Rap1B and Rap2B Translocation to the Cytoskeleton by von
Willebrand Factor Involves Fc
II Receptor-mediated Protein Tyrosine
Phosphorylation*
Mauro
Torti
§,
Alessandra
Bertoni
,
Ilaria
Canobbio
,
Fabiola
Sinigaglia¶,
Eduardo G.
Lapetina
, and
Cesare
Balduini
From the
Department of Biochemistry, University of
Pavia, via Bassi 21, 27100 Pavia, Italy, the ¶ Institute of
Biological Chemistry, University of Genoa, viale Benedetto XV 1, 16132 Genoa, Italy, and the
Molecular Cardiovascular Research Center,
Case Western Reserve University School of Medicine,
Cleveland, Ohio 44106
 |
ABSTRACT |
Stimulation of human platelets with von
Willebrand factor (vWF) induced the translocation of the small
GTPases Rap1B and Rap2B to the cytoskeleton. This effect was
specifically prevented by an anti-glycoprotein Ib monoclonal antibody
or by the omission of stirring, but was not affected by the peptide
RGDS, which antagonizes binding of adhesive proteins to platelet
integrins. Association of Rap2B with the cytoskeleton was very rapid,
while translocation of Rap1B occurred in a later phase of platelet
activation and was totally inhibited by cytochalasin D. vWF also
induced the rapid tyrosine phosphorylation of several proteins that was
prevented by the tyrosine kinases inhibitor genistein and by
cAMP-increasing agents. Under these conditions, also the association of
Rap1B and Rap2B with the cytoskeleton was prevented. Translocation of Rap proteins to the cytoskeleton induced by vWF, but not by thrombin, was inhibited by a monoclonal antibody against the Fc
II receptor. The same antibody inhibited vWF-induced tyrosine phosphorylation of
selected substrates with molecular masses of about 75, 95, and 150 kDa.
Three of these substrates were identified as the tyrosine kinase
pp72syk, the phospholipase C
2, and the inositol
5-phosphatase SHIP. Our results indicate that translocation of Rap1B
and Rap2B to the cytoskeleton is regulated by tyrosine kinases and
suggest a novel role for the Fc
II receptor in the mechanism of
platelet activation by vWF.
 |
INTRODUCTION |
Rap proteins are low molecular weight GTP-binding proteins that
share about 50% sequence homology with the product of the ras protooncogene. Human platelets express two members of
the Rap family of proteins, Rap1B and Rap2B, which are located at the
membrane as a consequence of post-translational modifications, including isoprenylation, proteolysis, and carboxylmethylation (1). The
role of Rap1B and Rap2B in platelet function is still poorly
understood. Rap1B is phosphorylated by the cAMP-dependent protein kinase A (PKA)1 (2)
and is rapidly activated upon platelet stimulation with extracellular
agonists (3, 4). By contrast, Rap2B is not a substrate for PKA (5), and
its activation upon cell stimulation has not been reported.
In thrombin-treated platelets both Rap1B and Rap2B translocate to the
cytoskeleton (6, 7). This actin-based structure is not only responsible
for the morphological changes of activated platelets, but also
represents a network connecting several molecules involved in signal
transduction processes, including protein-tyrosine kinases, lipid
metabolizing enzymes, and membrane glycoproteins (8). Translocation of
Rap2B to the cytoskeleton in thrombin-stimulated platelets requires
cell aggregation and is promoted by secondary signals generated by
binding of fibrinogen to the membrane glycoprotein IIb-IIIa (GP
IIb-IIIa) (9). Similarly, fibrinogen binding to GP IIb-IIIa strongly
supports Rap1B translocation to the cytoskeleton, although a small
amount of this protein associates with the actin-based structures, even
in activated cells in the absence of aggregation and ligand binding to
GP IIb-IIIa (10, 11). The production of secondary signals from GP
IIb-IIIa, generated during platelet aggregation and required for the
translocation of Rap proteins to the cytoskeleton, hampers the
possibility to elucidate the molecular mechanisms underlying this
process. In fact, many of the most common agents able to interfere with
specific signal transduction pathways, such as inhibitors of protein
kinases, Ca2+ chelating and cAMP-increasing agents, prevent
the agonist-induced conformational change of glycoprotein IIb-IIIa
required for the expression of the fibrinogen binding sites (12).
Therefore, their possible effects on Rap proteins translocation to the
cytoskeleton in thrombin-stimulated platelets may not be direct, but
may be a consequence of the prevention of platelet aggregation.
Searching for a more suitable model to investigate the mechanism
regulating Rap proteins interaction with the cytoskeleton, we
investigated the effect of a different platelet agonist, such as von
Willebrand factor (vWF). vWF is a large glycoprotein synthesized by
endothelial cells and megakaryocytes and plays an important role in
platelet adhesion and thrombus formation (13). The main platelet
receptor for vWF is the glycoprotein Ib-IX-V complex (GP Ib-IX-V
complex), a member of the leucine-rich glycoprotein gene family (14).
In activated platelets, vWF can also interact with GP IIb-IIIa through
a RGD-containing sequence (15). Binding of soluble vWF to GP Ib-IX-V
complex induces platelet activation associated with arachidonic acid
release, Ca2+ mobilization, and protein tyrosine
phosphorylation (16, 17). Moreover, it has been demonstrated that vWF
stimulates the association of pp60src and phosphatidylinositol
3-kinase with the intracellular cytoskeleton through a mechanism
independent of GP IIb-IIIa (18). Interestingly, in thrombin-stimulated
platelets, translocation of pp60src and phosphatidylinositol
3-kinase to the cytoskeleton occurs by a mechanism similar to that of
Rap proteins, since this process is regulated by fibrinogen binding to
GP IIb-IIIa and aggregation (19, 20).
Herein we investigated the ability of vWF to induce Rap1B and Rap2B
translocation to the cytoskeleton, and we found that this interaction
is actually promoted by the interaction of the agonist with the GP
Ib-IX-V complex. We also found that vWF-induced protein tyrosine
phosphorylation is partially mediated by the recruitment of the Fc
II
receptor (Fc
RII) on the cell surface, which is essential to promote
Rap proteins translocation to the cytoskeleton.
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EXPERIMENTAL PROCEDURES |
Materials--
Sepharose CL-2B was from Amersham Pharmacia
Biotech. Triton X-100 and bicinchoninic acid (BCA) kit for protein
determination were obtained from Pierce. Thrombin, ristocetin,
cytochalasin D, acetylsalicylic acid, indomethacin, prostaglandin
E1 (PGE1), RGDS peptide, protein A-Sepharose,
and mouse IgG were from Sigma. Iloprost was from Schering. von
Willebrand factor (Hemate P) was obtained from Behringwerke (Marburg,
Germany). Peroxidase-conjugated anti-human IgG antiserum was from Dako.
The monoclonal antibody M90 and the antisera against Rap1B and Rap2B
were described previously (2, 21). Ascites of the anti-glycoprotein Ib
monoclonal antibody AK2 were a gift of Dr. Patrizia Noris (IRCCS,
Policlinico San Matteo, Pavia, Italy). The monoclonal antibody IV.3
against the Fc
RII was obtained from Medarex. Anti-phosphotyrosine
antibody was purchased from Upstate Biotechnology Inc. The polyclonal
antiserum against SHIP was a gift from Dr. C. Erneux (Interdisciplinary Research Institute (IRIBHN), Université Libre de Bruxelles,
Brussels, Belgium). Polyclonal antibodies against pp72syk,
PLC
2 and pp125FAK were from Santa Cruz Biotechnology.
Platelet Preparation and Cytoskeleton Extraction--
Human
platelets from healthy donors were prepared by gel filtration on
Sepharose CL-2B and eluted with HEPES buffer (10 mM HEPES,
137 mM NaCl, 2.9 mM KCl, 12 mM
NaHCO3, pH 7.4) as described previously (7). Platelet
samples (0.4 ml, 109 platelets/ml) were incubated at
37 °C in an aggregometer (Chrono-Log) under constant stirring and
then stimulated with 10 µg/ml vWF and 0.5 mg/ml ristocetin or with
0.6 unit/ml thrombin. In some experiments, platelets were preincubated
with 10 µM cytochalasin D, 1 mM RGDS, 20 µg/ml IV.3 monoclonal antibody or 20 µg/ml control IgG for 2 min
before the addition of the agonists. Preincubation with 10 µM iloprost, 10 µM PGE1, 1 mM acetylsalicylic acid, 10 µM indomethacin,
100 or 200 µM genistein was performed for 30 min. Upon
addition of the agonists, aggregation was monitored continuously.
Except for the time course experiments, reaction was blocked after 5 min by addition of an equal volume of extraction buffer (HEPES buffer,
containing 2% Triton X-100, 10 mM EGTA, 4 mM
phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 2 mM Na3VO4). Lysates
were transferred to Eppendorf tubes, vigorously mixed, and placed on
ice for 15 min. Triton X-100-insoluble material was recovered by
centrifugation at 13,000 rpm for 5 min at 4 °C. The pellet was
washed twice with 1 ml of extraction buffer diluted 1:1 with HEPES
buffer and finally resuspended with 80 µl of 4% SDS. The protein
concentration of the cytoskeleton samples was determined by the
bicinchoninic acid assay. An equal volume of a mixture containing 1%
dithiothreitol, 20% glycerol, and 0.02% bromphenol blue was added to
the remaining of the samples and boiled for 5 min. For the analysis of
protein tyrosine phosphorylation of total platelet lysate, samples of
gel-filtered platelets were stimulated in the aggregometer as described
above. In this case, reaction was stopped by adding an equal volume of
SDS-sample buffer (25 mM Tris, 192 mM glycine,
pH 8.3, 4% SDS, 1% dithiothreitol, 20% glycerol, and 0.02%
bromphenol blue). Samples were boiled 5 min before being analyzed by
SDS-PAGE.
Immunoprecipitation--
Samples of gel-filtered platelets (0.25 ml, 109 platelets/ml), treated with buffer or stimulated
with 10 µg/ml vWF and 0.5 mg/ml ristocetin for 5 min in the absence
or in the presence of 20 µg/ml IV.3 monoclonal antibody, were lysed
with an equal volume of immunoprecipitation buffer two times (100 mM Tris/HCl, pH 7.4, 200 mM NaCl, 2 mM EGTA, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 2 mM phenylmethylsulfonyl fluoride, 2 mM
Na3VO4, 2 mM NaF, 2% Nonidet P-40,
0.5% sodium deoxycholate). Lysates were precleared for 1 h at
4 °C with 100 µl of protein A-Sepharose (50 mg/ml stock solution).
The cleared supernatants were incubated with 1 µg of anti-pp72syk, anti-PLC
2,
anti-pp125FAK or 5 µl of anti-SHIP antiserum
for 2 h at 4 °C, and the immunocomplexes were precipitated by
addition of 100 µl of protein A-Sepharose for 45 min. After brief
centrifugation, immunoprecipitates were washed three times with 1 ml of
immunoprecipitation buffer one time and finally resuspended in 25 µl
of SDS-sample buffer.
Electrophoresis and Immunoblotting--
Proteins from
cytoskeletal samples or total cell lysates, representing equal cell
number (1 × 108 and 1 × 107,
respectively), as well as immunoprecipitates, were separated on 7.5%
linear or 5-15% and 10-20% gradient acrylamide gels according to
Laemmli (22) and either stained with Coomassie Brilliant Blue or
transferred to nitrocellulose. Nitrocellulose membranes were blocked
overnight at 4 °C with 6% bovine serum albumin in Tris-buffered
salin (20 mM Tris/HCl, pH 7.5, 0.5 M NaCl) and
incubated with the appropriate antibodies for 2 h at room
temperature. The monoclonal antibody M90, the Rap1B, Rap2B, and the
SHIP antisera were used at 1:1,000 dilution, while the
anti-phosphotyrosine antibody was diluted 1 µg/ml. Antibodies against
pp72syk, PLC
2 and pp125FAK were
diluted 1:200. Membranes were extensively washed with 50 mM
Tris/HCl, pH 7.4, 0.2 M NaCl, 1 mg/ml polyethylene glycol
20,000, 1% bovine serum albumin, 0.05% Tween 20, and incubated with
peroxidase-conjugated secondary antibody (1:20,000 dilution) for 45 min. Upon extensive washing, reactive proteins were visualized with a
chemiluminescence reaction. In some experiments, nitrocellulose filters
were stripped by incubation in 62.5 mM Tris/HCl, pH 6.8, 2% SDS, 100 mM
-mercaptoethanol at 50 °C for 30 min,
blocked again with 6% bovine serum albumin, and reprobed with a
different antibody. All the experiments presented are representative of
at least three separated experiments.
 |
RESULTS |
Translocation of Rap1B and Rap2B to the Cytoskeleton in
vWF-stimulated Platelets--
Stimulation of human platelets with 10 µg/ml vWF and 0.5 mg/ml ristocetin induced the incorporation of
several proteins into the cytoskeleton, including actin-binding protein
(250 kDa), myosin (200 kDa), and
-actinin (99 kDa), as revealed the
Coomassie Blue-stained gel shown in Fig.
1A. Cytoskeleton reorganization induced
by vWF was very similar to that induced by 0.6 unit/ml thrombin (Fig. 1A). The ability of vWF to promote the translocation of Rap
proteins to the cytoskeleton was investigated by immunoblotting
experiments, using both the monoclonal antibody M90, which recognizes
all the members of the Rap family of proteins, and specific antisera
against Rap1B and Rap2B. As shown in Fig. 1, B-D, both
Rap1B and Rap2B translocated to the cytoskeleton upon stimulation with
vWF, and the amount of cytoskeletal-associated Rap1B and Rap2B was
comparable with that observed in thrombin-aggregated platelets. The
translocation of Rap proteins to the cytoskeleton in vWF-stimulated
platelets was not affected by the pretreatment of the cells with either acetylsalicylic acid or indomethacin, indicating that this effect was
not mediated by the production of thromboxane A2 (data not shown).

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Fig. 1.
Association of Rap1B and Rap2B with the
platelet cytoskeleton. Cytoskeleton was prepared from resting
platelets (Bas) and from platelets stimulated for 5 min with
either 10 µg/ml vWF and 0.5 mg/ml ristocetin (vWF) or 0.6 unit/ml thrombin (THR). A, proteins were
separated by SDS-PAGE on a 5-15% acrylamide gradient gel and stained
with Coomassie Brilliant Blue. B-D, proteins were separated
by SDS-PAGE on 10-20% acrylamide gradient gels, transferred to
nitrocellulose, and probed with the monoclonal antibody M90
(B), the Rap1B antiserum (C), or the Rap2B
antiserum (D). The amount of cytoskeletal proteins applied
to each gel lane was from 108 platelets.
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To analyze the kinetics of vWF-induced translocation of Rap proteins to
the cytoskeleton, platelets were stimulated in an aggregometer, and at
different times the Triton X-100-insoluble materials were analyzed with
the antisera against Rap1B and Rap2B. Fig.
2 shows that incorporation of Rap1B into
the cytoskeleton was detected after 1 min of stimulation, when
aggregation reached about 80%. By contrast, translocation of Rap2B was
detectable after 15 s upon addition of the agonist, when only 19%
of aggregation was measured. In both cases, incorporation of Rap1B and
Rap2B into the cytoskeleton was maximal after 5 min and was paralleled by maximal platelet aggregation.

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Fig. 2.
Time course of the translocation of Rap1B and
Rap2B to the cytoskeleton. Gel-filtered platelets were placed in
an aggregometer and stimulated with 10 µg/ml vWF and 0.5 mg/ml
ristocetin for increasing periods of time as indicated in the figure.
Aggregation was monitored continuously. Reactions were stopped by
addition of an equal volume of extraction buffer, and cytoskeletons
were immediately prepared. Cytoskeletal proteins (corresponding to
108 platelets) were separated on a 10-20% acrylamide
gradient gel, transferred to nitrocellulose, and probed with the Rap1B
and Rap2B antisera, as indicated. The percentage of platelet
aggregation measured for each sample is also reported.
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The role of agonist-induced actin polymerization in the translocation
of Rap proteins to the cytoskeleton was investigated by treating
platelets with 10 µM cytochalasin D for 2 min before stimulation with vWF. As reported previously with thrombin-stimulated platelets (23), we found that cytochalasin D did not affect platelet
aggregation induced by vWF (data not shown). Immunoblotting with the
specific antisera revealed that treatment of platelets with
cytochalasin D completely prevented the association of Rap1B to the
cytoskeleton and strongly reduced, but not totally inhibited, the
translocation of Rap2B to the Triton X-100-insoluble material (Fig.
3).

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Fig. 3.
Effect of cytochalasin D on vWF-induced
association of Rap1B and Rap2B with the cytoskeleton. Gel-filtered
platelets were incubated at 37 °C in the presence or absence of 10 µM cytochalasin D (cytD) for 2 min and
then stimulated with or without 10 µg/ml vWF and 0.5 mg/ml ristocetin
(vWF) for 5 min under constant stirring. Cytoskeletal
proteins were separated by SDS-PAGE on a 10-20% acrylamide gradient
gel, transferred to nitrocellulose, and probed with the Rap1B and Rap2B
antisera as indicated.
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Role of GP Ib-IX-V Complex and GP IIb-IIIa in the Translocation of
Rap Proteins to the Cytoskeleton--
In thrombin-stimulated
platelets, translocation of Rap proteins to the cytoskeleton is
regulated by fibrinogen binding to GP IIb-IIIa and aggregation (9, 11).
Fig. 4 shows that platelet aggregation
induced by vWF could be prevented by the omission of stirring.
Moreover, an antibody against the membrane glycoprotein Ib, AK2, also
caused total inhibition of aggregation. By contrast, preincubation of
platelets with 1 mM RGDS did not affect platelet aggregation induced by vWF (Fig. 4). The effect of the prevention of
vWF interaction with GP Ib-IX-V complex and platelet aggregation on the
translocation of Rap proteins to the cytoskeleton is reported in Fig.
5A. Preincubation of platelets
with the antibody AK2 completely blocked the association of both Rap1B
and Rap2B to the Triton X-100-insoluble material. Moreover, when
binding of vWF to platelets was allowed, but aggregation was prevented
by the omission of stirring, interaction of both Rap proteins with the
cytoskeleton was not observed (Fig. 5A). Thus, GP Ib-IX-V
complex-mediated aggregation of vWF-stimulated platelets is required
for the interaction of Rap1B and Rap2B with the cytoskeleton. To
investigate the role played by the receptor occupancy of GP IIb-IIIa,
we compared the effect of 1 mM RGDS peptide on thrombin-
and vWF-induced translocation of Rap proteins to the cytoskeleton. Fig.
5B shows that the ability of vWF to promote the interaction
of both Rap1B and Rap2B with the cytoskeleton was not affected by the
presence of the RGDS peptide. By contrast, Rap1B and Rap2B did not
associate with the cytoskeleton in platelets stimulated with thrombin
in the presence of the RGDS peptide.

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Fig. 4.
Analysis of platelet aggregation induced by
vWF. Samples of gel-filtered platelets were placed in an
aggregometer and stimulated with 10 µg/ml vWF and 0.5 mg/ml
ristocetin. Stimulation was performed under constant stirring
(trace A), in the absence of stirring (trace B),
in the presence of 1 mM RGDS peptide (trace C),
or in the presence of 1 µl of the anti-glycoprotein Ib monoclonal
antibody AK2 (trace D). The optical pattern of platelet
aggregation from a typical experiment is reported.
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Fig. 5.
Role of GP Ib-IX-V complex, GP IIb-IIIa, and
platelet aggregation on vWF-stimulated translocation of Rap proteins to
the cytoskeleton. A, platelet samples were treated with
buffer (Bas), 10 µg/ml vWF and 0.5 mg/ml ristocetin
(vWF), or 10 µg/ml vWF and 0.5 mg/ml ristocetin in the
presence of 1 µl of the AK2 ascitic fluid (AK2 + vWF) for
5 min under constant stirring. Stimulation with vWF and ristocetin was
also performed in the absence of sample stirring (vWF ns).
Cytoskeletons were extracted and analyzed by immunoblotting with the
antisera against Rap1B and Rap2B, as indicated. B, platelet
stimulation with 10 µg/ml vWF and 0.5 mg/ml ristocetin
(vWF) or with 0.6 unit/ml thrombin (THR) was
performed in the presence or absence of 1 mM RGDS peptide.
Immunoblotting analysis of the cytoskeletal proteins was performed with
the Rap1B and Rap2B antisera.
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Protein Tyrosine Phosphorylation and the Translocation of Rap
Proteins to the Cytoskeleton--
We next investigated the signal
transduction pathways mediating the translocation of Rap proteins to
the cytoskeleton in vWF-stimulated platelets, and we considered a
potential role for the agonist-induced protein tyrosine
phosphorylation. Immunoblotting with anti-phosphotyrosine antibodies
revealed that vWF induced the tyrosine phosphorylation of several
platelet substrates, including proteins with a molecular mass of 38, 75, 95, 116, 150, and 230 kDa (Fig. 6).
Tyrosine phosphorylation of these proteins was rapid, being already
detectable after 30 s of stimulation, and reached the maximal
level after 1 min. To analyze the role of vWF-induced protein tyrosine
phosphorylation on the translocation of Rap proteins to the
cytoskeleton, intact platelets were incubated with the tyrosine kinases
inhibitor genistein before stimulation with the agonist. Preincubation
with both 100 and 200 µM genistein did not inhibit
vWF-induced platelet aggregation (data not shown), but efficiently
blocked agonist-induced activation of tyrosine kinases, as demonstrated
by the total inhibition of protein tyrosine phosphorylation (Fig.
7A). Cytoskeletal samples of
platelets stimulated with vWF upon incubation with different concentrations of genistein were analyzed with the antisera against Rap1B and Rap2B. As shown in Fig. 7B, inhibition of tyrosine
kinases by genistein completely prevented the translocation of Rap1B to the cytoskeleton. By contrast, genistein, even at 200 µM,
caused only a 50% reduction of the association of Rap2B with the
cytoskeleton. Similar results were also obtained using different
inhibitors of tyrosine kinases, including tyrphostin A47 and erbstatin
(data not shown).

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Fig. 6.
Time course of protein tyrosine
phosphorylation induced by vWF. Platelet samples were stimulated
with 10 µg/ml vWF and 0.5 mg/ml ristocetin for the indicated times.
Reactions were stopped by addition of an equal volume of SDS-sample
buffer, and aliquots of the cell lysates, corresponding to
107 platelets, were loaded on a 5-15% acrylamide gradient
gel. Proteins were transferred to nitrocellulose and probed with the
anti-phosphotyrosine antibody. The positions of the molecular mass
markers are indicated on the left.
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Fig. 7.
Effect of genistein on protein tyrosine
phosphorylation and translocation or Rap proteins to the
cytoskeleton. Gel-filtered platelets were incubated with genistein
(0, 100, and 200 µM, as indicated) for 30 min at 37 °C
and then treated with buffer (Bas) or with 10 µg/ml vWF
and 0.5 mg/ml ristocetin (vWF) for 5 min under constant
stirring. A, total cell lysates were analyzed by
immunoblotting with the anti-phosphotyrosine antibody, upon proteins
separation on a 5-15% acrylamide gradient gel. The position of the
molecular mass markers are indicated on the left.
B, cytoskeletal proteins were separated by SDS-PAGE on a
10-20% acrylamide gradient gel, transferred to nitrocellulose, and
probed with the antisera against Rap1B and Rap2B as indicated.
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Increased levels of cAMP mediate the inhibition of platelet function
(12). The effect of cAMP on vWF-induced translocation of Rap proteins
to the cytoskeleton was investigated by treating platelets with the
prostacyclin analogue iloprost or with PGE1, both of which
are able to promote the activation of adenylate cyclase. Fig.
8A shows that pretreatment of
platelets with iloprost or PGE1 totally prevented the
ability of vWF to induce the translocation of Rap1B to the cytoskeleton
and caused a strong, but not total, inhibition of the translocation of
Rap2B. To correlate the inhibitory effect of high levels of cAMP with
that of the tyrosine kinases inhibitor genistein, the effect of
iloprost and PGE1 on protein tyrosine phosphorylation was
analyzed. Fig. 8B shows that increased cAMP levels
completely antagonized vWF-stimulated protein tyrosine phosphorylation.

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Fig. 8.
Inhibition of translocation of Rap proteins
to the cytoskeleton and protein tyrosine phosphorylation by cAMP.
Platelets were incubated with buffer (none), with 10 µM iloprost (Ilop), or with 10 µM PGE1 for 30 min at 37 °C and then
treated with buffer (Bas) or with 10 µg/ml vWF and 0.5 mg/ml ristocetin (vWF) for 5 min. A,
immunoblotting performed with the antisera against Rap1B and Rap2B of
cytoskeletal proteins obtained from the same number of cells.
B, immunoblotting with anti-phosphotyrosine antibody of
aliquots of total cell lysates upon separation of the proteins on a
5-15% acrylamide gradient gel. The position of the molecular mass
markers are reported on the left.
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Role of Fc
RII in Platelet Activation by vWF--
The physical
proximity between the vWF receptor, GP Ib-IX-V complex, and the
Fc
RII on the platelet surface has been reported recently (24).
Therefore, it was interesting to know whether the Fc
RII was related
to the transmembrane signaling leading to Rap proteins interaction with
the cytoskeleton. Platelets were treated with the anti-Fc
RII
monoclonal antibody IV.3, or with control IgG, and then stimulated with
either vWF or thrombin. In both cases, we found that the presence of
the IV.3 antibody did not affect platelet aggregation (data not shown).
Cytoskeletal samples were then analyzed with the Rap1B and Rap2B
antisera. Fig. 9 shows that vWF-induced
translocation of both Rap1B and Rap2B was strongly inhibited by the
preincubation of platelets with the IV.3 antibody, but not with control
IgG. Once again, while Rap1B was totally undetectable in the
cytoskeleton from IV.3-treated, vWF-stimulated platelets, a reduced
amount of Rap2B was still observed in the cytoskeleton from the same
samples. Fig. 9 also shows that the ability of thrombin to induce Rap1B and Rap2B translocation to the cytoskeleton was not affected either by
control IgG or by the IV.3 antibody.

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Fig. 9.
Role of the Fc RII on
the translocation of Rap1B and Rap2B to the cytoskeleton.
Platelets were incubated with buffer (none), with 20 µg/ml
control mouse IgG (IgG), or with 20 µg/ml IV.3 monoclonal
antibody (IV.3) for 2 min at 37 °C. Samples were then
stimulated with buffer (Bas), 10 µg/ml vWF and 0.5 mg/ml
ristocetin (vWF), or 0.6 unit/ml thrombin (THR)
for 5 min. Cytoskeletons were extracted upon lysis with Triton X-100
and analyzed by immunoblotting with the antisera against Rap1B and
Rap2B.
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To verify whether the vWF preparation used in this study could activate
the Fc
RII and lead to Rap proteins translocation to the cytoskeleton
through contaminating immunoglobulins, 1 µg of vWF, along with known
amounts of purified human IgGs, were analyzed by immunoblotting using
an anti-IgG antiserum. Fig.
10A shows that the vWF
preparation actually contained some contaminating immunoglobulins.
However, by densitometric analysis, we calculated that contaminating
IgGs represented about 2.4% of the vWF preparation. Thus, platelet
samples stimulated with 10 µg/ml vWF were actually incubated with
0.24 µg/ml immunoglobulins. Such a low concentration, and the absence
of secondary cross-linking antibodies, argues against a possible direct
activation of the Fc
RII. However, to completely rule out a possible
role of activation of Fc
RII by contaminating IgGs in the
translocation of Rap proteins to the cytoskeleton, 0.5 ml of the vWF
preparation was incubated with 10 mg of protein A-Sepharose, and the
supernatant was analyzed by immunoblotting with anti-human IgG
antibodies. As shown in Fig. 10A, this procedure completely
removed contaminating IgGs. The IgG-free vWF was as effective as the
crude vWF preparation in inducing platelet aggregation (data not shown)
as well as the translocation of both Rap1B and Rap2B to the
cytoskeleton (Fig. 10B). Therefore, we can exclude that the
involvement of Fc
RII in the relocation of Rap proteins is the
consequence of its recruitment by contaminating IgGs in the vWF
preparation.

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Fig. 10.
Translocation of Rap1B and Rap2B to the
cytoskeleton is not mediated by vWF-contaminating immunoglobulins.
A, 10 µl (1 µg) of vWF before (A) and after
(B) incubation with protein A-Sepharose, together with 50, 100, and 200 ng of purified human immunoglobulins, were analyzed by
immunoblotting using a peroxidase-conjugated goat anti-human IgG
antiserum. B, cytoskeletal proteins from resting platelets
(Bas) or platelets stimulated with 10 µg/ml vWF before
(A) or after (B) treatment with protein
A-Sepharose treated in combination with 0.5 mg/ml ristocetin were
separated on a 10-20% acrylamide gel, transferred to nitrocellulose,
and probed with the antisera against Rap1B and Rap2B as
indicated.
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Since our data demonstrated a role for tyrosine kinases in inducing Rap
proteins association with the cytoskeleton, the effect of platelet
preincubation with the IV.3 antibody on vWF- and thrombin-induced protein tyrosine phosphorylation was investigated. Fig.
11 shows that blockade of the Fc
RII
specifically caused a significant reduction of the number and intensity
of tyrosine-phosphorylated proteins in vWF-stimulated platelets. In
particular, the tyrosine phosphorylation of substrates with a molecular
mass of about 75, 95, and 150 kDa induced by vWF was prevented by the
incubation with the IV.3 antibody, but not with control IgG. Moreover,
the ability of thrombin to induce tyrosine phosphorylation of the same
substrates was not affected either by control IgG or by the IV.3
antibody.

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Fig. 11.
Inhibition of protein tyrosine
phosphorylation by the IV.3 monoclonal antibody. Platelet samples
were incubated and stimulated as described in the legend to Fig. 9.
Reactions were stopped by the addition of SDS-sample buffer, and equal
volumes of the total cell lysates were loaded on a 7.5% acrylamide
gel. Proteins were transferred to nitrocellulose and probed with the
anti-phosphotyrosine antibody. The position of the molecular mass
markers are reported on the left.
|
|
To identify some of the proteins whose tyrosine phosphorylation induced
by vWF was regulated by recruitment of Fc
RII, we immunoprecipitated
selected substrates with specific antibodies (Fig.
12). In agreement with a previous
report (25), we found that vWF stimulated the tyrosine phosphorylation
of pp72syk. In addition, Fig. 12 shows that vWF-induced
tyrosine phosphorylation of pp72syk was inhibited by
preincubation of platelets with the IV.3 monoclonal antibody. Tyrosine
phosphorylation of two different substrates with a molecular mass of
145-150 kDa, the PLC
2 and the inositol 5-phosphatase SHIP was then
investigated. We found that both these substrates were phosphorylated
in vWF-stimulated platelets and then this event was inhibited by the
blockade of the Fc
RII (Fig. 12). Finally, Fig. 12 shows the tyrosine
phosphorylation of pp125FAK in vWF-treated
platelets. However, in agreement with the evidence that the IV.3
monoclonal antibody did not significantly alter the phosphorylation of
any 125-kDa substrate (Fig. 11), we found that tyrosine phosphorylation
of pp125FAK was not affected by blockade of
Fc
RII.

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Fig. 12.
Effect of IV.3 monoclonal antibody on
vWF-induced tyrosine phosphorylation of pp72syk,
PLC 2, SHIP, and pp125FAK.
Gel-filtered platelets were incubated with or without 20 µg/ml IV.3
monoclonal antibody for 2 min at 37 °C and then treated with buffer
or 10 µg/ml vWF and 0.5 mg/ml ristocetin for 5 min. pp72syk,
PLC 2, SHIP, and pp125FAK were immunoprecipitated as
indicated on the left and analyzed by immunoblotting with
anti-phosphotyrosine antibodies (P-Tyr). Blots were then
stripped and reprobed with the same antibody used for the
immunoprecipitation, as indicated on the right.
|
|
 |
DISCUSSION |
vWF Causes the Translocation of Rap1B and Rap2B to the
Cytoskeleton--
Our present data demonstrate that stimulation of
human platelets with vWF induced the translocation of the low molecular
weight GTP-binding proteins Rap1B and Rap2B to the cytoskeleton. The amount of cytoskeletal-associated Rap1B and Rap2B in vWF-stimulated platelets was comparable with that measured in the cytoskeleton from
thrombin-aggregated platelets. However, several differences between the
effects of the two agonists were observed. Previous kinetic studies
revealed that, in thrombin-stimulated platelets, translocation of Rap1B
to the cytoskeleton preceded the translocation of Rap2B, which occurred
only in a late phase of platelet aggregation (6, 7, 10, 11).
Conversely, the interaction of Rap2B with the cytoskeleton in
vWF-stimulated platelets was observed after 15 s, when only about
19% of aggregation was measured. Moreover, translocation of Rap1B
occurred later on, when aggregation reached about 80%. It is likely
that these kinetics are the expression of different mechanisms
regulating the translocation of the two Rap proteins to the
cytoskeleton. In this regard, it is interesting to note that
pretreament of platelets with cytochalasin D, that prevents
agonist-induced actin polymerization, completely inhibited the
vWF-induced translocation of Rap1B to the cytoskeleton, but only
partially affected the translocation of Rap2B. This indicates that the
direct or indirect interaction with the newly polymerized actin
filaments totally mediates the incorporation of Rap1B into the
cytoskeleton, but it is not sufficient to explain the incorporation of
Rap2B. The cytochalasin D-insensitive interaction of Rap2B with the
cytoskeleton may indicate either the binding of the protein to
preformed actin filaments or the interaction with other structures, such as microtubules.
GP Ib-IX-V Mediates the Translocation of Rap1B and Rap2B--
Our
present information show that vWF-induced translocation of Rap proteins
to the cytoskeleton required platelet aggregation triggered by the
interaction of vWF with the GP Ib-IX-V complex. In thrombin-stimulated
platelets, interaction of Rap proteins with the cytoskeleton is
dependent on GP IIb-IIIa-mediated aggregation (9, 11). By contrast,
vWF-induced translocation of Rap proteins to the cytoskeleton is not
regulated by occupancy of the fibrinogen receptor, since no inhibitory
effects of the RGDS peptide were observed. In this regard, Rap proteins
behave much like pp60src and phosphatidylinositol 3-kinase,
whose translocation to the cytoskeleton in vWF-stimulated platelets is
independent of GP IIb-IIIa (18). We have also found that the antibody
AK2, which prevents binding of vWF to the glycoprotein Ib-IX-V, and the
omission of sample stirring, prevented vWF-induced translocation of Rap proteins to the cytoskeleton. This indicates that platelet aggregation mediated by interaction of vWF with the GP Ib-IX-V complex is sufficient to trigger this process.
Protein Tyrosine Phosphorylation Is Involved in the Signaling of GP
Ib-IX-V--
The evidence that, in vWF-stimulated platelets,
interaction of Rap proteins with the cytoskeleton was independent of
fibrinogen binding to GP IIb-IIIa allowed further investigations on
the signal transduction pathways mediating these events. We
successfully used an inhibitor of tyrosine kinases to demonstrate
that protein tyrosine phosphorylation is associated to the
translocation of Rap proteins to the cytoskeleton in vWF-stimulated
platelets. vWF induced the rapid tyrosine phosphorylation of several
substrates. By immunoprecipitation with specific antibodies we
identified the tyrosine kinases pp72syk and
pp125FAK and the lipid-metabolizing enzymes
PLC
2 and SHIP as four substrates that are tyrosine-phosphorylated
upon stimulation of human platelets with vWF. Tyrosine phosphorylation
and activation of pp72syk induced by vWF has been demonstrated
recently (25). In addition, our data provide the first evidence for a
possible involvement of pp125FAK, PLC
2, and
SHIP in the mechanism of platelet activation by this agonist. Protein
tyrosine phosphorylation induced by vWF was completely blocked by
genistein, which also totally prevented the interaction of Rap1B with
the cytoskeleton and reduced the amount of cytoskeletal-associated Rap2B without affecting platelet aggregation. Once again, we noticed that translocation of Rap1B and Rap2B to the cytoskeleton was differently sensitive to the inhibition of tyrosine kinases. This supports the hypothesis of the existence of two different mechanisms regulating Rap2B interaction with the cytoskeleton. The analysis of the
effect of increased amount of cAMP levels strengthens this conclusion.
In fact, both iloprost and PGE1 totally prevented the
association of Rap1B, but only partially that of Rap2B. The inhibitory
effect of cAMP on the translocation of Rap proteins to the cytoskeleton
is unlikely to be related to the PKA-mediated phosphorylation of Rap1B,
since previous works have demonstrated that both the phosphorylated and
nonphosphorylated form of the protein can interact with the actin
filaments (11). Moreover, we observed a partial inhibition of the
association of Rap2B to the cytoskeleton, even if this protein is not a
substrate for PKA (5). On the other hand, we have found that increased
levels of cAMP prevented vWF-induced protein tyrosine phosphorylation. Therefore, we propose that the inhibition of Rap proteins translocation to the cytoskeleton by incresed levels of cAMP is the consequence of
its inhibitory effects on the activation of tyrosine kinases induced by
vWF. Significantly, the different sensitivity of the translocation of
Rap1B and Rap2B to the cytoskeleton to cAMP-increasing agents is
similar to the effect of genistein. The mechanism of cAMP-mediated
inhibition of protein tyrosine phosphorylation induced by vWF is still
unclear, but the cross-talk between the two types of kinases shown
herein is most interesting. The evidence that iloprost and
PGE1 totally blocked this effect suggests that cAMP interfers with a very early event in the signal transduction pathway initiated by vWF. In this regard, it is interesting to note that one of
the best characterized substrate for the cAMP-dependent kinase is the
-chain of glycoprotein Ib, an essential component of
the vWF receptor on the platelet surface (26).
Signaling by GP Ib-IX-V Is Mediated by Fc
RII--
Although
protein tyrosine phosphorylation in vWF-stimulated platelets has been
reported previously, the mechanism coupling GP Ib-IX-V complex
occupancy to the activation of tyrosine kinases has not been
identified. Recently evidence was advanced for the association of the
14-3-3 signaling protein with the intracellular domain of glycoprotein
Ib and its translocation to the cytoskeleton upon stimulation with vWF
(27, 28). We now provide evidence that signaling initiated by binding
of vWF to GP Ib-IX-V complex is partially mediated by the Fc
RII.
Blockade of the Fc
RII by the monoclonal antibody IV.3 selectively
prevented tyrosine phosphorylation of specific substrates in
vWF-stimulated, but not in thrombin-stimulated, platelets. A previous
work reported the physical proximity of GP Ib-IX-V complex with the
Fc
RII (24). Here it is demonstrated for the first time that Fc
RII
participates in platelet activation induced by vWF. In this respect the
mechanism of action of vWF resembles that of collagen. In this case it
has been shown that tyrosine phosphorylation of some proteins,
including the tyrosine kinase pp72syk, is promoted by binding
of collagen to glycoprotein VI and is mediated by the association with
the Fc receptor
chain (29). Moreover, a direct involvement of the
Fc
RII in collagen-induced platelet activation was suggested by Keely
and Parise (30). It is interesting to note that both collagen and vWF
are multimeric adhesive proteins expressing different binding sites for
specific receptors and are able to promote the formation of
glycoproteins clusters upon binding to the platelet surface. The
inhibition of vWF-induced tyrosine phosphorylation by blockade of the
Fc
RII appeared to be limited to a selected number of substrates,
which include pp72syk, PLC
2, and SHIP, but not
pp125FAK. The exact mechanisms by which Fc
RII
participate in vWF-initiated signaling, as well as the possibility that
Fc
RII itself becomes tyrosine-phosphorylated, deserves further
investigations. Interestingly, we found that blockade of the Fc
RII
by the IV.3 antibody totally inhibited the association of Rap1B to the
cytoskeleton. In light of similar inhibitory effect observed in
genistein-treated platelets, our results suggest that translocation of
Rap1B to the cytoskeleton is mediated by signaling through tyrosine
phosphorylation induced by vWF in a Fc
RII-dependent
mechanism. In this regard, it is interesting to note that the partial
inhibition of the translocation of Rap2B to the cytoskeleton in
genistein-treated platelets closely correlates with that observed in
platelets incubated with the IV.3 antibody.
Conclusion--
We have demonstrated that binding of vWF to the GP
Ib-IX-V complex induced the translocation of both Rap1B and Rap2B to
the platelet cytoskeleton through a GP IIb-IIIa-independent mechanism. The evidence shows a novel role of Fc
RII in regulating both protein tyrosine phosphorylation and translocation of Rap proteins to the
cytoskeleton. These results provide new insights into the mechanisms of
platelet activation by vWF.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Patrizia Noris Istituto di
Ricovero e Cura a Carattere Scientifico (IRCCS), Policlinico San
Matteo, Pavia, Italy) for the AK2 monoclonal antibody, Dr. Christophe
Erneux (Interdisciplinary Research Institute (IRIBHN), Université
Libre de Bruxelles, Brussels, Belgium) and Dr. Bernard Payrastre
(INSERM U326, Toulouse, France) for the SHIP antiserum, and Marco
Bellaviti (Department of Biochemistry, University of Pavia, Italy) for
technical assistance in the preparation of the figures.
 |
FOOTNOTES |
*
This work was supported by grants from the Ministero dell'
Università e della Ricerca Scientifica e Tecnologica (MURST,
Progetti di Ricerca di Interesse Nazionale) and Consiglio Nazionale
delle Ricerche (CNR, Target Project on Biotechnology).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.
§
To whom correspondence should be addressed: Dept. of Biochemistry,
University of Pavia, via Bassi 21, 27100 Pavia, Italy. Tel.:
39-0382-507238; Fax: 39-0382-507240; E-mail: mtorti{at}unipv.it.
 |
ABBREVIATIONS |
The abbreviations used are:
PKA, protein kinase
A;
vWF, von Willebrand factor;
GP, glycoprotein;
Fc
RII, Fc
II
receptor;
PGE1, prostaglandin E1;
PAGE, polyacrylamide gel electrophoresis;
PLC, phospholipase C;
SHIP, SH2
domain-containing inositol 5-phosphatase.
 |
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