From the Department of Molecular Oncology, General
Surgery, Witten/Herdecke University, 42117 Wuppertal, Germany and the
¶ Department of Pharmacology, University of Oxford, Oxford OX1
3QT, United Kingdom
Received for publication, August 18, 2000, and in revised form, October 11, 2000
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ABSTRACT |
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It has recently been shown that the monoclonal
antibody JAQ1 to murine glycoprotein VI (GPVI) can cause
aggregation of mouse platelets upon antibody cross-linking and that
collagen-induced platelet aggregation can be inhibited by preincubation
of platelets with JAQ1 in the absence of cross-linking
(Nieswandt, B., Bergmeier, W., Schulte, V., Rackebrandt, K., Gessner,
J. E., and Zirngibl, H. (2000) J. Biol. Chem.
275, 23998-24002). In the present study, we have shown that
cross-linking of GPVI by JAQ1 results in tyrosine phosphorylation of
the same profile of proteins as that induced by collagen, including the
Fc receptor (FcR) The platelet collagen receptor
GPVI1 plays a pivotal role in
platelet activation following receptor cross-linking by collagen. This
is highlighted by the impaired response to collagen in GPVI-deficient patients (2-4) and the bleeding disorders associated with this. GPVI
is a 60-65-kDa type I transmembrane glycoprotein belonging to the
immunoglobulin superfamily (5), which forms a complex with the FcR
These signaling events occur upon collagen-induced GPVI cross-linking
and following GPVI binding to collagen-related peptides (CRPs)
(15-17). CRPs contain Gly-Pro-Hyp triplet repeats (where Hyp is
hydroxyproline) in a triple-helical conformation, which are
formed into a quarternary structure by cysteine or lysine cross-linking
at the N- and C-terminal ends (18). Platelets can also be activated
through GPVI via interaction with the snake venom toxin
convulxin (Cvx), isolated from the venom of the tropical rattlesnake Crotalus durissus terrificus (19, 20).
Cvx-induced platelet activation is associated with a similar pattern of
tyrosine phosphorylation as that induced by collagen (20).
We have recently shown that a monoclonal antibody (JAQ1) to murine GPVI
can cause aggregation of mouse platelets upon antibody cross-linking
and that collagen-induced platelet aggregation can be inhibited by
preincubation of platelets with JAQ1 (1). In the present study, we
demonstrate that JAQ1 inhibits aggregation by low concentrations of
collagen and CRP, and this effect is overcome by high concentrations of
collagen, but not CRP. This study presents a novel motif within
collagen that is mediating platelet activation.
Animals--
Specific pathogen-free mice (NMRI strain) 6-10
weeks of age were obtained from Charles River, Sulzfeld, Germany or
(CD1 strain) from Harlan SERA-LAB Ltd (Belton, Leics., UK).
Materials--
Convulxin was obtained from the venom of the
tropical rattlesnake Crotalus durissus terrificus, which was
kindly donated by Drs. Mireille Leduc and Cassian Bon (Unite des
Venins, Insitut Pasteur, Paris, France). Collagen (predominantly type
1, derived from Equine achilles tendon) was from Nycomed (Munich,
Germany). CRP (GKO(GPO)10GKOG, single letter amino acid
code where O is hydroxyproline) was synthesized by Tana Laboratories,
L.C., TX and was cross-linked with 0.25% glutaraldehyde for 3 h
on ice and then dialysed into phosphate-buffered saline. All salts and Nonidet P-40 were purchased from BDH-Merck. PP1 was purchased from
Calbiochem-Novabiochem (Nottingham, UK). The mAb JAQ1 was produced as
described previously (1). Anti-SLP-76 sheep polyclonal anti-serum was
generously provided by Dr. Gary Koretsky (Abramson Family Cancer
Center, University of Pennsylvania). All other reagents were from
previously described sources (15). Modification of antibodies was
performed as follows. Fab fragments from JAQ1 were generated by 12-hour
incubation of 10 mg/ml mAb with immobilized papain (Pierce, Rockford,
IL), and the preparations were then applied to an immobilized protein A
column followed by an immobilized protein G column (Amersham Pharmacia
Biotech) to remove Fc fragments and any undigested IgG. The purity of
the Fab fragments was checked by SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) and silver staining of the gel.
Preparation of Platelets--
Mice were bled under ether
anesthesia from the retro-orbital plexus. Blood was collected in a tube
containing 7.5 units/ml heparin, and platelet-rich plasma (PRP) was
obtained by centrifugation at 300 × g for 10 min at
room temperature. Isolated platelets did not show any signs of
activation as shown by flow cytometry (staining for P-selectin and
surface-bound fibrinogen). Alternatively, blood was taken by cardiac
puncture following carbon dioxide asphyxiation using acidic citrate
dextrose (120 mM sodium citrate, 110 mM
glucose, 80 mM citric acid) as anticoagulant. Blood was
diluted with 200 µl of modified Tyrodes-HEPES buffer (134 mM NaCl, 0.34 mM
Na2HPO4, 2.9 mM KCl, 12 mM NaHCO3, 20 mM HEPES, 5 mM glucose, 1 mM MgCl2, pH 6.6) and
centrifuged at 200 × g for 8 min at room temperature.
Platelets were removed from PRP by pipetting the cells above the
plasma/red blood cell interface. PRP was centrifuged at 1000 × g in the presence of prostacyclin (0.1 µg/ml) for 7 min at
room temperature. Pelleted platelets were resuspended in modified
Tyrodes-HEPES buffer pH 7.3 to the required concentration and left for
30 min at 30 °C prior to stimulation. All experiments were performed at 37 °C in siliconized glass tubes with continuous stirring. Agonists were added as 10-100-fold concentrates.
Aggregometry--
Platelet aggregation was monitored in PRP and
in washed platelets. Similar results were obtained in both cases. To
determine aggregation, light transmission (200 µl with 0.5 × 106 platelets/µl) was measured relative to platelet-poor
plasma (PPP). Transmission was recorded on a Fibrintimer 4-channel
aggregometer (APACT Laborgeräte und Analysensysteme, Hamburg,
Germany) over 10 min and was expressed as percent transmission relative
to PPP. Platelet aggregation was induced by addition of collagen (1-30 µg/ml) or CRP (1-100 µg/ml).
Protein Phosphorylation Studies--
Platelet stimulation was
carried out using 500 µl of platelet suspension containing between
0.7-1 × 108 cells/ml. All experiments were performed
in the presence of EGTA (100 µM) and indomethacin (10 µM) to prevent secondary events and facilitate protein
isolation. Reactions were stopped by addition of an equal volume of
ice-cold Nonidet P-40 lysis buffer (20 mM Tris, 300 mM NaCl, 10 mM EDTA, 2% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 2 mM
Na3VO4, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 1 µg/ml pepstatin A, pH 7.3). Antibody (4 µl of anti-LAT
antibody or 1 µl of anti-SLP-76 antiserum or 2 µl of anti-PLC
Proteins were separated by SDS-PAGE on 10% acrylamide gels under
reducing or nonreducing conditions and transferred to PVDF membrane.
Membranes were blocked by incubation with TBS-T containing 10% (w/v)
bovine serum albumin. Antibodies were diluted in blocking buffer
(dilutions: 4G10, 1:1000; anti-Syk, 1:1000; anti-PLC The Inhibitory Effect of JAQ1 Is Overcome by High Concentrations of
Collagen--
We recently reported the generation of the first mAb
(JAQ1, rat IgG2a) against mouse GPVI and demonstrated that JAQ1
inhibits platelet aggregation in response to collagen (5 µg/ml).
Whereas binding of JAQ1 alone did not induce platelet activation,
cross-linking of the surface bound antibody induced irreversible
platelet aggregation (1).
Subsequent to this first report, we performed a series of studies to
further characterize the interaction of JAQ1 with GPVI on platelets. To
our surprise, we found that the inhibitory effect of JAQ1 against
collagen could be overcome by increasing the concentration of the
adhesion molecule. Thus, in the presence of intact JAQ1 (10 µg/ml) or
monovalent Fab fragments of the mAb (10 µg/ml), collagen at a
concentration of 10 µg/ml stimulated partial aggregation, which
reached almost maximal levels at 20 µg/ml (Fig.
1, A and B). Flow
cytometric preincubation studies demonstrated that bound fluorescein
isothiocyanate-labeled JAQ1 (JAQ1FITC) was not displaced by
a 50-fold excess of unlabeled JAQ1 for at least 30 min (not shown),
indicating that JAQ1 binds essentially irreversibly to GPVI over the
time course of this study. Therefore, collagen must be able to bind
either to a second site on GPVI or to a second surface receptor to
mediate activation.
To help distinguish between these two possibilities, we investigated
the effect of JAQ1 on collagen-induced tyrosine phosphorylation of key
proteins in the GPVI signaling cascade. We found that in the absence of
cross-linking JAQ1-IgG, but not control IgG2a (not shown), stimulated a
low level of tyrosine phosphorylation of the adapters, LAT and SLP-76,
and PLC
Despite this low level of stimulation, JAQ1 inhibited collagen-induced
tyrosine phosphorylation of LAT, SLP-76, and PLC JAQ1 Completely Inhibits Platelet Activation to CRP--
These
results provide evidence for a second epitope on the collagen molecule
that is capable of binding to GPVI in the presence of JAQ1 or that the
adhesion molecule is binding to a second activatory receptor. In the
latter case, it is possible that binding is mediated through the same
epitope in the collagen molecule. To investigate this, we examined the
effect of JAQ1 on responses to the collagen-related peptide (CRP),
which is made up of repeat GPO motifs cross-linked via lysine residues
in the N- and C-terminals. We have previously shown that CRP is a more
powerful agonist than collagen on phosphorylation and functional
responses in platelets, presumably because of the greater number of GPO
repeat motifs that enables a greater degree of cross-linking of GPVI
(16).
Intact JAQ1 as well as Fab fragments (10 µg/ml) completely blocked
platelet aggregation in response to CRP at concentrations up to 100 µg/ml (Fig. 2, A and
B). CRP stimulated a marked increase in tyrosine
phosphorylation in the whole cell lysate, which was reduced to basal in
the presence of the antibody (Fig. 2C). These results
demonstrate that JAQ1 is able to fully inhibit responses to CRP, in
contrast to the result for collagen, despite the fact that CRP is
generally recognized as a stronger agonist than collagen (16). This
result rules out the possibility that the high concentrations of
collagen overcome the inhibitory effect of JAQ1 through interaction of
the GPO motif with a second site on GPVI or on a receptor other than
GPVI. This demonstrates the presence of a second epitope in collagen
that mediates activation in the presence of a saturating concentration
of JAQ1. This epitope could bind either to a second site in GPVI or to
a second receptor for collagen. Bearing in mind that aggregation in
response to collagen is completely inhibited in mice deficient in the
FcR Cross-linking of GPVI by JAQ1 Stimulates Aggregation and Protein
Phosphorylation--
We have previously reported that cross-linking of
JAQ1 by addition of anti-rat IgG antibodies (10 µg/ml) stimulates
platelet aggregation (1). In view of the ability of JAQ1 alone to
stimulate a minimal increase in protein-tyrosine phosphorylation and
also the absence of tyrosine phosphorylation of LAT in response to high
concentrations of collagen in the presence of JAQ1, we were interested
in examining the effect of cross-linking JAQ1 on phosphorylation. The
aim of this set of studies was to examine whether cross-linking JAQ1
induces a similar pattern of tyrosine phosphorylation to that seen in
response to classical activation of GPVI, and specifically whether LAT
is phosphorylated.
Cross-linking of surface-bound JAQ1 on platelets stimulated a similar
pattern and time course of tyrosine phosphorylation in whole cell
lysates to those detected in platelets activated by the GPVI selective
agonist, convulxin (5 µg/ml), albeit with a lower intensity of
phosphorylation (Fig. 3A).
Phosphorylation in response to convulxin was maintained for all
proteins up to 90 s, with the exception of a band of 25 kDa
(nonreducing conditions), which comigrates with FcR Conclusion--
The present results demonstrate that high
concentrations of collagen induce platelet aggregation in the presence
of an antibody to GPVI, which completely blocks aggregation in response
to the selective agonist, CRP. This strongly suggests that there is a second activatory sequence within collagen, which is able to mediate platelet activation. It appears that the binding site for this novel
epitope within collagen is located on a receptor that is coupled to the
FcR -chain, Syk, LAT, SLP-76, and phospholipase C
2.
In contrast, platelet aggregation and tyrosine phosphorylation of these
proteins were inhibited when mouse platelets were preincubated with
JAQ1 in the absence of cross-linking and were subsequently stimulated
with a collagen-related peptide (CRP) that is specific for GPVI and low
concentrations of collagen. However, at higher concentrations of
collagen, but not CRP, aggregation of platelets and tyrosine
phosphorylation of the above proteins (except for the adapter LAT) is
re-established despite the presence of JAQ1. These observations suggest
that a second activatory binding site, which is distinct from the CRP binding site on GPVI on mouse platelets, is occupied in the presence of
high concentrations of collagen. Although this could be a second site
on GPVI that is activated by a novel motif within the collagen molecule, the absence of LAT phosphorylation in response to collagen in
the presence of JAQ1 suggests that this is more likely to be caused by
activation of a second receptor that is also coupled to the FcR
-chain. The possibility that this response is mediated by a receptor
that is not coupled to FcR
-chain is excluded on the grounds that
aggregation is absent in platelets from FcR
-chain-deficient mice.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-chain at the cell surface in human and mouse platelets (1, 6, 7).
Signaling through GPVI occurs via a similar pathway to that used by
immunoreceptors (8) as revealed by the tyrosine phosphorylation of the
FcR
-chain immunoreceptor tyrosine-based activation motif (ITAM) by
a Src-like kinase (9, 10). The Src-like kinases Fyn and Lyn are
believed to be involved in this phosphorylation (9-11). Syk is able to
bind to the tyrosine-phosphorylated ITAM of the FcR
-chain via its
tandem SH2 domains (12) leading to autophosphorylation and activation.
Syk plays a critical role in the regulation of PLC
2 through
phosphorylation of a number of key proteins including the adapters LAT
and SLP-76 (13-15).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2
antiserum or 2 µl of anti-Syk antibody per sample) was added to the
lysate followed by 20 µl of protein A-Sepharose CL-4B. The mixture
was incubated overnight at 4 °C on a rotating carousel and
centrifuged to pellet the protein-A-Sepharose. The pellets were washed
in lysis buffer followed by Tris-buffered saline (TBS-T; 20 mM Tris, 137 mM NaCl, 0.1% (v/v) Tween 20, pH
7.6) before the addition of Laemmli sample treatment buffer in
preparation for SDS-PAGE. A GST fusion protein containing the tandem
SH2 domains of Syk was prepared and used to precipitate the FcR
-chain as described previously (12).
2, 1:1000;
anti-SLP-76, 1:500) and incubated with blots for 1 h at room
temperature. Membranes were washed twice for 30 min in TBS-T before
incubation for 1 h with an appropriate HRP-conjugated secondary antibody diluted 1:10,000 in TBS-T. Following washing in TBS-T as
above, the membranes were developed using an enhanced chemiluminescence (ECL) detection system. Densitometric analysis of the results was
performed using Quantity One (version 4) densitometry software (Bio-Rad).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (36K):
[in a new window]
Fig. 1.
The inhibitory effect of JAQ1 is overcome by
high concentrations of collagen. A and B,
heparinized PRP was incubated with stirring in the presence of
irrelevant rat IgG2a (10 µg/ml, circles), Fab fragments of
JAQ1 (10 µg/ml, triangles), or JAQ1 (10 µg/ml,
squares) for 5 min before addition of the indicated
concentrations of collagen. Results are given as mean ± S.D.
(n = 3-6). C, washed platelets were
incubated for 5 min with stirring in the presence or absence of JAQ1
(10 µg/ml) followed by stimulation with various concentrations of
collagen. After 2 min, samples were dissolved in reducing SDS buffer
following immunoprecipitation (IP) for LAT, PLC 2, and
SLP-76 as described under "Experimental Procedures." After
separation by 12.5% SDS-PAGE and transfer to PVDF membranes, the
proteins were incubated with the anti-phosphotyrosine antibody 4G10
(anti-PY) and detected by anti-mouse IgG-HRP and ECL. The
membranes were partly stripped and reprobed for the immunoprecipitated
proteins. The results are representative of four experiments.
D, densitometric quantitation of tyrosine phosphorylation in
LAT in the presence (black bars) or absence (gray
bars) of JAQ1.
2 (Fig. 1C), despite the fact that it inhibits
aggregation to low concentrations of collagen. JAQ1 alone also
stimulated tyrosine phosphorylation of FcR
-chain and Syk as shown
later in this manuscript. A similar low level of protein-tyrosine
phosphorylation in the absence of aggregation is induced in human
platelets by a mAb to the Fc
RIIA receptor, IV.3, in the absence of
cross-linking (not shown); Fc
RIIA also signals through an
ITAM-regulated pathway. JAQ1-Fab did not induce detectable tyrosine
phosphorylation of any of these proteins (not shown), suggesting that
antibody-mediated dimerization of GPVI is required to induce this
subliminal signaling.
2 (Fig.
1C). This inhibition was more clearly seen at higher
concentrations of collagen because of the weak stimulatory effect of
the antibody itself. Nevertheless, higher concentrations of collagen
(10 and 30 µg/ml) were able to stimulate significant tyrosine
phosphorylation of SLP-76 and PLC
2 in the presence of JAQ1,
consistent with the restoration of aggregation to collagen under these
conditions (Fig. 1, A and B). Densitometric
analysis demonstrated that the increase in tyrosine phosphorylation of
PLC
2 and SLP-76 induced by high concentrations of collagen was
similar in the presence or absence of JAQ1 (not shown). However, this
was not the case for LAT, which did not increase in phosphorylation in
response to higher concentrations of collagen in the presence of JAQ1
(Fig. 1, C and D). These observations demonstrate
that higher concentrations of collagen are able to overcome the
inhibitory effect of JAQ1 on aggregation and phosphorylation. The
minimal tyrosine phosphorylation of LAT, however, in contrast to the
substantial phosphorylation of SLP-76 and PLC
2 that is observed in
the presence of JAQ1, raises the possibility that this is mediated by a
receptor other than GPVI. In this context, it is noteworthy that
tyrosine phosphorylation of PLC
2 and platelet activation in response
to CRP are heavily reduced but not abolished in LAT-deficient platelets
(15). Thus, there is a pathway of regulation of PLC
2 and platelet
activation by collagen which is independent of LAT.
-chain (not shown) and that tyrosine phosphorylation is
predominantly inhibited (8), it would appear that this second receptor
is also associated with the FcR
-chain.
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Fig. 2.
JAQ1 completely inhibits platelet aggregation
and decreases protein-tyrosine phosphorylation induced by CRP.
A and B, heparinized PRP was incubated with
stirring in the presence of irrelevant rat IgG2a (10 µg/ml,
circles), Fab fragments of JAQ1 (10 µg/ml,
triangles), or JAQ1 (10 µg/ml, squares) for 5 min before addition of CRP (1-100 µg/ml). Results are shown as
mean ± S.D. (n = 3-6). C, washed
platelets were incubated for 5 min with stirring in the absence or
presence of JAQ1 (10 µg/ml) followed by stimulation with 100 µg/ml
CRP. After 2 min, samples were dissolved in nonreducing SDS buffer.
After separation by 12.5% SDS-PAGE and transfer to PVDF membranes,
blots were incubated with the anti-phosphotyrosine (anti-PY)
antibody 4G10, and proteins were detected by anti-mouse IgG-HRP and
ECL. The arrows indicate the positions of 145- and 36-kDa
proteins, which have previously been shown to represent PLC 2 and
LAT, respectively. Results are representative of two experiments.
-chain. In
contrast, tyrosine phosphorylation in response to cross-linking of JAQ1
declined more rapidly, most notably for protein bands of 25 and 36 kDa
(Fig. 3, B and C). Immunoprecipitation studies
confirmed that cross-linking of JAQ1 stimulated tyrosine
phosphorylation of the same pattern of proteins as seen with convulxin,
including namely FcR
-chain, Syk, SLP-76, LAT, and PLC
2 (Fig.
4). Tyrosine phosphorylation of all of
these proteins was less than that seen in response to convulxin and was
particularly weak for FcR
-chain, which may partly be explained by
the more rapid decline in tyrosine phosphorylation in response to
cross-linking of JAQ1. Aggregation and protein phosphorylation in
response to cross-linking of JAQ1 were completely inhibited in the
presence of the Src family kinase inhibitor, PP1 (not shown), as
previously reported for activation by CRP (10). These results confirm
that cross-linking of GPVI by JAQ1 stimulates a similar pattern of
protein-tyrosine phosphorylation and aggregation to that seen in
response to activation of GPVI by convulxin.
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Fig. 3.
Cross-linking of GPVI by JAQ1 mimics platelet
activation by convulxin. Washed platelets were incubated with
stirring at 37 °C and were stimulated with JAQ1 (10 µg/ml), JAQ1
and polyclonal rabbit anti-rat IgG (both 10 µg/ml), or convulxin (2 µg/ml). A, for the time-course assay aliquots were removed
at the indicated times and dissolved in reducing SDS buffer.
Protein-tyrosine phosphorylation was determined as described in the
legend to Fig. 2. The arrows indicate the migration of 36- and 25-kDa proteins, which have previously been identified as LAT and
the FcR -chain, respectively. Densitometric quantitation of tyrosine
phosphorylation was performed with bands corresponding to LAT
(B) and the FcR
-chain (C). Black
bars, JAQ1 cross-linked; gray bars, convulxin.
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Fig. 4.
JAQ1 and convulxin induce tyrosine
phosphorylation of FcR -chain, Syk, SLP-76,
LAT, and PLC
2. Washed platelets were
incubated as described in the legend to Fig. 3. Samples for
immunoprecipitations (IP) were lysed after 2 min by addition
of an equal volume of ice-cold Nonidet P-40 buffer and were
immunoprecipitated for FcR
-chain, Syk, SLP-76, LAT, and PLC
2 as
described under "Experimental Procedures." After separation by
SDS-PAGE (12.5% acrylamide, nonreducing conditions) and transfer to
PVDF membranes, the proteins were incubated with the
anti-phosphotyrosine antibody 4G10 (anti-PY) and were
detected by anti-mouse IgG-HRP and ECL. The membranes were stripped and
reprobed for the immunoprecipitated proteins. Results are from a
representative experiment (n = 3).
-chain bearing in mind that aggregation to collagen is
completely inhibited in FcR
-chain-deficient platelets. This site
could be located on GPVI or on a novel receptor, which also signals
through the FcR
-chain. Evidence for the latter is provided by the
distinct profile of tyrosine phosphorylation, characterized by the
absence of phosphorylation of LAT that is seen in response to high
concentrations of collagen in the presence of JAQ1. Studies on mice
platelets in which GPVI has been depleted through gene targeting are
required to confirm this conclusion.
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ACKNOWLEDGEMENT |
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We thank K. Rackebrandt for excellent technical assistance. The support given by U. Barnfred is very much appreciated.
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FOOTNOTES |
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* This work was supported in part by Grant Ni 556/2-1 (to B. N.) from the Deutsche Forschungsgemeinschaft, the BAYER AG, and the British Heart Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
A British Heart Foundation Senior Research Fellow.
** To whom correspondence should be addressed: Ferdinand-Sauerbruch Klinikum Wuppertal Universität Witten/Herdecke Arrenbergerstr. 20, Haus 10 D-42117 Wuppertal, Germany. Tel.: 49 202 896 5280; Fax: 49 202 896 5283; E-mail: nieswand@klinikum-wuppertal.de.
Published, JBC Papers in Press, October 17, 2000, DOI 10.1074/jbc.M007536200
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ABBREVIATIONS |
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The abbreviations used are:
GP, glycoprotein;
CRP, collagen-related peptides;
Cvx, convulxin;
FcR, Fc receptor;
IP, immunoprecipitation;
ITAM, immunoreceptor tyrosine-based activation
motif;
LAT, linker for activation of T cells;
mAb, monoclonal antibody;
PLC2, phospholipase C
2;
PRP, platelet-rich plasma;
PVDF, polyvinylidene difluoride;
HRP, horseradish peroxidase;
PAGE, polyacrylamide gel electrophoresis.
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