From the Theodor Kocher Institute, University of
Berne, Freiestrasse 1, CH-3012 Berne, Switzerland, ¶ Klinik und
Polyklinik für Anästhesiologie und Operativ
Intensivmedizin, Experimental and Clinical Haemostasis, University of
Münster, Mendelstrasse 11, D-48149 Münster, Germany, and
the
Serono Pharmaceutical Research Institute SA, 14, chemin des
Aulx, CH-1228 Plan-les-Ouates, Geneva, Switzerland
Received for publication, February 20, 2001, and in revised form, March 27, 2001
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ABSTRACT |
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Aggretin, a potent platelet activator, was
isolated from Calloselasma rhodostoma venom, and 30-amino
acid N-terminal sequences of both subunits were determined. Aggretin
belongs to the heterodimeric snake C-type lectin family and is thought
to activate platelets by binding to platelet glycoprotein
Platelet-collagen interactions are integral to primary hemostasis
(1, 2). Resting platelets using several receptors adhering to
subendothelium of damaged blood vessels are activated and spread to
provide finally a new nonthrombogenic surface until vasculature
regeneration occurs. Reversible binding between
GPIb-V-IX1 and von Willebrand
factor, associated with collagen, is crucial to slow down the
platelet (especially under high shear) so that it can bind more firmly
via other receptors (3, 4). This mechanism strongly parallels that of
the selectins in leukocyte adhesion (5). Another important receptor is
the Because adhesion is rapid, the function of the individual receptor
classes is difficult to assess. Inhibition of any of them can prevent
platelet activation but does not indicate which synergies provide the
final activating signal. In analyzing the mechanisms of each of these
steps, reagents that bind to and activate platelets via individual
receptor types are important tools. Convulxin, a C-type lectin from the
venom of Crotalus durissus terrificus, the tropical
rattlesnake, activates platelets by binding to and clustering the
GPVI/Fc Materials--
Lyophilized C. rhodostoma venom was
from ICN Biomedicals GmbH (Eschwege, Germany) and CV Herpafauna
(Indonesia). Protein A-Sepharose, peroxidase-conjugated goat anti-mouse
and anti-rabbit antibodies, bovine serum albumin, and
biotinamidocaproate N-hydroxysuccinimide ester were from
Sigma. The BCA protein assay kit and the SuperSignal chemiluminescence detection system were from Pierce. Methylated Type I
calf skin collagen was a kind gift from Dr. J. Rauterberg. Anti-phosphotyrosine MoAb (4G10) was from Upstate Biotechnology Inc.
(Lake Placid, NY); anti-p72SYK (4D10) MoAb and polyclonal
antibodies against PLC Purification of Aggretin--
Lyophilized C. rhodostoma venom dissolved in 300 mM NaCl, 100 mM NH4HCO3, pH 7.2, was separated
by gel filtration on a Fractogel EMD BioSEC 650 (S) column (16 × 1200 mm; Merck). The fractions were analyzed by SDS-PAGE/silver
staining and assayed for their ability to induce platelet aggregation.
Active fractions showing strong 60- and 28-kDa (nonreduced) and 12- and
14-kDa (reduced) bands were dialyzed against 50 mM sodium
acetate, pH 5.0, and separated on a Fractogel EMD TMAE column (10 × 150 mm; Merck) with a linear gradient of sodium chloride from 0 to 1 M. Active fractions were pooled, dialyzed against 50 mM sodium acetate, pH 5.0, and loaded on a BioScale Q2
column (7 × 52 mm; Bio-Rad). Alternatively, fractions containing
aggretin were separated by reverse phase HPLC on a wide pore C4 column
(4.6 × 250 mm; J. T. Baker, Phillipsburg, NJ) using an
acetonitrile gradient (0.1% trifluoroacetic acid). Peak fractions were
lyophilized and stored at 4 °C until used.
Purification and Sequence Analysis of Aggretin
Subunits--
Dithiothreitol (1/20 volume of 650 mM) was
added to 1 volume of 300 µg/ml aggretin in 6 M guanidine
HCl, 0.1 M Tris, pH 8.0, and incubated at 45 °C for 30 min, followed by 1/80 volume of 4-vinylpyridine and incubation at room
temperature for 1 h. 1/20 volume of 10% trifluoroacetic acid was
added to the sample, and modified aggretin subunits were isolated by
reverse phase HPLC on a wide pore C4 column (4.6 × 250 mm;
J. T. Baker) using an acetonitrile gradient (0.1% trifluoroacetic
acid). N-terminal sequencing of S-pyridylethylated Biotinylation of Aggretin--
Purified aggretin was dialyzed
against 10 mM sodium phosphate buffer, pH 8.0. Biotinamidocaproate N-hydroxysuccinimide ester in
Me2SO (2 mg/ml) was added to aggretin at a molar ratio 2:1. The mixture was incubated at room temperature for 2 h.
Biotin-aggretin conjugate was separated from free biotin by gel
filtration on a Sephadex G-10 column.
Preparation of Washed Platelets, Platelet Aggregation, and
Immunoprecipitations--
Human platelets were isolated from buffy
coats, less than 20 h after collection, obtained from the Central
Laboratory of the Swiss Red Cross Blood Transfusion Service (15).
Platelets were resuspended at 5 × 108 platelets/ml in
20 mM Hepes, 140 mM NaCl, 4 mM KCI,
1 mM MgCl2,1 mM CaCl2,
5.5 mM glucose, pH 7.4. For immunoprecipitation, aliquots (700 µl, 5 × 108 platelets/ml) of control, resting
as well as activated platelets were solubilized in Hepes buffer
containing 1.2% Triton X-100 with 1 mM
phenylmethylsulfonyl fluoride, 2 mM EDTA, 2 mM
N-ethylmaleimide, and 2 mM sodium orthovanadate.
After centrifugation, platelet lysates precleared with protein
A-Sepharose were stirred for 2 h with specific antibodies before
adding 20 µl of protein A-Sepharose followed by 6 h of incubation.
Preparation of Triton X-100 Platelet Lysates, Wheat Germ
Agglutinin Affinity Chromatography, and Biotinylated
Aggretin-Avidin-Sepharose Affinity Chromatography--
Human platelets
were isolated from buffy coats as described above but in the presence
of 10 µM Iloprost. Washed platelets were diluted
with phosphate-buffered saline to 5 × 109/ml and
solubilized in phosphate-buffered saline containing 1.2% Triton
X-100,1 mM phenylmethylsulfonyl fluoride, 100 µM leupeptin, 2 mM
N-ethylmaleimide, and 2 mM sodium orthovanadate
with or without 5 mM EDTA. After centrifugation
(40,000 × g, 1 h, 4 °C) the supernatant was
applied to a column of wheat germ agglutinin-Sepharose 4B equilibrated
with 130 mM NaCl, 10 mM Tris/HCl, pH 7.4 (buffer D). The column was washed thoroughly with buffer D containing 0.2% octanoyl-N-methylglucamide. The bound material was
eluted with 2.5% N-acetylglucosamine in 10 mM
Tris/HCl, 30 mM NaCl, pH 7.4 (buffer E) containing 0.2%
octanoyl-N-methylglucamide. Biotinylated aggretin A was
added to the pooled fractions containing eluted membrane glycoproteins,
and after 2 h of incubation avidin-Sepharose was added to the
mixture. After further incubation avidin-Sepharose was washed
thoroughly with buffer D containing 0.2%
octanoyl-N-methylglucamide. The avidin-Sepharose with bound
biotinylated aggretin A and platelet proteins was boiled for 1 min with
buffer E containing 1% SDS. Eluted proteins were separated by
electrophoresis and transferred to the PVDF membrane.
FcR Flow Cytometric Analysis of Fibrinogen-FITC Binding to Aggretin,
Convulxin, and Collagen-activated Mouse Platelets--
Mouse
platelet-rich plasma was diluted to 2.5 × 107
platelets/ml with Tyrode's solution, buffered to pH 7.4, to minimize
the formation of platelet aggregates and was preincubated for 3 min at
room temperature with 150 µg/ml fibrinogen-FITC (saturating concentration). Platelet suspension (100 µl) was added to 20 µl of
the platelet agonist solution (aggretin, convulxin, or collagen). Platelet activation was stopped after 120 s by fixing with
formaldehyde in phosphate-buffered saline for 30 min. Platelets were
washed and resuspended in 100 µl of phosphate-buffered saline, and
104 single platelets were analyzed by flow cytometry
(FACScan; Becton Dickinson, Heidelberg, Germany). Nonspecific
background labeling was determined using control platelets treated with
10 mM GRGDPS (Novabiochem, Bad Soden, Germany) to
prevent specific fibrinogen binding.
Aggretin Is a Heterodimeric C-type Lectin--
Aggretin was
purified from lyophilized C. rhodostoma venom by gel
filtration and ion-exchange chromatography. The final product gave 60-, 28-, and 13-14-kDa bands under nonreduced conditions and bands at 14 and 12 kDa under reduced conditions, by SDS-PAGE/silver staining
analysis (Fig. 1). Gel filtration under
nondenaturing conditions gave a peak at 60 kDa, suggesting that a
tetrameric form (dimer of heterodimer) is the principle native form.
Reverse phase HPLC gave two forms, one of which was more hydrophobic
and eluted later from the HPLC column. Reduced and
S-pyridylethylated aggretin was separated by HPLC into Aggretin Acts via GPIb--
Aggretin is a powerful platelet
agonist and induced maximal platelet aggregation at concentrations in
the 40-60 ng/ml range. Apart from convulxin, most venom C-type lectins
investigated so far that affect platelet responses do so by binding to
GPIb so that it was important to investigate this as receptor.
Antibodies to GPIb (lb-23, SZ-2, and AP-1) did not inhibit
aggretin-induced platelet activation. Echicetin, a snake venom C-type
lectin that binds to GPIb and blocks platelet agonists acting via GPIb
showed slight inhibitory effects in about 30% of the experiments.
However, the MoAb VM16d, directed against the thrombin-binding site in GPIb, was able to inhibit aggretin-induced platelet aggregation completely in a dose-dependent way (Fig.
3A) that was reproducible with
platelets from different donors. The protein-tyrosine phosphorylation occurring in platelets after activation with aggretin was also blocked
by VM16d (Fig. 3B).
Aggretin Involves Aggretin Binds Both Aggretin Activates Mouse Platelets from the
Fc Tyrosine Phosphorylation in Platelets Induced by Aggretin Compared
with Collagen and Convulxin--
Fig. 6
shows a tyrosine phosphorylation time range for platelets activated by
100 ng/ml aggretin compared with 1.5 µg/ml collagen and 30 ng/ml
convulxin. Comparison of the proteins phosphorylated on tyrosine showed
several differences between platelets activated by aggretin and the
others. A band at 90 kDa is persistently phosphorylated in aggretin,
rapidly transiently phosphorylated in convulxin-activated, but not
phosphorylated in collagen platelets. The bands at 36-38 kDa, which
are closely associated with signaling via GPVI/Fc Signal Transduction by Aggretin involves p72SYK and
PLC Aggretin Causes Strong Activation of p125FAK, Even When
GPIIb-IIIa Is Blocked--
Platelets activated by aggretin, convulxin,
or collagen in the presence or the absence of GPIIb-IIIa inhibitor
(Ro44-9883, 1 µM) were solubilized in Triton X-100 and
centrifuged, and the supernatant was used for immunoprecipitation with
antibodies to p125FAK (Fig. 8C). Convulxin with
GPIIb-IIIa inhibitor gave little phosphorylation of
p125FAK, but collagen and aggretin induced phosphorylation
of p125FAK in the presence of the inhibitor, with aggretin
having the strongest effect.
Several earlier studies reported proteins from the venom of
C. rhodostoma that activate platelets, and it was suggested
that one of these, called aggregoserpentin or aggretin, acts via
In all previous reports (17, 18) the authors showed that aggretin acts
via the Shin and Morita (19) isolated a C-type lectin that activates platelets
from venom of C. rhodostoma that they called rhodocytin. They did not establish an activation mechanism but showed that it was
not via GPIb. The N-terminal sequences that they determined are
identical with the first 22 and 19 amino acids, respectively, of
aggretin. Recently, the same group reported that the In earlier studies, lack of involvement of GPIb as a receptor for
aggretin or rhodocytin was based on the failure of monoclonal antibodies or echicetin to affect platelet activation. In fact, Shin
and Morita (19) do show an increase in the lag phase response to
rhodocytin in the presence of echicetin, and the slope of aggregation was also slightly inhibited. We also found that echicetin had a minor
inhibitory effect on platelet responses to low concentrations of
aggretin (data not shown). The monoclonal antibodies that had no effect
on aggretin were AP-1 and 6D1 (17). Thus, the binding site for aggretin
on GPIb must partly overlap with the echicetin-binding site but not
with the epitopes of AP-1, 6D1, SZ-2, or Ib-23. On the other hand,
VM16d shares the same binding site as aggretin. VM16d is a well
characterized monoclonal antibody to GPIb with its epitope in the
double-loop region (27) that inhibits thrombin-binding but not von
Willebrand factor binding (28) and must therefore bind to one face of
the GPIb Considerable evidence has accumulated that among the potential
candidates for collagen receptors, at least
Convulxin, a hexameric C-type lectin from C. durissus
terrificus, clusters GPVI/Fc Surprisingly, although the normal mouse platelets reacted strongly, the
Fc Earlier studies on Fc Suzuki-Inoue et al. (21) showed that rhodocytin induced
aggregation of Fc In platelets stimulated by aggretin, p125FAK was strongly
activated. In contrast to platelets treated with convulxin, where the activation of p125FAK is a consequence of the secondary
involvement of GPIIb-IIIa (15) in aggretin-treated platelets, the
activation of p125FAK was only slightly blocked by
GPIIb-IIIa inhibitors and is thus a primary consequence of the
interaction between aggretin and It is still controversial whether The main platelet activation pathway by both collagen and convulxin was
shown to involve phosphorylation of Fc2
1. We now show that binding to
glycoprotein (GP) Ib is also required. Aggretin-induced platelet activation was inhibited by a monoclonal antibody to GPIb as well as by
antibodies to
2
1. Binding of both of
these platelet receptors to aggretin was confirmed by affinity
chromatography. No binding of other major platelet membrane
glycoproteins, in particular GPVI, to aggretin was detected. Aggretin
also activates platelets from Fc receptor
chain (Fc
)-deficient
mice to a greater extent than those from normal control mice, showing
that it does not use the GPVI/Fc
pathway. Platelets from
Fc
-deficient mice expressed fibrinogen receptors normally in
response to collagen, although they did not aggregate, indicating that
these platelets may partly compensate via other receptors including
2
1 or GPIb for the lack of the Fc
pathway. Signaling by aggretin involves a dose-dependent lag phase followed by rapid tyrosine phosphorylation of a number of
proteins. Among these are p72SYK, p125FAK, and
PLC
2, whereas, in comparison with collagen and convulxin, the Fc
subunit neither is phosphorylated nor coprecipitates with p72SYK. This supports an independent, GPIb- and
integrin-based pathway for activation of p72SYK not
involving the Fc
receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 integrin, which is essential for
anchoring the platelet to collagen in the subendothelium (6) and for
linking to the platelet cytoskeleton to prevent the receptor being torn
from the membrane by the forces that it has to withstand. Activation
induces the release of storage granules and the expression of new
receptors on the platelet surface (7) as well as changes in other
receptors such as the fibrinogen receptor,
IIb
3, which is critical for
spreading. Although GPIb-V-IX and
2
1 also participate in signaling to the
platelet interior (8, 9), recent studies, particularly in patients with
platelet receptor deficiencies, have implicated GPVI/Fc
as a major
collagen receptor for platelet activation (10-12). Patients with
platelets lacking any one of these receptors (GPIb-V-IX,
2
1, or GPVI/Fc
) have increased
bleeding times, and platelet adhesion to subendothelium or collagen is
defective under flow conditions (13, 14).
receptor (15). Several other snake venom proteins are potent
platelet activators and may act via collagen-like mechanisms. Some of
these, including trimucytin from Trimeresurus mucrosquamatus
(16) and aggretin from Calloselasma rhodostoma (17), have
been reported to involve
2
1 on platelets
as receptor. We isolated a protein from C. rhodostoma venom
that is a powerful platelet activator. This is most likely aggretin as
described by Huang et al. (17), based on the molecular mass,
sequence of subunits (18) and properties in activating platelets. Like aggretin it also belongs to the snake C-type lectin family. A C-type
lectin from the same species, termed rhodocytin, was described later
(19) with N-terminal sequences identical to those of aggretin, but it
was reported to have some different properties. The mechanism of
rhodocytin action on platelets was recently investigated (20, 21),
showing that rhodocytin interacts with the
2-subunit of
2
1 on the platelet surface. On the other
hand, Eble et al. (22) have reported that rhodocytin does
not bind to a recombinant
2
1 complex. We
now show that aggretin activates platelets via interaction with GPIb as
well as
2
1; however, GPVI/Fc
is not
required. This mechanism via GPIb and
2
1
may also be relevant to activation of platelets via collagen.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 and p125FAK were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA); BHA2.1 anti-
2
1 MoAb was from Chemicon
International Inc. (Temecula, CA); AP-1 anti-GPIb MoAb was a kind gift
from Dr. T. J. Kunicki; SZ-2 anti-GPIb MoAb was a kind gift from
Dr. C. Ruan; Ib-23 anti-GPIb MoAb and Ro44-9883, a GPIIb-IIIa
inhibitor, were kind gifts from Dr. B. Steiner; VM16d anti-GPIb MoAb
was a kind gift from Dr. A. V. Mazurov; MoAb 6F1 against
2
1 was a kind gift from Prof. B. Coller;
polyclonal antibody against Fc
RI
was a kind gift from Prof. J.-P.
Kinet. Echicetin and convulxin were purified from lyophilized
Echis carinatus sochureki venom (Latoxan, Rosans, France)
and C. terrificus durissus venom (Sigma), respectively, as
previously described (15, 23). Highly purified human fibrinogen (Enzyme
Research Laboratories, South Bend, IN) was conjugated with FITC using
the FITC-celite (Calbiochem, Bad Soden, Germany) method (24) but using
pH 7.8 buffer for 48 h at 4 °C, resulting in a F/P ratio of
5.0-5.2. Sephadex G-10 and Sepharose 4B were from Amersham Pharmacia
Biotech. Iloprost was a kind gift from Schering AG (Zürich,
Switzerland). Autoradiography (Fuji RX) films were from Fujifilm
(Dielsdorf AG, Switzerland). PVDF membranes were PolyScreen from
PerkinElmer Life Sciences.
- and
-subunits of aggretin was done on an Applied Biosystem model 477 A
pulsed liquid phase protein sequencer with model 120 A on-line
phenylthiohydantoin amino acid analyzer.
Chain-deficient Mice--
FcR
chain-deficient C57BL/6
(B6) mice have been previously described (25) and were kindly provided
by Dr. Olle Korsgren (26). Normal B6 mice were used as controls. Whole
blood was collected from normal and Fc
-deficient mice anesthetized
with phenobarbital by puncturing the inferior vena cava with
heparinized syringes at a final concentration of 25 units of heparin/ml
of blood.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-
and
-subunits (Fig. 1). Both the less and the more hydrophobic forms
of aggretin gave similar subunits, but these forms also showed
differences in hydrophobicity. The
-subunits and
-subunits,
respectively, of the two forms had identical N-terminal sequences. The
assignment to the
-subunit and the
-subunit was based upon the
nomenclature used for the other heterodimeric snake C-type lectins
where the
-subunit is defined as the larger. N-terminal sequencing
gave the 30-amino acid sequences GLEDCDFGWSPYDQHCYQAFNEQKTWDEAE
for the
-subunit and DCPSGWSSYEGHCYKPFNEPKNWADAERFC for the
-subunit. The sequences of both subunits shows full identity to the
sequence of the cloned aggretin established by Chung et al.
(18). Aggretin is a heterodimeric C-type lectin with strong sequence
similarity to other venom C-type lectins interacting with platelet
receptors (Fig. 2).
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Fig. 1.
Separation of aggretin subunits on reverse
phase HPLC column. Aggretin was reduced with dithiothreitol, then
treated with vinylpyridine, and loaded on a C-4 HPLC column. Peak
1, aggretin -subunit, hydrophilic form; peak 2,
aggretin
-subunit, hydrophobic form; peak 3, aggretin
-subunit, hydrophilic form; peak 4, aggretin
-subunit,
hydrophobic form. The two forms of each subunit had the same N-terminal
sequence and may be due to differential glycosylation. The peaks in the
3-10-min interval originated from the reduction and
S-pyridylethylation. mAu, milliabsorbance units
at 216 nm; %B, percentage of 80% acetonitrile. The
inset shows starting material and fractions 1-3 analyzed by
SDS-PAGE/silver staining.
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Fig. 2.
A comparison of the N-terminal sequences of
the subunits of aggretin with members of the snake venom C-type lectin
family. The sequences are highly similar, and aggretin clearly
belongs to the same protein family. Assignment to - and
-subunits
was on the basis of the subunit size. The positions with amino acids
conserved in at least four of the six subunit sequences, respectively,
are shown with bold letters.
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Fig. 3.
Antibodies against
2
1
and GPIb inhibit aggretin-induced platelet aggregation and
protein-tyrosine phosphorylation. A, platelet
aggregation was induced by 40 ng/ml of aggretin.
Anti-
2
1 antibody (6F1, left
panel) or anti-GPIb antibody (VM16d, right panel) at
various concentrations were added to platelets 1 min before aggretin.
The amounts of antibodies added are indicated. B, washed
human platelets (700 µl, 5 × 108 platelets/ml) were
stirred at 1000 rpm at 37 °C. Aggretin at 40 ng/ml was added, and
aliquots were withdrawn at the times indicated and dissolved in SDS
buffer containing inhibitors. After separation by SDS-PAGE (7-17%
acrylamide gradient) and transfer to PVDF membranes, the proteins were
incubated with anti-phosphotyrosine antibody 4G10 before detection by
peroxidase-linked second antibody and chemiluminescence.
Anti-
2
1 antibody (6F1, 50 µg/ml) or
anti-GPIb antibody (VM16d, 20 µg/ml) were added to the platelets 1 min before aggretin. The left panel shows changes in
protein-tyrosine phosphorylation in platelets activated by aggretin
without any antibodies; the middle panel shows changes in
platelets pretreated with 6F1; and the right panel shows
changes in platelets pretreated with VM16d.
2
1 as
Receptor--
The monoclonal antibody to
2
1, 6F1, blocked platelet aggregation to
aggretin in a dose-dependent way with the addition of 2.5, 12.5, or 50 µg/ml, giving 58, 36, or 11%, respectively, of the
initial aggregation (Fig. 3A). The monoclonal antibody
BHA2.1 against
2
1 also inhibited
aggretin-induced platelet aggregation. The protein-tyrosine
phosphorylation occurring in platelets after activation with aggretin
was completely blocked by antibodies against
2
1 (Fig.
3B).
2
1 and GPIb from
Platelet Lysates--
No platelet receptors bound specifically to
biotinylated aggretin/avidin-Sepharose or avidin-Sepharose as a control
following affinity chromatography of platelet lysate prepared in the
presence of EDTA. However, affinity chromatography of platelet lysate
prepared in the absence of EDTA gave specific binding of
2
1 and GPIb to biotinylated
aggretin/avidin-Sepharose. Avidin-Sepharose 4B alone did not bind any
membrane proteins from the platelet lysate (Fig.
4).
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Fig. 4.
Platelet surface glycoproteins binding to
biotinylated aggretin-avidin Sepharose 4B. Platelets were lysed
with 1.2% Triton X-100 in buffer containing inhibitors (without EDTA),
and glycoproteins were isolated by wheat germ agglutinin-affinity
chromatography. Biotinylated aggretin was added to aliquots and
incubated together for 2 h. Then avidin-Sepharose 4B was added,
and the mixture was incubated for a further 4 h. As a negative
control, avidin-Sepharose was incubated with an aliquot of platelet
glycoproteins without adding biotinylated aggretin. After intensive
washing both avidin-Sepharose samples were boiled in 1% SDS. Eluted
proteins were separated by SDS-PAGE, transferred to a PVDF membrane,
and treated with anti- 2 (lanes 1-3) or
anti-GPIb (lanes 4-6) monoclonal antibodies before
detection by peroxidase-linked second antibody and chemiluminescence.
Lanes 1 and 4, platelet glycoproteins;
lanes 2 and 5, eluate from avidin-Sepharose
without biotinylated aggretin; lanes 3 and 6,
eluate from biotinylated aggretin-avidin-Sepharose.
-deficient Line and Therefore Does Not Act via
GPVI/Fc
--
GPVI requires Fc
for platelet activation by
collagen, and platelets from mice rendered deficient for Fc
were
reported not to respond to collagen or collagen-like peptides (25).
Aggretin was therefore tested on normal and Fc
-deficient mouse
platelets. A flow cytometric method was used to measure activation of
GPIIb-IIIa (fibrinogen-binding sites) as a parameter of platelet
stimulation using FITC-labeled fibrinogen. As expected the
Fc
-deficient mouse platelets did not respond to convulxin, the
GPVI-specific C-type lectin, as agonist, whereas the control platelets
were activated (Fig. 5A).
The Fc
-deficient mouse platelets gave a stronger response to
aggretin than the control platelets (Fig. 5B) and also
aggregated, whereas although both types of platelets gave a similar
FITC-fibrinogen binding response with collagen (Fig. 5C),
the Fc
-deficient platelets did not give detectable aggregates. The
data shown are the means of results from three different
experiments.
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Fig. 5.
Flow cytometry analysis of fibrinogen-binding
sites exposed on Fc -deficient and control
platelets activated by aggretin, convulxin, and collagen.
Platelets from control (
) or Fc
-deficient mice (
) were
activated with convulxin (A), aggretin (B), and
collagen (C) in the presence of fibrinogen-FITC. After
120 s the platelets were fixed, washed, and analyzed by flow
cytometry. The data shown are the means of the results of three
different experiments.
are very strongly
phosphorylated in platelets activated by convulxin and more weakly in
those activated by collagen as already described (15). In
aggretin-activated platelets either the tyrosine-phosphorylated bands
in the 36-38-kDa range were very weakly phosphorylated or a different,
slightly lower band was phosphorylated in response to this agonist.
Unlike platelet responses to convulxin, neither the aggregation nor the
tyrosine phosphorylation response to aggretin is virtually
instantaneous. There is a clear dose-dependent lag phase
during which the phosphorylation of p72SYK increases slowly
(Fig. 7).
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Fig. 6.
Time dependence of tyrosine phosphorylation
in proteins from platelets activated by aggretin, collagen, or
convulxin. Washed platelets (700 µl, 5 × 108
platelets/ml) were stirred at 1000 rpm at 37 °C. Agonist was added,
and aliquots were withdrawn at the times indicated and dissolved in SDS
buffer containing inhibitors. After separation by SDS-PAGE (7-17%
acrylamide gradient) and transfer to PVDF membranes, the proteins were
incubated with anti-phosphotyrosine antibody 4G10 before detection by
peroxidase-linked second antibody and chemiluminescence.
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Fig. 7.
Dose dependence of lag phase of aggregation
and tyrosine phosphorylation in proteins from platelets activated by
aggretin. Upper panel, washed human platelets (500 µl, 5 × 108 platelets/ml) were stirred at 1,100 rpm
at 37 °C, and aggregation was induced by aggretin at 2 µg/ml
(line A), 0.4 µg/ml (line B), or 0.08 µg/ml
(line C) (an arrow marks the point of addition).
Lower panel, aliquots of aggretin-activated platelets were
lysed with SDS at the times shown, and proteins were separated by
SDS-PAGE, transferred to a PVDF membrane, and detected with
anti-phosphotyrosine antibody (4G10), peroxidase-linked second
antibody, and chemiluminescence.
2 but Not Fc
--
Platelets activated by either aggretin,
convulxin, or collagen as above were solubilized in Triton X-100 and
centrifuged, and the supernatant was used for immunoprecipitation with
antibodies to p72SYK and PLC
2 (Fig.
8, A and B). Both
p72SYK and PLC
2 were activated and
tyrosine-phosphorylated by all three agonists. However,
tyrosine-phosphorylated Fc
was directly co-immunoprecipitated with
p72SYK platelets activated by collagen and convulxin but
not in those activated by aggretin.
View larger version (24K):
[in a new window]
Fig. 8.
Identification of tyrosine-phosphorylated
bands implicated in aggretin-induced platelet activation. Control
platelets or platelets activated by aggretin (AGN), collagen
(COLL), or convulxin (CVX) were used.
A, platelets were lysed in Triton X-100 buffer containing
inhibitors. Aliquots were immunoprecipitated with
anti-p72SYK antibody. The immunoprecipitates were separated
by SDS-PAGE. After transfer to PVDF membrane, the proteins were
incubated with 4G10 anti-phosphotyrosine antibody and detected by
peroxidase-linked second antibody and chemiluminescence. The membrane
was stripped and reprobed for p72SYK and for Fc .
B, platelets were lysed in Triton X-100 and
immunoprecipitated with anti-PLC
2 antibodies. After SDS-PAGE and
transfer to PVDF membranes, the proteins were detected with 4G10
anti-phosphotyrosine antibody. The membrane was stripped and treated
with anti-PLC
2 antibodies. C, platelets activated in the
absence (
) or the presence (+) of 1 µM Ro-44-9883, a
GPIIb-IIIa inhibitor, were immunoprecipitated with
anti-pp125FAK antibodies. After SDS-PAGE separation and
transfer to PVDF membrane, the immunoprecipitates were incubated with
4G10 anti-phosphotyrosine antibody. The membrane was stripped and
reprobed for pp125FAK.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
1 (17). Aggretin was recently cloned,
and the full amino acid sequence was established (18). We isolated a
protein from C. rhodostoma venom that has N-terminal
sequences of
- and
-subunits identical to aggretin. This protein
has a molecular mass of 28 kDa with subunits of 12 and 14 kDa on
SDS-PAGE. Under nondenaturing conditions by gel filtration this protein
has a molecular mass around 60 kDa. This implies that two subunits of
the protein are linked together by disulfide bridges and that two
heterodimers interact noncovalently to form tetramers. Aggretin
aggregated platelets in a dose-dependent manner with a long
lag phase as previously described (17).
2
1 receptor. We confirmed that
aggretin binds to
2
1 but showed that it
binds to GPIb as well. GPIb involvement in activation of platelets via
aggretin was shown by affinity chromatography and inhibition of
aggretin-induced platelet aggregation using an anti-GPIb monoclonal
antibody. The monoclonal antibody VM16d completely inhibited
aggretin-induced platelet aggregation as well as changes in
protein-tyrosine phosphorylation. Other monoclonal antibodies against
GPIb (lb-23, SZ-2, and AP-1) did not inhibit aggretin-induced platelet
activation. This indicates that the aggretin-binding site on GPIb
is
limited to the same region as the epitope for VM16d, on the complex
double-loop domain (27). Both GPIb and
2
1
were detected in eluted material specifically bound to biotinylated
aggretin-avidin-Sepharose. These receptors bound to aggretin only when
the platelet lysate was prepared in the absence of EDTA or EGTA. When
the platelet lysate was prepared with EDTA, proteins were not
specifically bound to aggretin, showing that divalent cations are
important for aggretin interactions with platelet receptors. It is not
yet clear whether divalent cations are necessary for the active
structure of aggretin or whether they are necessary for receptors to
maintain a structure that is recognized by aggretin.
2
subunit of
2
1 is involved in the platelet
interaction with rhodocytin and investigated some aspects of signal
transduction occurring in platelets after activation with rhodocytin
(20, 21). The situation has been complicated by a recent report that
rhodocytin does not bind to a recombinant
2
1 complex, whereas another C. rhodostoma venom C-type lectin, rhodocetin, composed of
noncovalently associated
- and
-subunits with different sequences
than those of aggretin or rhodocytin, bound strongly to the recombinant
complex and blocked its binding to collagen (22).
molecule. These results suggest that the previous studies
did not detect aggretin (rhodocytin) binding to GPIb because the
anti-GPIb reagents used did not bind to the same region (or face) of GPIb.
2
1 and GPVI/Fc
are critical. It was
suggested earlier that
2
1 is essentially an adhesion receptor, whereas GPVI/Fc
is the main activation receptor for collagen, although this is controversial (11, 29-31). Collagen-related peptides based on a repetitive GP*P sequence (where *P
is hydroxyproline) signal via Fc
to activate p72SYK and
PLC
2 (31-33).
to activate platelets via a
tyrosine phosphorylation pathway (15). To establish whether or not
aggretin requires the GPVI/Fc
receptor, the effects of aggretin on
platelets from Fc
-negative mice were compared with those on normal
mouse platelets (Fig. 5). Platelet binding of fluorescein-labeled
fibrinogen was used to monitor the activation of GPIIb-IIIa as a marker
of platelet stimulation. Control experiments with convulxin, showed a
clear difference between the two types of mouse platelets with a strong activation of GPIIb-IIIa on the control mouse platelets, whereas the
Fc
-negative platelets were not activated (Fig. 5A). Our
results with convulxin on the Fc
-deficient mouse platelets support
those obtained by Gibbins et al. (34) as well as earlier
studies with collagen-related peptides and GPVI-deficient human
platelets (31) and confirm our earlier conclusions that convulxin acts
via GPVI (15). In the case of aggretin, platelets from both types of mice responded.
-deficient platelets responded even more powerfully (Fig.
5B), suggesting either that more
GPIb/
2
1 is expressed or that signaling
via these receptors is up-regulated by the loss of the Fc
pathway.
Both types of platelet also aggregated in response to aggretin. The
GPIIb-IIIa responses of both the Fc
-deficient platelets and the
normal platelets to collagen were almost identical (Fig. 5C)
(note that total GPIIb-IIIa activation after 2 min was measured here;
therefore differences in rates were not studied). However, neither
Fc
-deficient mouse nor GPVI-deficient human platelets (31) aggregate
to collagen. Therefore, a signal from GPVI/Fc
is essential for the
aggregation response to collagen. The clear results obtained with
convulxin as well as Western blot studies with anti-Fc
antibodies
(data not shown) confirmed the Fc
-deficient status of these
platelets. Keely and Parise (35) reported that Fc
RIIA played an
essential role in activation of platelets via cross-linking with
antibodies to
2
1. Because mouse platelets
do not express Fc
RIIA (36), it is unlikely that this receptor is
involved in platelet activation by aggretin.
-deficient mouse platelets reported that
they show only low responses to collagen (37) including a remnant low activation of p72SYK and PLC
2. Although the
exposure of the fibrinogen-binding sites is not identical to
aggregation, a close correlation might be expected between these two
parameters. However, Poole et al. (37) did not study
aggregation to collagen with the Fc
-deficient mouse platelets. In
the case of human platelets from a patient with GPVI deficiency, also
lacking Fc
(38), the platelets also still responded to collagen but
much more slowly than normal human platelets (31). It was suggested
that the remnant response of GPVI-deficient human platelets to collagen
could be due to its interactions with
2
1 alone.
-deficient mouse platelets and that
2 bound to a rhodocytin affinity column. Rhodocytin
binding to liposomes containing recombinant
2
1 was not inhibited by EDTA (21). Eble
et al. (22), however, found no rhodocytin binding to
recombinant
2
1 whether in an
enzyme-linked immunosorbent assay or in competition with
2
1 binding to collagen. The question of
whether aggretin and rhodocytin are identical or variants with the same
N-terminal sequences is not yet clear.
2
1 and GPIb.
2
1 is
constitutively active or needs to be activated by inside-out signaling
to bind collagen. This is difficult to test with collagen because of
its numerous recognition sequences that can interact with different
receptors. In the two-step, two-receptor model (39), which is the
simplest version proposed for collagen-platelet interactions in primary hemostasis, it was suggested that the platelet binds through
2
1 first and then is activated via
interactions with GPVI/Fc
. However, platelet activation via
GPVI/Fc
may be necessary to modulate
2
1 to allow it to bind to collagen. In
adhesion of platelets to the subendothelium, the situation may be
further complicated because under high shear stress conditions it may
be the GPIb-V-IX receptor that causes platelet activation via von
Willebrand factor bound to collagen to bring
2
1 into action. Another piece of evidence
supporting at least a basal level of constitutive activation comes from
the results with the GPVI-deficient platelets from the Japanese
patients where collagen is still able to induce a weak response;
however, other collagen receptors, including GPIb/von Willebrand
factor, may participate in this process.
, leading to activation of
p72SYK and hence of PLC
2. A role for
2
1 in activation of p72SYK
and of PLC
2 by pathways requiring Fc
RIIA was also suggested by
studies using specific antibodies and inhibitors (35), and Polanowska-Grabowska et al. (40) showed that
2
1 signals via dephosphorylation of a
heat shock protein complex. However, these investigations are limited
by the use of collagen as agonist where interactions with more than one
receptor cannot be excluded or by the use of activating antibodies
where a role of Fc
RIIA was critical. It has been suggested that
aggretin or rhodocytin may provide a good tool for investigating
signaling via
2
1. However, because
aggretin binds to both
2
1 and GPIb the
question about the role of other receptors in preactivation of
2
1 before it can bind agonists such as
collagen is still open. Because aggretin is again an agonist with more
than one binding site for platelet receptors, this suggests that
binding and clustering of
2
1 may not be
enough to activate platelets, and therefore GPIb or GPVI (in the case
of collagen) is necessary to give adequate stimulation of signaling
pathways. Platelet activation with aggretin-induced activation of
p72SYK and of PLC
2 but not Fc
co-immunoprecipitated
with p72SYK. The absence of a direct, early, signaling role
for Fc
in the platelet response to aggretin supports the results
obtained using platelets from Fc
-deficient mice where the GPIIb-IIIa
activation response to aggretin was stronger than that obtained with
platelets from normal, control mice. Aggretin, by activating and
clustering
2
1 and GPIb, gives a powerful
signaling response in platelets. This strongly supports a major
signaling role for
2
1 integrin in
collagen-induced platelet activation as well. Results measuring "soluble collagen" binding to resting and activated platelets (41)
suggest that, although there is a residual binding to resting platelets, this is considerably enhanced in activated platelets. Because the structure of soluble collagen is poorly defined, a role for
GPVI/Fc
in resting platelets in binding this collagen can still not
be excluded. In the platelet response to collagen,
2
1 acts synergistically with GPVI/Fc
and modulates and controls the response to this receptor. This is
clearly seen in the strength of the platelet response to convulxin
compared with collagen. In this,
2
1 shows
a number of parallels to GPIIb-IIIa. Normally, GPIIb-IIIa needs to be
activated by signaling from other receptors to change conformation and
allow fibrinogen binding. However, platelets can adhere to and be
activated by a fibrinogen-coated surface (42), so there must be a
recognition potential even in the "nonactivated" conformation.
Clustering of GPIIb-IIIa leads to activation of p72SYK
without obvious Fc
involvement; however, the mechanism for this is
not yet known (43). Aggretin clustering of
2
1 and GPIb appears to induce a similar
signaling pathway that may be common to several integrins. The
availability of aggretin as a specific reagent, activating platelets
via
2
1 and GPIb independently of
GPVI/Fc
, should allow further analysis of the signaling pathways from these receptors. Together with reagents specific for GPVI/Fc
, such as convulxin (15) or collagen-related peptides (32), it should
provide the tools to analyze how these major receptors work together in
collagen-induced platelet activation.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank the Central Laboratory of the Swiss Red Cross Blood Transfusion Service in Berne for the supply of buffy coats. We are grateful to the many colleagues listed under "Experimental Procedures" for the generous supply of antibodies and noncommercial reagents.
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FOOTNOTES |
---|
* This work was supported in part by Grant 31-52396.97 (to K. J. C.) from the Swiss National Science 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.
§ Present address: Harvard School of Public Health, Cardiovascular Biology Lab., Huntingdon Ave., Boston, MA 02115.
** To whom correspondence should be addressed: Theodor Kocher Inst., University of Berne, Freiestrasse 1, CH-3012 Berne, Switzerland. Tel.: 41-31-631-41-48; Fax: 41-31-921-54-43; E-mail: clemetson@tki.unibe.ch.
Published, JBC Papers in Press, April 3, 2001, DOI 10.1074/jbc.M101585200
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ABBREVIATIONS |
---|
The abbreviations used are:
GP, glycoprotein;
PLC2, phospholipase C
2;
MoAb, monoclonal antibody;
Fc
, Fc
receptor
chain;
FITC, fluorescein isothiocyanate;
PVDF, polyvinylidene difluoride;
PAGE, polyacrylamide gel electrophoresis;
HPLC, high pressure liquid chromatography.
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