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Prepublished online as a Blood First Edition Paper on August 29, 2002; DOI 10.1182/blood-2002-03-0806.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Clinical and Laboratory
Medicine, Faculty of Medicine, University of Yamanashi, Tamaho,
Nakakoma, Yamanashi, Japan, and the Department of
Biochemistry and Molecular Biology, Monash University, Victoria,
Australia.
The binding of von Willebrand factor (VWF) to glycoprotein (GP)
Ib-IX-V stimulates transmembrane signaling events that lead to platelet
adhesion and aggregation. Recent studies have implied that activation
of Src family kinases is involved in GPIb-mediated platelet activation,
although the related signal transduction pathway remains poorly
defined. This study presents evidence for an important role of Src and
GPIb association. In platelet lysates containing Complete, a
broad-spectrum protease inhibitor mixture, Src and Lyn dynamically
associated with GPIb on VWF-botrocetin stimulation. Cytochalasin D,
which inhibits translocation of Src kinases to the cytoskeleton,
further increased Src and GPIb association. Similar results were
obtained with botrocetin and monomeric A1 domain, instead of intact
VWF, with induction of both Src activation and association between GPIb
and Src. These findings suggest that ligand binding of GPIb, without
receptor clustering, is sufficient to activate Src. Immunoprecipitation
studies demonstrated that Src, phosphoinositide 3- kinase (PI
3-kinase), and GPIb form a complex in GPIb-stimulated
platelets. When the p85 subunit of PI 3-kinase was immunodepleted,
association of Src with GPIb was abrogated. However, wortmannin, a
specific PI 3-kinase inhibitor, failed to block complex formation
between Src and GPIb. The Src-SH3 domain as a glutathione S-transferase
(GST)-fusion protein coprecipitated the p85 subunit of PI 3-kinase
and GPIb. These findings taken together suggest that the p85 subunit of
PI 3-kinase mediates GPIb-related activation signals and activates Src
independently of the enzymatic activity of PI 3- kinase.
(Blood. 2003;101:3469-3476) Platelet adhesion to subendothelial structures is
an early and critical event in hemostasis and thrombosis. von
Willebrand factor (VWF) is a major adhesive glycoprotein (GP) required
for normal hemostasis under conditions of high shear stress, such as
those that occur in small arterioles and arterial
capillaries.1 In the presence of high shear stress or
modulators such as botrocetin or ristocetin, VWF binds to the platelet
membrane GPIb-IX-V complex and initiates intracellular signals leading
to platelet activation.2 These include protein tyrosine
phosphorylation, activation of protein kinase C, activation of
phosphoinositide 3-kinase (PI 3-kinase), elevation of the
intracellular calcium ion concentration, and synthesis of thromboxane
A2.3-8 These intracellular signaling events
play an essential role in the reorganization of platelet cytoskeletal
actin filaments leading to platelet adhesion and spreading, and
activation of integrin Recently, the events related to protein-tyrosine phosphorylation have
emerged as important signals mediated by GPIb.3-6,12-14 GPIb-mediated platelet activation in response to VWF plus botrocetin, shear stress, or VWF from patients with von Willebrand disease type
IIb, induces tyrosine phosphorylation of multiple proteins, suggesting
that VWF binding to GPIb causes the activation of tyrosine kinases such
as Syk and Src.5 Coassociated transmembrane proteins, FcR The GPIb-IX-V complex is composed of 4 different polypeptides, GPIb With a number of receptors, especially those for growth factors and
adhesive molecules, clustering of receptors is a prerequisite for
signal transduction; simple binding of ligand is insufficient. It is
widely accepted that VWF, with its multimeric binding sites, induces
clustering of GPIb molecules on the platelet membrane, thereby
eliciting downstream signals. In accord with this notion, neither
dispase-treated VWF nor the monomeric A1 domain of VWF can induce
platelet aggregation.27,28 There are, however, several reports suggesting that simple ligand binding of GPIb can partially elicit some activation events.29,30 In this report, we
therefore also sought to address this issue by using recombinant A1
domain of VWF and cytochalasin D, an inhibitor of actin polymerization.
Materials
VWF and botrocetin were purified as described
previously.5,31 The recombinant A1 domain of VWF linked to
maltose-binding proteins was generously donated by the Ajinomoto
Company (Kawasaki, Kanagawa, Japan).32 The noninhibitory
anti-GPIb MoAb, WGA3, was provided by Dr M. Handa (Keio University,
Tokyo, Japan). Glutathione S-transferase (GST)-fusion proteins
containing the tandem SH2 domains of Syk or the SH3 domain of Src were
obtained from Dr C.-L. Law (University of Washington, Seattle) and Dr
Ashley Dunn (Ludwig Institute, Melbourne, Australia), respectively.
Jararaca GPIb-binding protein was donated by Dr Y. Fujimura (Nara
Medical University, Nara, Japan).
Preparation and stimulation of platelets
Immunoprecipitation and GST-fusion protein precipitation After the indicated time intervals of activation, platelets were solubilized with an equal volume of 2 × ice-cold lysis buffer (100 mM Tris [tris(hydroxymethyl)aminomethane]/HCl, pH 7.4, 5 mM EGTA [ethylene glycol tetraacetic acid], 2 mM PMSF, 2 mM Na3VO4, 100 µg/mL leupeptin, 2% Triton X-100, and 1 tablet of Complete in 25 mL lysis buffer) and kept on ice for 30 minutes. The lysis buffer used in this study contained Complete, a broad-spectrum protease inhibitor mixture, unless otherwise stated. All subsequent steps were performed at 4°C. The lysates were centrifuged at 15 000g for 5 minutes, and the resulting supernatants were precleared by incubation for 30 minutes with protein A-Sepharose for immunoprecipitation experiments, or with glutathione-Sepharose for GST-fusion protein precipitation. For immunoprecipitation, the supernatants were then incubated with the appropriate antibodies, followed by the addition of protein A-Sepharose. For GST-fusion protein precipitation, the supernatants were incubated with the indicated fusion proteins, followed by the addition of glutathione-Sepharose beads. The precipitates obtained after centrifugation were washed 3 times with 1 × lysis buffer before the addition of Laemmli sample buffer.Immunoblotting Precipitated proteins from equal number of platelets were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidine difluoride (PVDF) membranes. The membranes were blocked with 10% BSA in phosphate-buffered saline (PBS). After extensive washing with PBS containing 0.4% Tween-80, the immunoblots were incubated with the appropriate antibodies for 2 hours at room temperature, or overnight at 4°C. Antibody binding was detected using peroxidase-conjugated secondary antibodies diluted at 1:7500, and visualized with enhanced chemiluminescence (ECL) reagent. For reprobing with other antibodies, the antibody bound to PVDF membranes was removed with a stripping buffer (2% SDS, 62.5 mM Tris/HCl, pH 6.8, 100 µM 2-mercaptoethanol) at 50°C for 20 minutes. After washing, the membranes were blocked with 1% BSA and reprobed with the indicated antibodies. Where indicated, the level of proteins detected by immunoblotting was quantitatively analyzed using a PDI420oe scanner (PDI, New York, NY) with Quantity One 2.5a software for Macintosh (PDI).Immunoprecipitation kinase assay The immunoprecipitates were washed 3 times with 1 × lysis buffer, and once with HEPES buffer (10 mM HEPES/NaOH, 1 mM Na3VO4, pH 8.0), followed by further processing for an in vitro kinase assay, according to the method described previously.5 Briefly, the beads were incubated with 50 µL kinase reaction buffer (100 mM HEPES/NaOH, pH 8.0, 5 mM MnCl2, 50 mM MgCl2, and 1 mM Na3VO4) containing 10 µg acid-treated enolase. The reaction was initiated by the addition of 2.0 µM -32P] adenosine triphosphate (ATP; 10 µCi
[0.37 MBq]). After 10 minutes of incubation at 20°C,
reactions were stopped by the addition of Laemmli buffer and then
subjected to boiling for 3 minutes. After Western blotting, the
membrane was treated with 1 M KOH for 60 minutes and then dried. The
radioactivity was quantified with a BAS-2000 phosphorimage analyzer
(Fuji Film, Tokyo, Japan).
Subcellular fractionation of platelets After the indicated time of activation, platelets were lysed with an equal volume of 2 × ice-cold lysis buffer containing Complete and kept on ice for 30 minutes. All subsequent steps were performed at 4°C. The lysates were centrifuged at 15 000g for 5 minutes, and the resulting supernatant (the Triton-soluble fraction) was discarded. After washing, the pellet (the Triton-insoluble fraction) was solubilized in 1 × SDS sample buffer. Alternatively, the pellet was solubilized in RIPA buffer (10 mM Tris/HCl, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 158 mM NaCl, 2.5 mM EGTA, 50 µg/mL leupeptin, 1 mM PMSF, 1 mM Na3VO4, and Complete) for 60 minutes at 4°C. This lysate was centrifuged at 15 000g for 5 minutes. The supernatant (the Triton-insoluble/RIPA-soluble fraction) was collected, and subjected to immunoprecipitation analysis as described in the previous paragraph.Presentation of data Unless stated otherwise, the results shown are from a single experiment representative of at least 3 separate experiments. Results are shown as mean ± SEM of these experiments.
Stimulation-dependent association of Src and Lyn with GPIb in platelets Previous studies have shown that Src and Lyn are specifically involved in GPIb signaling. They have been shown to form a complex with the FcR chain or Syk, as well as to translocate to the
cytoskeleton.5,12 Although we previously reported that a
tyrosine kinase activity associates with GPIb on VWF-GPIb interaction,
the identity of this tyrosine kinase was not determined.5
The failure to identify tyrosine kinases by Western blotting was
attributed to the insensitivity of this method relative to the in vitro
kinase assay. Alternatively, it is possible that tyrosine kinases were
dissociated from GPIb by proteases during the process of cell lysis and
immunoprecipitation. Calpain activation occurs in GPIb-mediated
platelet activation, resulting in the cleavage of multiple signaling
molecules such as Src, PTP1B, FAK, and talin, as well as GPIb
itself.33 To see whether activated calpain affects the
association of tyrosine kinases with GPIb, especially Src family
kinases, we added 50 µg/mL calpeptin, a specific calpain inhibitor,
to the lysis buffer in addition to the protease inhibitors, PMSF and
leupeptin, used in our previous study. There was, however, little
increase in the amount of Src coimmunoprecipitated with GPIb (lanes 1-3 versus 4-6 in Figure 1A). Recently,
however, there has been increasing evidence that proteases other than
calpain, such as matrix metalloproteinases and caspases, are also
present in platelets and can modulate platelet function.34-39 We therefore added Complete, a
broad-spectrum protease inhibitor mixture able to inhibit a wide range
of proteases, to the lysis buffer. As shown in Figure 1A (lanes 7-9), a
significant amount of Src was recovered in GPIb immunoprecipitates of
VWF-stimulated platelets in the lysates containing Complete, but not
with control platelet lysates (lanes 1-3), suggesting that Src
associates with GPIb on GPIb-VWF interaction. These findings suggest
that the interaction between Src and GPIb is more sensitive to
proteolysis than ordinary protein-protein interactions related to
tyrosine phosphorylation. It is likely that the Src-GPIb interaction is not direct, probably mediated by an intermediate signaling molecule. Hereafter, all experiments were performed with lysis buffer containing Complete, unless otherwise stated.
We also found in VWF-stimulated platelets that association of Src with GPIb is a rapid process; it increased as soon as 15 seconds after stimulation and peaked at 1 minute, followed by a decrease at 5 minutes (3.1 ± 0.6 fold [n = 3] increase over the control at a maximum point; Figure 1B). In a pattern similar to Src, Lyn also associated with GPIb, albeit to a lesser degree (Figure 1B). In contrast to these 2 tyrosine kinases, Fyn and other members of the Src kinase family were not detected (Figure 1B; data not shown). When platelets were pretreated with jararaca GPIb-binding protein (GPIb-BP), which competitively inhibits VWF binding to GPIb,40 Src and Lyn association with GPIb were both blocked (Figure 1B), suggesting that the increased level of Src and Lyn association with GPIb is specific for GPIb-mediated platelet activation. Consistent with the increased level of GPIb-associated Src, the inclusion of Complete in the lysis buffer also allowed us to observe in immunoprecipitation kinase assays the pronounced autophosphorylation of a 60-kDa protein associated with GPIb, as well as the included enolase substrate (Figure 1C), implying that Src is the main candidate responsible for GPIb-associated tyrosine kinase activity. GPIb stimulates redistribution of Src to the cytoskeleton, with resultant loss of its activity in the Triton-soluble fraction We have found in this study that GPIb stimulates association of Src with GPIb. However, we were not able to detect an increase in Src kinase activity in anti-Src immunoprecipitates in the Triton-soluble fraction5 (Figure 2A). It has been well documented that members of the Src family kinases are redistributed to the cytoskeleton during platelet activation.41-43 Yuan and colleagues and we have found that GPIb stimulation induces translocation of Src as well as Lyn to the cytoskeleton as an early event during platelet activation.5,32 Thus, it is possible that GPIb associated with Src translocates to the reorganized cytoskeleton thereby depleting Src in the Triton-soluble fraction. However, although we attempted to measure Src kinase activity in the RIPA-extracted actin cytoskeletal fraction, an increase in Src activity was not detectable. It is likely that the presence of SDS in the RIPA buffer interfered with the enzyme activity of Src. We therefore sought to recover Src in the Triton-soluble fraction by preventing its translocation with cytochalasin D, which binds to the growing ends of actin filaments and inhibits actin polymerization by blocking the further addition of monomeric actin molecules. As shown in Figure 2B, pretreatment of platelets with 1 µM cytochalasin D indeed reduced cytoskeletal translocation of Src and GPIb mediated by VWF plus botrocetin stimulation to approximately 22% ± 5% (n = 3) and 24% ± 6% (n = 3) of control, respectively. The amounts of GPIb or Src in Triton-soluble fractions were little affected by cytochalasin D treatment (Figure 2C). Furthermore, the inhibition by cytochalasin D of cytoskeletal translocation of Src or GPIb allowed us to observe an increase in autophosphorylation of Src and its kinase activity in the Triton-soluble fraction, as early as 15 seconds after stimulation (Figure 2A).
Ligand binding of GPIb is sufficient to activate Src, but its clustering is necessary for downstream signaling It has been widely held that GPIb clustering with multimeric VWF is a prerequisite for full activation of platelets. Our finding that Src is activated by GPIb stimulation in cytochalasin D-pretreated cells in which receptor clustering would be predicted to be blocked disagress with this notion and suggests that at least part of GPIb-mediated signal transduction does not require GPIb clustering. The recombinant A1 domain of VWF is monomeric.32 Although it can interact with GPIb in the presence of botrocetin, it fails to engage multiple GPIb receptors and to induce aggregation or actin polymerization as well as subsequent cytoskeletal relocation of Src and Lyn.30 When platelets were stimulated with 6 µg/mL botrocetin plus 20 µg/mL A1 domain, Src activation, as detected by its autophosphorylation and enolase phosphorylation, gradually increased in a time-dependent manner (Figure 3A). Densitometric analysis of enolase phosphorylation revealed that Src activity was increased 8.3-fold 5 minutes after stimulation (Figure 3B). In contrast, we did not detect a significant change in the kinase activity of Lyn, at least over this time course (Figure 3A-B). A1 domain interaction with GPIb also induced Src association with GPIb (Figure 3C). These results suggest that, without receptor clustering, occupancy of GPIb with the A1 domain of VWF is sufficient to stimulate Src association with GPIb and to activate Src.
The physical association of Src with GPIb as well as its activation
indicates that Src is the most likely candidate responsible for
GPIb-related protein tyrosine phosphorylation events. However, despite
Src activation, A1 domain of VWF interaction with GPIb failed to
mediate downstream signaling events, such as tyrosine phosphorylation
of FcR
Interaction of VWF with GPIb stimulates complex formation between Src, PI 3-kinase, and GPIb Although we found that Src associates with GPIb on stimulation, the effect of protease inhibitors implies that this association is probably indirect. Furthermore, the GPIb complex lacks motifs to which Src can bind. Recently, Munday et al15 reported that PI 3-kinase associates with GPIb in platelets. We therefore sought to determine whether Src, PI 3-kinase, and GPIb form a complex following GPIb stimulation. PI 3-kinase is a heterodimeric phospholipid kinase composed of a regulatory subunit, p85, and a catalytic subunit, p110.24,44 It is known that the p85 subunit coassociates with p110 at a ratio of 1:1 in platelets.45 Thus, the heterodimer of p85 and p110 PI 3-kinase can be obtained by immunoprecipitation with an anti-p85 antibody. GPIb and PI 3-kinase immunoprecipitates were analyzed separately by Western blotting with anti-Src and anti-p85, or anti-Src and anti-GPIb antibodies. As shown in Figure 5A-B, the association between p85 and GPIb decreased on VWF stimulation, which is in agreement with the report of Munday et al.15 However, the association between GPIb and Src, and that of PI 3-kinase and Src, increased as early as 15 seconds after stimulation with VWF plus botrocetin. These associations peaked at 1 minute followed by a decrease at 5 minutes (Figure 5A-B). Taking into account that VWF-GPIb interaction stimulates GPIb translocation to the cytoskeleton,46 it is possible that the decreased association between GPIb and PI 3-kinase is due to their relocation in a complex form to the cytoskeleton.15 To test this possibility, the reorganization of the actin-based cytoskeleton was prevented by pretreatment with 1 µM cytochalasin D. As shown in Figure 5A-B, neither the recovery of PI 3-kinase in GPIb immunoprecipitates nor that of GPIb in p85 immunoprecipitates was altered on VWF stimulation in the presence of cytochalasin D, implying that GPIb constitutively associates with PI 3-kinase, and that the level of their association is maintained throughout platelet activation. On the other hand, cytochalasin D pretreatment augmented the level of association between Src and GPIb and that between Src and PI 3-kinase (Figure 5A-B), suggesting that Src translocates to the cytoskeleton in a complex form with GPIb and PI 3-kinase. Consistent with this conclusion, Src and GPIb were coprecipitated with anti-p85 antibody from RIPA extracts of the cytoskeleton of VWF-botrocetin-activated platelets, suggesting that Src, PI 3-kinase, and GPIb indeed form a complex after GPIb stimulation (Figure 5C). Taken together, these results suggest that PI 3-kinase constitutively associates with GPIb, and that GPIb-mediated platelet activation does not change the level of PI 3-kinase interaction with GPIb. Src, however, associates with GPIb-PI 3-kinase to form a complex in a stimulation-dependent manner, which is upstream of actin polymerization.
Involvement of PI 3-kinase in GPIb-mediated platelet activation is not dependent on its enzyme activity PI 3-kinase plays a central role in the regulation of multiple cellular events.44,47 Many of these effects are mediated by the production of D-3 lipids that act to recruit various proteins to cell membranes. The products of PI 3-kinase also regulate the activity of a number of tyrosine kinases.16,19,24 Several lines of evidence indicate that PI 3-kinase is activated by GPIb-VWF interaction.7,15 We also found in this study that Src associates with the PI 3-kinase-GPIb complex on platelet activation. We therefore asked whether the enzyme activity of PI 3-kinase is required for GPIb-mediated Src activation. Wortmannin is known to inactivate the catalytic p110 subunit of PI 3-kinase by covalently modifying it at Lys802, a residue involved in phosphate transfer by this enzyme.48 Pretreatment of platelets with 100 nM wortmannin did not affect Src association with GPIb after GPIb-VWF interaction (Figure 6A). Additionally, it had no inhibitory effect on tyrosine phosphorylation of the FcR
chain, Syk, and PLC2 stimulated by VWF plus botrocetin
(Figure 6B). A structurally unrelated PI 3-kinase inhibitor, LY
294002, also failed to inhibit tyrosine phosphorylation of FcR
chain, Syk, and PLC2, induced by GPIb-VWF interaction (data not shown). These data suggest that the functional role of
PI 3-kinase in Src activation and the resultant downstream signaling
and platelet aggregation induced by GPIb-VWF interaction are
independent of its catalytic activity.
Association of Src with GPIb is mediated by p85/PI 3-kinase The subunits of the GPIb complex, as well as its associated proteins, ABP-280 and 14-3-3 , all lack potential tyrosine
phosphorylation sites and other related binding structures such as SH3
domains, proline-rich motifs, and SH2 domains. Thus, p85, which has 2 proline-rich motifs, is a good candidate to serve as a docking protein
for Src. To test this hypothesis, we performed an immunodepletion experiment of p85/PI 3-kinase, as shown in Figure
7A-B. After platelets pretreated with
cytochalasin D were activated, the platelets were lysed and then 2 rounds of immunoprecipitation were performed with anti-p85/PI3-kinase
antibody. p85 was completely depleted in the whole cell lysates (Figure
7A lane 3), and p85 could be no longer be detected in GPIb
immunoprecipitates (Figure 7B lane 3 of blot for p85). Depletion of p85
completely eliminated Src recovery in GPIb immunoprecipitates (Figure
7B lane 3 of blot for Src). There are no major changes in the amount of
GPIb in studies between immunoprecipitation and immunodepletion (Figure 7B lane 3 of blot for GPIb). These findings suggest that Src
association with GPIb is mediated by PI 3-kinase, rather than GPIb,
and that only a small population of GPIb is included in forming the
GPIb-PI3-kinase/Src complex. We next used a GST-fusion protein of the
Src SH3 domain to see whether Src and p85/PI 3-kinase association is
mediated by the binding between the Src SH3 domain and p85/PI
3-kinase. As shown in Figure 7C, GPIb and p85 were detected to some
extent in GST-Src-SH3 precipitates in the resting state, and their
recovery increased on stimulation at 15 seconds. It was then followed
by a decrease at 1 minute and a complete loss at 5 minutes. This phenomenon is compatible with the notion that on stimulation Src transiently associates with PI 3-kinase linked to GPIb and that they
redistribute as a complex to the Triton-insoluble cytoskeleton (Figure
5). These findings further suggest that PI 3-kinase functions as an
adaptor protein to recruit Src to GPIb, independent of its catalytic activity.
The findings presented in this study demonstrate for the first time that Src kinases physically associate with GPIb and that they are activated by the interaction between GPIb and VWF. In a previous report, we found that an unidentified tyrosine kinase activity coprecipitated with GPIb on VWF-GPIb interaction.5 In this study, we modified the lysis buffer to include Complete, a mixture of protease inhibitors with activity toward a wide range of proteases. With the use of this new lysis buffer, we found that Src and Lyn associate with GPIb on VWF-GPIb interaction. This relative lability of the association between Src family kinases and GPIb implies that the mode of their interaction is probably indirect. We have found that a large amount of active Src was associated with GPIb after VWF-GPIb interaction, suggesting that interaction of VWF with GPIb may first stimulate Src association with GPIb, and consequently activate it. Alternatively, active Src may preferentially link to GPIb. This issue remains to be resolved. On the other hand, even with the use of Complete, we still failed to
detect an increase in Src activity in the Triton-soluble fraction of
VWF-stimulated platelets. Previous studies have shown that clustering
of GPIb receptors by multimeric VWF induces actin polymerization10 and stimulates Src and Lyn translocation
to the reorganized cytoskeleton as an early event in platelet
activation.5,32 In this study, we found that translocation
of Src kinases was inhibited by pretreatment with cytochalasin D. This
procedure allowed us to observe an increase in Src autophosphorylation
and its kinase activity. Thus, the rapid redistribution of Src kinases appears to have resulted in an underestimate of the increased activity
of Src in the Triton-soluble fraction. These findings also imply that
GPIb-stimulated Src activation is not dependent on receptor clustering.
This conclusion is at odds with the widely held notion that GPIb
clustering is a prerequisite for its downstream signaling. To address
this issue, we used recombinant A1 domain instead of intact VWF to
stimulate platelets, because the monomeric A1 domain would not be
expected to cluster GPIb with subsequent reorganization of actin
filaments. With platelets stimulated by A1 domain plus botrocetin, Src
rather than Lyn is selectively activated in a time-dependent manner.
Moreover, A1 domain plus botrocetin also stimulates Src association
with GPIb, demonstrating that binding of the A1 domain alone to GPIb is
indeed sufficient to activate Src. To the best of our knowledge, this
is the first report showing that the interaction between GPIb and VWF
specifically activates Src without receptor clustering. Given that PP1,
a specific inhibitor of Src family kinases, completely blocks
tyrosine phosphorylation of Syk, LAT, PLC The VWF multimer is a major physiologic ligand for GPIb. Its
multivalency is believed to induce GPIb clustering on VWF-GPIb interaction, and it is widely held that GPIb clustering is a
prerequisite for platelet activation. In this study, we found that GPIb
ligand binding without receptor clustering, as induced by A1 domain
plus botrocetin, is sufficient to initiate intracellular signaling such
as Src association with GPIb as well as Src activation. However, it was
insufficient to induce tyrosine phosphorylation of the FcR The constituents of the GPIb-IX-V complex lack tyrosine-phosphorylated residues and other special binding motifs such as proline-rich domains and SH2 domains, which would allow the binding of Src to GPIb. Thus, it is likely that the association of Src with GPIb is not direct, but is mediated by another signaling molecule. This is supported by the finding that recovery of Src in GPIb immunoprecipitates was only made possible by use of a lysis buffer containing Complete, although the target proteases inhibited by Complete in this setting remain unknown. A potential role for PI 3-kinase in GPIb signaling has been suggested by the observation that VWF binding to GPIb can induce the cytoskeletal association and activation of the p85/p110 form of PI 3-kinase.7 More interestingly, PI 3-kinase has been shown to associate with GPIb.15 In this study, we detected a complex of Src, PI 3-kinase, and GPIb in lysates of platelets activated by VWF and botrocetin, suggesting that PI 3-kinase links Src with GPIb. On GPIb stimulation, the complex of GPIb, PI 3-kinase, and Src appears to translocate to the cytoskeleton, a conclusion supported by the observation that Src and GPIb were recovered in p85 immunoprecipitates of RIPA extracts of cytoskeletal proteins. We then asked what could be the mode of interaction between GPIb, PI 3-kinase, and Src. On GPIb stimulation, PI 3-kinase recovered in GPIb immunoprecipitates decreased in amount. Correspondingly, GPIb recovered in PI 3-kinase immunoprecipitates was also reduced, suggesting that these 2 molecules translocate to the cytoskeleton in a complex. When their translocation to the cytoskeleton was blocked by cytochalasin D pretreatment, the level of their association remained constant, irrespective of stimulation, which implies that GPIb and PI 3-kinase constitutively associate and that their association remains constant even after platelet activation. On the other hand, Src association with GPIb or PI 3-kinase is dynamically dependent on GPIb stimulation, especially in platelets pretreated with cytochalasin D. These findings taken together suggest that Src is recruited to an already-existing complex of GPIb and PI 3-kinase on platelet activation. It is of note that the overall recovery of GPIb in anti-GPIb
immunoprecipitates was virtually the same from control and PI 3-kinase-depleted platelet lysates (Figure 7B), suggesting that only
a small portion of the GPIb molecules participate in the formation of
the GPIb/PI 3-kinase/Src complex. It is possible that the rest of GPIb
molecules are linked to signaling molecules other than PI 3-kinase and
Src, and that the GPIb/PI 3-kinase/Src signaling constitutes only one
of several signaling pathways involved in GPIb-related platelet
activation. It is also possible that only a portion of the GPIb
molecules are recruited to form a complex with Src/PI 3-kinase at a
given moment, taking into consideration the fast on and off rate of the
GPIb-VWF interaction. Alternatively, the limited association of GPIb
and PI 3-kinase may simply reflect their equilibrium redistribution as
a consequence of PI 3-kinase immunodepletion of the platelet lysate
and the relative avidity of their association. Although a small portion
of the GPIb molecules appear to participate in the signaling pathway
related to PI 3-kinase and Src, we suggest that signaling through
Src/Fc We then sought to determine which molecule, GPIb or PI 3-kinase, directly binds Src. Using an immunodepletion method with anti-p85/PI 3-kinase antibody, we found that PI 3-kinase and Src always coexist in GPIb immunoprecipitates, which suggests that Src binds to PI 3-kinase, but not to GPIb. PI 3-kinase plays a central role in the regulation of multiple
cellular events, such as cell growth, vesicular trafficking, cytoskeletal organization, proliferation, and
apoptosis.44,47 In particular, a number of tyrosine
kinases appear to be regulated by PI 3-kinase via its production of
D-3 lipids that act to recruit proteins to cell membranes. In this
study, wortmannin had no effects on GPIb-mediated Src activation, the
formation of the GPIb/Src/PI 3-kinase complex, or on tyrosine
phosphorylation of the FcR In conclusion, our study demonstrates for the first time that VWF-GPIb interaction stimulates Src association with GPIb and Src activation. The GPIb-associated p85 subunit of PI 3-kinase functions as a scaffolding protein, recruiting Src to GPIb, thereby leading to its activation. The physical interaction of Src with GPIb and its early activation imply that Src activation constitutes the paramount pathway for GPIb-related tyrosine phosphorylation events.
We are grateful to Prof Y. Fujimura for kindly supplying jararaca-GPIb-binding protein, Dr M. Handa for the generous donation of anti-GPIb MoAb, WGA3, Dr C.-L. Law for the kind gift of GST-Syk-SH2, and Dr A. Dunn for provision of the GST-Src-SH3 construct.
Submitted March 15, 2002; accepted July 25, 2002.
Prepublished online as Blood First Edition Paper, August 29, 2002; DOI 10.1182/blood-2002-03-0806.
Y.W. and N.A. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Yukio Ozaki, Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, 1110 Shimokatoh, Tamaho, Nakakoma, Yamanashi 409-3898, Japan; e-mail: yozaki{at}res.yamanashi-med.ac.jp.
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Glycoprotein Ib-von Willebrand factor interactions activate tyrosine kinases in human platelets.
Blood.
1997;90:4789-4798 6. Canobbio I, Bertoni A, Lova P, et al. Platelet activation by von Willebrand factor requires coordinated signaling through thromboxane A2 and Fc | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Abstract
Introduction
IIb
3, with
resultant platelet aggregation.
) and anti-Lyn (
)
immunoprecipitates shown in panel A was quantified by densitometry, and
represented as a fold-increase over the control (0 seconds). The data
are expressed as mean ± SEM of 3 separate experiments. (C) The
samples were prepared as described in panel A and the subsequent
lysates immunoprecipitated with anti-GPIb MoAb. The immunoprecipitates
were separated by SDS-PAGE and analyzed by immunoblotting with anti-Src
and anti-GPIb MoAbs, respectively.
