. Biol. Chem.,
January 5, 2001;
276(2):
1643 - 1652.
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M. Achison, C. M. Elton, P. G. Hargreaves, C. G. Knight, M. J. Barnes, and R. W. Farndale
Integrin-independent Tyrosine Phosphorylation of p125fak in Human Platelets Stimulated by Collagen
J. Biol. Chem.,
January 26, 2001;
276(5):
3167 - 3174.
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A. Navdaev, J. M. Clemetson, J. Polgar, B. E. Kehrel, M. Glauner, E. Magnenat, T. N. C. Wells, and K. J. Clemetson
Aggretin, a Heterodimeric C-type Lectin from Calloselasma rhodostoma (Malayan Pit Viper), Stimulates Platelets by Binding to alpha 2beta 1 Integrin and Glycoprotein Ib, Activating Syk and Phospholipase Cgamma 2, but Does Not Involve the Glycoprotein VI/Fc Receptor gamma Chain Collagen Receptor
J. Biol. Chem.,
June 8, 2001;
276(24):
20882 - 20889.
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Abstract
Introduction
Abstract