From the Department of Molecular Oncology, General
Surgery, Witten/Herdecke University, 42117 Wuppertal, Germany, the
§ Department of Experimental Pathology, Lund University,
22185 Lund, Sweden, and the
Institute for Physiological
Chemistry and Pathobiochemistry, Universität Münster,
48149 Münster, Germany
Received for publication, May 1, 2001
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ABSTRACT |
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Although Collagen is one of the major components of the vessel wall and
contributes to platelet activation and adhesion at sites of vascular
injury. The interaction between platelets and collagen can either occur
indirectly via immobilized von Willebrand factor (vWf)1 binding to platelet
receptors glycoprotein (GP) Ib-V-IX and/or activated
Although GPVI has been established as the major activating platelet
collagen receptor, the way Snake venom-derived proteins are frequently used to study mechanisms of
platelet activation and aggregation because many of them specifically
bind to platelet surface glycoprotein receptors and interfere with
their function. Rhodocytin (also termed aggretin (6)), purified from
the venom of Calloselasma rhodostoma belongs to the family
of C-type lectins and induces aggregation of human as well as mouse
platelets (7). Recent studies gave rise to conflicting results on the
mechanisms underlying this activation process. Several experiments
suggested that rhodocytin activates platelets in a collagen-like
manner. First, both processes are sensitive to inhibition of
thromboxane A2 formation by treatment with acetylsalicylic
acid, and, second, both can be inhibited by mAbs against
Both hypotheses, however, were challenged by our finding that
rhodocytin does not bind recombinant, soluble
To directly test whether rhodocytin induces platelet activation by
mechanisms similar to those induced by collagen, we now examined the
effects of this agonist on platelets lacking
Animals: Generation of Mice with Depletion of Platelet GPVI--
Mice were injected with 100 µg
of JAQ1 intraperitoneally, and platelets were isolated on day 7. As
reported previously, GPVI was not detectable on those platelets by flow
cytometry and Western blotting (16).
Reagents--
High molecular weight heparin (Sigma),
Purification of Rhodocytin--
C. rhodostoma
(Malayain pit viper) venom was purchased from Sigma. Dissolved in 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.02% sodium azide, the venom was
separated by gel filtration chromatography on a Superose 6 column. The
eluate fractions of respective size were pooled. After dilution with
Mono S-buffer A (20 mM MES/NaOH, pH 6.5), the pooled
fractions were applied to a Mono S HR5/5 column (Amersham Pharmacia
Biotech). Not binding to Mono S resin under these conditions, the
rhodocytin in the flow-through was dialyzed against Mono Q buffer A (20 mM Tris/HCl, pH 8.0, 0.02% sodium azide) and applied to a
Mono Q HR5/5 column (Amersham Pharmacia Biotech). Rhodocytin was eluted
from the Mono Q column in a linear NaCl gradient at sodium chloride
concentrations above 300 mM. The rhodocytin-containing
eluate fraction was concentrated by centrifugal ultrafiltration in a
Centricon 10 tube. Finally, the concentrated solution of rhodocytin was
purified by gel filtration on a tandem array of TSK G3000 SWXL and TSK
G2000 SWXL (TosoHass, Stuttgart, Germany). Purity was determined by
SDS-polyacrylamide gel electrophoresis in a 12-18% acrylamide
separating gel. Protein concentration was assayed by the BCA method
according to the manufacturer's protocol (Pierce).
Antibodies--
The rat anti-mouse P-selectin mAb RB40.34 was
kindly provided by D. Vestweber (Münster, Germany) and modified
in our laboratories. FITC hamster anti- Platelet Preparation--
Mice were bled under ether anesthesia
from the retroorbital plexus. The blood was collected in a tube
containing 10% (v/v) 7.5 units/ml heparin, and platelet-rich
plasma was obtained by centrifugation at 300 × g for
10 min at room temperature. The platelets were washed twice in
Tyrode's buffer (137 mM NaCl, 2 mM KCl, 12 mM NaHCO3, 0.3 mM
NaH2PO4, 1 mM MgCl2, 2 mM CaCl2, 5.5 mM glucose, 5 mM Hepes, pH 7.3) containing 0.35% bovine serum albumin
and finally resuspended at a density of 2 × 105
platelets/µl in the same buffer in the presence of 0.02 unit/ml of
the ADP scavenger apyrase, a concentration sufficient to prevent desensitization of platelet ADP receptors during storage. Platelets were kept at 37 °C throughout all experiments.
Immunoblotting--
Platelets (108) were solubilized
in 1 ml of lysis buffer (Tris-buffered saline containing 20 mM Tris/HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, and 0.5% Nonidet P-40; all
from Roche Molecular Biochemicals). After lysis, whole cell extract was
run on a 9% SDS-polyacrylamide gel and transferred onto a
polyvinylidene difluoride membrane. The membrane was first incubated
with 5 µg/ml FITC-labeled mAb followed by rabbit
anti-FITC-horseradish peroxidase (1 µg/ml). The proteins were
visualized by ECL.
Flow Cytometry--
Washed platelets (2 × 106)
were incubated with the indicated amounts of agonists for 5 min
followed by staining with fluorophore-conjugated Abs (5 µg/ml) for 10 min at 37 °C and immediately analyzed on a FACScalibur (Becton
Dickinson). Platelets were gated by forward scatter/side scatter characteristics.
Treatment of Platelets with O-Sialoglycoprotein
Endopetidase--
The washed platelets (2 × 109/ml)
were resuspended in Tyrode's buffer (1 mM
MgCl2, 1 mM CaCl2) and incubated at
37 °C for 30 min with 100 µg/ml O-sialoglycoprotein
endopeptidase. Aliquots of the platelet suspensions were analyzed in
flow cytometry and Western blotting to estimate markers of platelet
activation and alterations in platelet glycoproteins.
Aggregometry--
To determine platelet aggregation, light
transmission was measured using washed platelets (200 µl with
0.5 × 106 platelets/µl). Transmission was recorded
on a Fibrintimer 4 channel aggregometer (LAbor, Hamburg,
Germany) over 10 min and was expressed as arbitrary units with 100%
transmission adjusted with Tyrode's buffer.
It has been reported that rhodocytin induces platelet aggregation
by interacting with the collagen receptor
To directly investigate whether rhodocytin activates platelets through
mechanisms similar to those induced by collagen, we compared the
effects of rhodocytin and fibrillar collagen on mouse platelets lacking
the collagen receptors 2
1
integrin (glycoprotein Ia/IIa) has been established as a platelet
collagen receptor, its role in collagen-induced platelet activation has
been controversial. Recently, it has been demonstrated that rhodocytin
(also termed aggretin), a snake venom toxin purified from the venom of
Calloselasma rhodostoma, induces platelet activation that
can be blocked by monoclonal antibodies against
2
1 integrin. This finding suggested that
clustering of
2
1 integrin by rhodocytin
is sufficient to induce platelet activation and led to the hypothesis
that collagen may activate platelets by a similar mechanism. In
contrast to these findings, we provided evidence that rhodocytin does
not bind to
2
1 integrin. Here we show
that the Cre/loxP-mediated loss of
1 integrin on mouse
platelets has no effect on rhodocytin-induced platelet activation, excluding an essential role of
2
1
integrin in this process. Furthermore, proteolytic cleavage of the
45-kDa N-terminal domain of glycoprotein (GP) Ib
either on normal or
on
1-null platelets had no significant effect on
rhodocytin-induced platelet activation. Moreover, mouse platelets
lacking both
2
1 integrin and the
activating collagen receptor GPVI responded normally to rhodocytin.
Finally, even after additional proteolytic removal of the 45-kDa
N-terminal domain of GPIb
rhodocytin induced aggregation of these
platelets. These results demonstrate that rhodocytin induces platelet
activation by mechanisms that are fundamentally different from those
induced by collagen.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
IIb
3 integrin (1) or by direct
recognition of collagen by specific receptors expressed on the platelet
surface. Several receptors for collagen have been identified on
platelets, most importantly the Ig-like receptor GPVI (2) and
2
1 integrin (3). In contrast to earlier
reports, we have recently shown with
1 integrin-null
platelets that
2
1 integrin is not
essential for platelet adhesion to fibrillar collagen. GPVI, however,
was found to be indispensable for this process (4).
2
1 integrin
modulates the activation process is still unclear. Experimental
evidence suggests that collagen contains two distinct epitopes
contributing to activation of (murine) platelets. One of these epitopes
specifically binds to GPVI, and this interaction is blocked by the
anti-GPVI mAb JAQ1 (5). In contrast, activation through the second
epitope is not blocked by JAQ1 and involves GPVI,
2
1 integrin, and high concentrations of
fibrillar collagen (4). The mechanisms underlying this activation
pathway and the role of
2
1 integrin are
unclear. In addition to
2
1 and GPVI,
other receptors may be involved in this activation pathway. One
candidate is GPIb
, because this receptor indirectly interacts with
collagen via vWf (1).
2
1 integrin (7, 8). Based on these
results, the authors concluded that rhodocytin activates platelets by
interacting with
2
1 integrin (8). Others
reported that rhodocytin activates platelets through
2
1 integrin and GPIb
(9), results that were also based on experiments with inhibitory antibodies against both receptors.
2
1 (10), and the same result has
meanwhile been obtained with wild-type
2
1 integrin isolated from human
platelets.2 Additionally,
rhodocytin activates platelets from FcR
chain-deficient mice (7, 9),
which lack GPVI (11) and do not respond to collagen (4, 12), suggesting
that rhodocytin uses other mechanisms than collagen to induce
aggregation. Moreover, we have recently shown that
1-null platelets fail to express
2
1 integrin but display no reduced
response to fibrillar collagen, indicating that
2
1 integrin is not a major signaling
collagen receptor on platelets (4).
2
1 integrin, GPVI, the ligand-binding
domain on GPIb
, or all three of them. The results of these studies
demonstrate that none of these receptors is required for platelet
activation by rhodocytin.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-null
Platelets--
Mice carrying the
1-null allele in
megakaryocytes were generated as described previously (4). Briefly,
1(fl/fl) mice (13) were crossed with transgenic mice
carrying the Mx-cre transgene (mx-cre+) (14). Deletion of the
1 gene was induced in 4-5-week-old (
1(fl/fl)/Mx-cre+) mice by three intraperitoneal
injections of 250 µg of polyinosinic-polycytidylic acid at 2-day
intervals. Control mice
1(fl/fl) received the same
treatment and were derived from same litters. For experiments, mice
were used at least 2 weeks after polyinosinic-polycytidylic acid
injection. The absence of the
2 and
1
integrin subunits on the platelets from these mice was always confirmed
by flow cytometry and Western blotting as described (4). C57Bl/6 mice
deficient in the FcR
chain (15) were obtained from Taconics
(Germantown, NY). C57Bl/6 × SV129 mice deficient in GPV were
kindly provided by F. Lanza (Strasbourg, France).
-thrombin (Roche Molecular Biochemicals), collagen (Nycomed GmbH,
Munich, Germany), and O-sialoglycoprotein endopeptidase
(Cedarlane, Hornby, Canada) were purchased. Apyrase was purified
from potatoes as described previously (17). Acetylsalicylic acid was
from Sanofi-Synthelabo (Paris, France). Botrocetin and purified human
vWf were kindly provided by F. Lanza (Strasbourg, France) and G. Dickneite (Marburg, Germany), respectively.
1 integrin
(Ha31/8), FITC hamster anti-
2 integrin, and rat
anti-
1 integrin (9EG7) were from BD Pharmingen.
Horseradish peroxidase-labeled rabbit anti-FITC, polyclonal rabbit
anti-fibrinogen, and polyclonal rabbit anti-vWf were purchased from
DAKO. All other antibodies were generated, produced, and modified
in our laboratories: JAQ1 (anti-GPVI) (11), JON1 (anti-GPIIb/IIIa)
(18), p0p4 (anti-GPIb
) (18), DOM1 (anti-GPV) (19), and ULF1
(anti-CD9) (19).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 integrin. Based on these results, it
was suggested that collagen might also activate platelets via
2
1 integrin (7). These findings were challenged by our result showing that rhodocytin does not bind to a
recombinant soluble form of
2
1 integrin
(10).
2
1 integrin and
GPVI. As reported previously, FcR
chain-deficient platelets lack
GPVI but express normal amounts of
2
1
integrin (Fig. 1a) and do not respond to collagen (11, 12) (Fig. 1b). In contrast,
platelets from mice with a Cre/loxP-mediated deletion of the
1 gene in megakaryocytes lack all
1
integrins but express normal amounts of GPVI (Fig. 1a).
1-null platelets display delayed but not reduced aggregation to fibrillar collagen (4) (Fig. 1b).
View larger version (22K):
[in a new window]
Fig. 1.
Characterization of collagen
receptor-deficient platelets. a, platelets from the
indicated mice were stained with fluorophore-conjugated mAbs against
GPVI or 1 integrin at a final concentration of 5 µg/ml
for 10 min at 37 °C and analyzed directly. b, washed
platelets from the indicated mice were stimulated by the addition of
fibrillar collagen (2 µg/ml), and light transmission was recorded on
a standard aggregometer. wt, wild type.
The effects of rhodocytin on mouse platelets were similar to those
reported for human platelets (7-9). Aggregation occurred with a
dose-dependent lag phase (Fig.
2a) and was sensitive to inhibitors of the thromboxane A2-producing system (not
shown). Platelet activation by rhodocytin occurred independently of
GPVI/FcR (Fig. 2a) and was accompanied by marked
degranulation (as shown by P-selectin expression) and strong fibrinogen
binding (Fig. 2b).
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Strikingly, rhodocytin also induced aggregation of
1-null platelets. The extent of degranulation and
fibrinogen binding was indistinguishable between
1-null
and control platelets (Fig. 2b). Furthermore, no differences
were found in the dose-response characteristics between normal and
1-null platelets (not shown). These results demonstrate
that
1 integrins, including
2
1, are not essential for
rhodocytin-induced platelet activation.
It has been shown that snake venom toxins may induce platelet
activation by interacting with multiple receptors (20). Thus, blocking
only one of them would not inhibit platelet activation/aggregation. Therefore, we examined the effects of rhodocytin on platelets lacking
both 2
1 and GPVI. To generate such
platelets, GPVI was depleted in mice with
1-null
platelets by injection of JAQ1 (100 µg/mouse). This treatment induces
a virtually complete internalization and proteolytic degradation of
GPVI in circulating platelets but does not affect other receptors,
including
IIb
3, GPIb-V-IX, and CD9 (16).
GPVI-depleted platelets do not respond to collagen, whereas activation
by other agonists is not affected (16). As shown in Fig.
3a, platelets from
JAQ1-treated
1-null mice lacked both collagen receptors.
However, these
1/GPVI-deficient platelets responded
normally to rhodocytin as demonstrated by aggregometry and flow
cytometric analysis (Fig. 3, b and c). This
finding excludes an essential role of
2
1
and GPVI in rhodocytin-induced platelet activation.
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A very recent report showed that rhodocytin (aggretin)-induced platelet
activation was inhibited by a mAb against the 45-kDa N-terminal domain
on GPIb (9). This domain contains the binding sites for all known
ligands, including vWf, thrombin (21), P-selectin (22), and MAC-1 (23),
as well as snake venom-derived C-type lectins like jararaca GPIb-BP
(24), alboaggregin A (20), and echicetin (25). Based on their results,
Navdaev et al. (9) concluded that GPIb
plays an
essential role in rhodocytin-induced platelet activation. To directly
test this hypothesis, we treated platelets with
O-sialoglycoprotein endopeptidase (26). This treatment
resulted in complete proteolytic removal of the 45-kDa N-terminal
domain of GPIb
as demonstrated by flow cytometric analysis (Fig.
4a). Interestingly, Western
blot analysis revealed that, in addition to cleavage of the 45-kDa
N-terminal domain, the truncated remainder of GPIb
(105 kDa) was
further cleaved in close vicinity to the transmembrane region of
GPIb
, resulting in the release of glycocalicin lacking the 45-kDa
N-terminal region (~85 kDa) (Fig. 4b). Because of the
complete lack of the 45-kDa N-terminal domain of GPIb
,
botrocetin-induced vWf binding was abolished in
O-sialoglycoprotein endopeptidase-treated platelets (Fig.
4c). However, these platelets responded normally to
rhodocytin (Fig. 4, d and e), demonstrating that
the ligand-binding domain on GPIb
is not essential for this
activation process.
|
Although our results demonstrated that neither the collagen receptors
2
1 integrin and GPVI nor GPIb
are
essential for rhodocytin-induced platelet activation, it could not be
excluded that rhodocytin binds to all three receptors that
independently elicit aggregation. Therefore, we removed the 45-kDa
N-terminal region of GPIb
from
1- and
1/GPVI-deficient platelets and examined their response to rhodocytin. As shown in Fig. 5, even
the absences of
2
1, GPVI, and the 45-kDa
N-terminal domain of GPIb
had no significant effect on
rhodocytin-induced platelet activation and aggregation.
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DISCUSSION |
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The mechanism underlying platelet activation by rhodocytin
(aggretin) has been debated. Several investigators suggested that rhodocytin activates platelets in a collagen-like manner by interacting with 2
1 integrin or
2
1 integrin and GPIb
(7, 20). In contrast to these hypotheses, we reported that rhodocytin does not bind
to
2
1 integrin (10).
In the present study we demonstrate that rhodocytin induces activation
of murine platelets in the absence of 2
1
integrin, GPVI, and the ligand-binding domain of GPIb
. These are the
three major receptors that directly or indirectly interact with
collagen. Our findings exclude an essential role for these receptors in rhodocytin-induced activation and demonstrate that the activation process follows mechanisms that are fundamentally different from those
induced by collagen. Our present findings are in contrast to those
reported by others (7, 9), who showed that mAbs against
2
1 blocked rhodocytin-induced platelet
aggregation and concluded that the integrin plays an essential role in
the activation process. An explanation for this discrepancy could be
that treatment of platelets with mAbs against
2
1 may have different effects than the
absence of the receptor. Antibodies may exert steric effects on other
cell surface proteins or may elicit inhibitory signals. Previously,
results from inhibition studies with some antibodies against
2
1 could not be confirmed in a genetic
model in which the
1 gene is deleted in all
hematopoietic cells, including megakaryocytes. It was shown that
antibodies against
2
1 integrin markedly
reduced or abolished platelet adhesion and aggregate formation on
collagen in stasis and flow (27) as well as collagen-induced platelet
aggregation (7). Using the Cre/loxP technology, we ablated the
1 gene in megakaryocytes and showed that
2
1 integrin is not required for platelet
adhesion and thrombus formation on fibrillar collagen under static as
well as low and high shear flow conditions.
1-null
platelets display a delayed but not reduced aggregation in response to
collagen, demonstrating that
2
1 integrin is not a major signaling collagen receptor on platelets (4). The
investigations with convulxin provide another example in which antibody
inhibition and gene deletion studies showed conflicting results.
Whereas the action of convulxin can be inhibited with some antibodies
against
2
1 (28), Cre/loxP-mediated
ablation of
1 integrin on platelets revealed no
detectable role of
2
1 for platelet
activation by this agonist (4). Together, these findings strongly
suggest that certain antibodies against
2
1 integrin induce inhibitory effects
that are not based on blockage of the integrin. Therefore, treatment of
platelets with anti-
2
1 antibodies may not
be suitable for determining dependence on
2
1 integrin.
Another striking difference between rhodocytin- and collagen-mediated
platelet aggregation is the central role of GPVI for collagen but not
for rhodocytin. We showed recently that GPVI is the major collagen
receptor for platelet activation and that GPVI is essential for
collagen-induced platelet aggregation (16). Therefore, GPVI-independent
aggregation processes are different from collagen-induced aggregation.
Both FcR-null platelets, which lack GPVI (11), and GPVI-depleted
platelets (16) fail to activate
1 and
3
integrins in response to collagen. Consequently, these platelets
neither adhere to collagen nor do they bind adhesive ligands or
aggregate in response to this agonist (4, 12, 16). From these data, we
conclude that rhodocytin activates platelets by mechanisms that are
different from those induced by collagen.
Navdaev and co-workers (9) proposed a mechanism for rhodocytin
(aggretin)-induced platelet activation that involves two platelet
receptors, 2
1 integrin and GPIb
. The
importance of GPIb
for rhodocytin-induced aggregation was concluded
from a dose-dependent inhibitory effect of a mAb directed
against the thrombin-binding site on GPIb
, which is located in the
45-kDa N-terminal region of the receptor (29). This finding was in contrast to observations made by other investigators who found no role
of GPIb in rhodocytin-induced activation (30, 31) or no binding of
rhodocytin to GPIb (7). The discrepancies in the binding studies are
difficult to explain. They may be related to different experimental
conditions used in these studies. In our present study we show that
platelets lacking the 45-kDa N-terminal domain on GPIb
respond
normally to rhodocytin (Fig. 5). This finding excludes an essential
role for the ligand-binding region of GPIb
in the rhodocytin-induced
activation process and is in clear contrast to the finding by Navdaev
and co-workers (9). Steric hindrance or elicitation of inhibitory
signals by the GPIb
-specific antibody could be an explanation for
the conflicting results. It is known that occupancy of GPIb
induces
tyrosine phosphorylation of different signaling molecules in
vitro (32-34) and that dimerization of GPIb
by certain mAbs
affects platelet function by yet undefined mechanisms in
vitro and in vivo (18, 35, 36).
Our results do not exclude the possibility that rhodocytin interacts
with an epitope on the GPIb-V-IX complex that is distinct from the
45-kDa N-terminal region of GPIb. However, in studies using mAbs
against different epitopes on either GPIX, GPV, or GPIb
/
,
we were unable to alter rhodocytin-induced activation/aggregation. In
addition, GPV-deficient mouse platelets respond normally to rhodocytin
(not shown). These results suggest that the GPIb-V-IX complex has no
essential role in rhodocytin-induced platelet activation.
Using genetic ablation of 2
1,
antibody-mediated depletion of GPVI, and proteolytic digestion of GPIb,
we show that platelets lacking all three major receptors that directly
or indirectly interact with collagen (
2
1,
GPVI, and GPIb) respond normally to rhodocytin. Because
rhodocytin-induced aggregation of human and mouse platelets occurs with
a dose-dependent lag time and independently of the FcR
chain and both processes are sensitive to acetylsalicylic acid, it is
unlikely that species-specific differences explain the contrasting
results presented here and in other studies (7, 9, 20, 31).
Furthermore, other snake venom-derived toxins like convulxin (16, 37),
botrocetin (38), and alboaggregin A
(20)3 also show similar
activities on human and mouse platelets. Therefore, it appears that
receptor(s) other than
2
1, GPVI, and GPIb
mediate rhodocytin-induced platelet activation. Rhodocytin induced
marked degranulation, suggesting that it stimulates signaling pathways normally used by strong platelet agonists (e.g. thrombin).
Alternatively, the strong effects of rhodocytin could be explained by
the synergism between two or more platelet receptors. Such synergistic
effects have previously been shown for a variety of agonists that
stimulate different G-protein-coupled receptors (39-41) and Ig-like
receptors and Gi-coupled receptors (42-44). Possible
target receptors for rhodocytin on the platelet membrane include
Ig-like receptors like the recently cloned F11 receptor (45), G-protein
coupled receptors, as well as CD9, CD36, CD47 (integrin-associated
protein), or
IIb
3 integrin, all of which
may transduce activation signals when stimulated appropriately. Further
studies will be needed to reveal how rhodocytin induces platelet aggregation.
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ACKNOWLEDGEMENTS |
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We thank Kirsten Rackebrandt and Alison Pirro for excellent technical assistance and U. Barnfred for constant support throughout the study. We also thank F. Lanza for providing GPV-deficient mice.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft (to B. N.) and the Swedish Research Foundation (to C. B. and R. F).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.
¶ Wenner Gren Fellow.
** To whom correspondence should be addressed: Dept. of Molecular Oncology, Ferdinand Sauerbruch Klinikum Elberfeld, Arrenbergerstr. 20, Haus 10, 42117 Wuppertal, Germany. Tel.: 49-202-896-5280; Fax: 49-202-896-5283; E-mail: nieswand@klinikum-wuppertal.de.
Published, JBC Papers in Press, May 14, 2001, DOI 10.1074/jbc.M103892200
2 J. A. Eble, unpublished results.
3 B. Nieswandt, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are: vWf, von Willebrand factor; FcR, Fc receptor; FITC, fluoresceine isothiocyanate; GP, glycoprotein; Ab, antibody; mAb, monoclonal antibody; PE, R-phycoerythrin; MES, 4-morpholineethanesulfonic acid.
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