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J. Biol. Chem., Vol. 278, Issue 35, 32880-32891, August 29, 2003
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Signaling Role for Phospholipase C
2 in Platelet Glycoprotein Ib
Calcium Flux and Cytoskeletal Reorganization
INVOLVEMENT OF A PATHWAY DISTINCT FROM FcR
CHAIN AND FcRIIA*
¶ || **
From the
INSERM U.311, Etablissement
Français du Sang-Alsace, 10 rue Spielmann, BP 36, 67065 Strasbourg
cedex, France and the ||Department of Medicine,
Australian Centre for Blood Diseases, Monash University, Victoria 3127,
Australia
Received for publication, March 6, 2003 , and in revised form, June 12, 2003.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Interaction of the platelet GPIb-V-IX complex with surface immobilized von Willebrand factor (vWf) is required for the capture of circulating platelets and their ensuing activation. In previous work, it was found that GPIb/vWf-mediated platelet adhesion triggers Ca2+ release from intracellular stores, leading to cytoskeletal reorganization and filopodia extension. Despite the potential functional importance of GPIb-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for phospholipase C (PLC)-dependent phosphoinositide turnover for GPIb-dependent cytoskeletal remodeling. This is supported by the findings that the vWf-GPIb interaction induced a small increase in inositol 1,4,5-triphosphate (IP3) and that treating platelets with the IP3 receptor antagonist APB-2 or the PLC inhibitor U73122 [GenBank] blocked cytosolic Ca2+ flux and platelet shape change. Normal shape change was observed in G
q–/– mouse
platelets, excluding a role for PLC
isoforms in this process. However,
decreased shape change and Ca2+ mobilization were
observed in mice lacking PLC2, demonstrating that this isotype played
an important, albeit incomplete, role in GPIb signaling. The signaling
pathways utilized by GPIb involved one or more members of the Src kinase
family as platelet shape change and Ca2+ flux were
inhibited by the Src kinase inhibitors PP1 and PP2. Strikingly, shape change
and Ca2+ release occurred independently of
immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors,
because these platelet responses were normal in human platelets treated with
the anti-FcRIIA blocking monoclonal antibody IV.3 and in mouse
platelets deficient in the FcR chain. Taken together, these studies
define an important role for PLC2 in GPIb signaling linked to platelet
shape change. Moreover, they demonstrate that GPIb-dependent calcium flux and
cytoskeletal reorganization involves a signaling pathway distinct from that
utilized by ITAM-containing receptors. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Platelets are specialized blood cells that display unique adhesive properties relevant to hemostasis and thrombosis. Platelet adhesion is critically dependent on the synergistic interaction between multiple platelet receptors and adhesive ligands, with the involvement of specific receptor-ligand pairs dependent on the prevailing blood flow conditions (1). Under conditions of high shear stress, von Willebrand factor (vWf)1 binding to the platelet adhesive receptor, glycoprotein (GP) Ib/V/IX is critical for the initiation of platelet-vessel wall and platelet-platelet interactions and as such is a key adhesive event promoting thrombus growth (2). In addition to its adhesive function, the GPIb-V-IX complex also plays an important role in regulating the organization of the platelet cytoskeleton (3). For example, it has long been recognized from the study of platelets deficient in GPIb-V-IX (Bernard-Soulier syndrome) that this complex is important for maintaining the compact structure of the membrane skeleton, due at least in part, to the physical linkage between GPIb and actin-binding protein (ABP) 280 (ABP-280 or filamin-1) (4). More recently it has been demonstrated that vWf engagement of GPIb can induce cytoskeletal reorganization (5) and that these cytoskeletal changes play a potentially important role in regulating the adhesive function of GPIb (6). The physiological importance of vWf in inducing cytoskeletal reorganization has previously been highlighted from the study of von Willebrand disease pigs (7). Platelets from these animals adhere to sites of vascular injury; however, they exhibit defective cytoskeletal remodeling leading to reduced filopodial extension and poor spreading.
Despite the potential importance of GPIb in initiating platelet activation,
the mechanisms by which GPIb transduces signals linked to cytoskeletal
remodeling remains incompletely understood. We have previously demonstrated a
potentially important role for intracellular Ca2+
mobilization in this process
(5); however, to date, the
proximal signaling molecules utilized by GPIb-V-IX to induce cytosolic
Ca2+ flux have not been identified. The GPIb-V-IX
receptor consists of four protein subunits belonging to the leucine-rich
repeat superfamily, GPIb, GPIb, GPIX, and GPV
(8). The receptor intracellular
region does not have catalytic activity, nor does it bind GTP-binding proteins
or become phosphorylated by tyrosine kinases. Previous studies have suggested
that GPIb may signal directly as a consequence of its association with
the cytoskeletal structural protein, actin-binding protein (ABP-280)
(3), and/or signaling molecules
such as 14-3-3
(9–11),
calmodulin (12), Src kinase
(13), and phosphoinositide
(PI) 3-kinase (14).
Alternatively, GPIb may transduce signals indirectly through the physical
association with the ITAM-bearing receptors, FcR chain
(15,
16), or FcRIIA
(17,
18). FcR and
FcRIIA receptor signaling is initiated by Src kinase-dependent tyrosine
phosphorylation of the receptors' ITAM motif, leading to the recruitment of
p72syk, PI 3-kinase, and the adaptor proteins (LAT,
SLP-76, and vav), which ultimately promote the activation of phospholipase
C2 (PLC2). The subsequent hydrolysis of phosphatidylinositol
4,5-diphosphate, leading to IP3 generation and intracellular
calcium release is a critical event for efficient platelet activation
(19,
20).
A consensus model for GPIb signaling has yet to emerge, despite intense
investigation from a number of groups. In fact, there remains considerable
controversy over a number of basic aspects with regards to GPIb signaling,
including the contribution of direct and indirect pathways for platelet
activation. One possible reason for the discrepancies in results from
different studies reflects the variability in experimental approaches used to
examine this process. For example, a wide variety of ligands (venom peptides,
soluble human or bovine vWf, recombinant vWf fragments, and immobilized vWf),
artificial modulators (ristocetin and botrocetin), cell types (human and mouse
platelets, GPIb-V-IX-transfected CHO and K562 cell lines), and functional
assays (suspension-based aggregation studies, shear-induced platelet
aggregation, and static or flow-based adhesion assays) have been utilized to
examine GPIb signaling. In many of these studies it is unclear what the direct
contribution of the cytoplasmic tails of the GPIb-V-IX complex are for signal
generation relative to indirect signaling mediated through the FcR
chain and FcRIIA receptor, soluble agonist release, and integrin
IIb3 outside-in signaling. Furthermore, it
is not always clear that the end-point used to examine GPIb signaling is in
fact a direct functional response linked to this receptor.
We have previously established that the vWf-GPIb interaction per
se is sufficient to induce cytosolic calcium spikes that are necessary
for platelet cytoskeletal remodeling, independent of integrin
IIb3
(5). In the current study we
have utilized platelet shape change and cytosolic calcium transients as
bona fide GPIb-dependent responses to examine the proximal signaling
events linked to this receptor. Through the use of various pharmacological
inhibitors and knock-out mouse models linked to PLC signaling, our studies
have demonstrated an important role for Src kinase-dependent activation of the
PLC2 isoform in GPIb-dependent calcium flux and cytoskeletal
remodeling. In contrast to many other studies, our results do not support an
important role for the FcR chain and FcRIIA receptors in GPIb
signaling.
|
EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Materials—2-Aminoethoxydiphenyl borate (2-APB), prostaglandin I2, bovine serum albumin, TRITC-phalloidin, fatty acid-free human serum albumin, bovine type I collagen, and serotonin were purchased from Sigma Chemical Co. (St. Louis, MO). U73122 [GenBank] and U73343 [GenBank] were from Calbiochem (San Diego, CA), PP1 and PP2 were from BIOMOL (Plymouth, PA). Ristocetin was obtained from ICN (Costa Mesa, CA), and botrocetin was a kind gift from Prof. Michael Berndt (The Baker Institute, Melbourne, Australia). 1-Paraformaldehyde was purchased from Electron Microscopy Sciences (Washington, PA), and glass coverslips were from Polylabo (Strasbourg, France). Anti-
IIb3 chimeric Fab fragment of
monoclonal antibody (mAb) 7E3 (c7E3 Fab-abciximab) was from Eli-Lilly
(Centocor, Leiden, Netherlands). The anti-FcRIIA mAb, IV.3, was kindly
donated by Dr. Ben Chong (Royal Prince of Wales Hospital, New South Wales,
Australia), the anti-phosphotyrosine mAb PY 20 was purchased from BD
Biosciences Transduction Laboratories (Lexington, KY), and the
anti-PLC2 was from Santa Cruz Biotechnologies (Santa Cruz, CA). Human
vWf (HvWf) was purified from plasma cryoprecipitate according to the method of
Montgomery and Zimmerman (21),
and human fibrinogen was from Kabi (Stockholm, Sweden). Apyrase was purified
from potatoes as previously described
(22).
Mouse Strains—C57BL/6 Gq-deficient
(Gq–/–) and wild type (Gq+/+)
mouse colonies were established at the animal facilities of the Etablissement
Français du Sang-Alsace by breeding the Gq
heterozygotes (Gq+/–) provided by Prof. Stephan
Offermanns (Universität Heidelberg, Germany)
(23). C57BL/6 FcR
chain-deficient mice (FcR–/–)
(24) were obtained from
Taconic (Germantown, NY). C57BL/6 PLC2-deficient mice
(PLC2–/–) were provided by Prof. J. Ihle (St. Jude
Children's Research Hospital, Memphis, TN)
(25).
Cell Line—Chinese hamster ovary (CHO) cells, expressing the GPIb-IX complex, were established as described previously (26).
Platelet Preparation—Blood was drawn from healthy volunteers who had not taken an anti-platelet medication in preceding 2 weeks and collected in an acid citrate dextrose anticoagulant. Platelets were isolated by sequential centrifugation of the blood and washed as previously described (22). Platelets were finally resuspended in Tyrode's buffer (12 mM NaHCO3, 0.3 mM NaH2PO4, 5 mM Hepes, pH 7.3, 137 mM NaCl, 2 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 5.5 mM glucose) containing human serum albumin and apyrase (0.02 unit/ml) and kept at 37 °C. Aspirinated platelets were obtained by treating cells with 1 mM aspirin for 15 min, prior to final suspension in Tyrode's buffer. Washed mouse platelets were prepared according to the method described by Moog et al. (27). Mouse blood was taken from the abdominal aorta of anesthetized animals and collected in an acid citrate dextrose anticoagulant.
Platelet Aggregation Studies—Aggregation was performed using
a dual-channel Payton aggregometer (Payton Associates, Scarborough, Ontario,
Canada). Platelets (3 x 108 platelets/ml) were stimulated at
37 °C by collagen (1.25 µg/ml) in the presence of human fibrinogen (275
µg/ml), in a final volume of 500 µl. Aggregation was initiated by
stirring the platelet mixture at 1100 rpm. When vWf-induced platelet
agglutination was examined, aspirinated platelets (3 x 108
platelets/ml) were pre-treated with cFab 7E3 (20 µg/ml) for 10 min or with
EDTA (5 mM), then stirred in the presence of human vWf (20
µg/ml) and ristocetin (1 mg/ml). In control studies, the pharmacological
activity of the IP3 receptor inhibitor, 2-APB, was confirmed by
complete inhibition of thrombin (1 unit/ml)-induced platelet aggregation at 50
µM. The ability of IV.3 (5 µg/ml) to block FcRIIA
function was confirmed by inhibition of heat-aggregated immunoglobulins (800
µg/ml)-induced platelet aggregations.
Cell Adhesion and Morphology Analysis—Human platelet
adhesion studies were performed as previously described
(5). Briefly, platelets (2
x 107/ml) in Tyrode's buffer were treated with c7E3 Fab (20
µg/ml), then allowed to adhere to a HvWf (10 µg/ml) matrix for 30 min at
37 °C, in the presence of 2 units apyrase. Where indicated, platelets were
also preincubated for 10 min with the inhibitors of phospholipase C (U73122
[GenBank]
(10 µM)), IP3 receptor (2-APB (20 µM)),
Src kinases (PP1 (10 µM) and PP2 (10 µM)), and
FcRIIA blocking Ab IV.3 (5 µg/ml), prior to application to vWf
matrices. In some studies, the treated platelets were then allowed to adhere
to vWf in the presence of Ca2+ ionophore A23187
[GenBank]
(40
nM) to induce extracellular Ca2+ influx.
Non-adherent cells were removed, and adherent cells were fixed with 3.7%
formaldehyde for 20 min. In control studies, platelets were fixed in
suspension then applied onto a vWf matrix. To examine the cell morphology, the
coverslips were mounted onto glass slides with Mowiol 4-88 solution (France
Biochem, Meudon, France), and platelets subjected to differential interference
contrast microscopy (Leica TCSNT) (63x objective). Alternatively, the
fixed adherent platelets were stained with TRITC-phalloidin (2 µg/ml) for
30 min and then subjected to fluorescence microscopy (UV illumination at 570
nm, Leica DMDL microscope) (63x objective). The number of adherent cells
was scored in five to eight random fields. Platelet shape change was defined
by the transformation of platelet morphology from discoid with no filopodial
projection to spherical with filopodial projections greater than 0.2 µm in
length.
When adhesion assays were performed using mouse platelets (3 x 107/ml) or CHO cells (1 x 106/ml), identical experimental conditions were used except that botrocetin (2 µg/ml) was present to support cell adhesion to HvWf, and the integrin-vWf interaction was prevented by the addition of EDTA (5 mM) or c7E3 Fab (20 µg/ml), respectively.
Scanning Electron Microscopy—Adherent platelets were fixed for 45 min with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.3, 305 mosM/kg) containing 2% sucrose. The fixed cells were washed three times in 0.9% saline and dehydrated sequentially in increasing concentrations of ethanol solutions. Samples were air-dried with 1,1,1,3,3,3-hexamethyldisilazan, sputtered with gold, and examined under an Hitachi S-800 scanning electron microscope (Hitachi, Tokyo, Japan) (5 kV) (28).
Immunoprecipitation and Western Blotting Studies—Washed
platelets (2 x 108/ml) were treated with c7E3 Fab (20
µg/ml) and allowed to adhere to immobilized HvWf (10 µg/ml) for 20 min
at 37 °C. Where indicated, platelets were also pretreated with the
inhibitor of Src family kinases, PP2 (5 µM), prior to adhesion.
The adherent platelets were lysed in a radioimmunoprecipitation assay buffer
(10 mM Tris, pH 7.4, 1% Triton X-100, 1% sodium deoxycholate, 0.1%
SDS, 158 mM NaCl, 2 mM EDTA, 1 mM
Na3VO4, 1 mM phenylmethylsulfonyl fluoride,
and 2 mM benzamidine), scrapped off, and centrifuged at 15,000
x g for 5 min. The supernatant was incubated for 2 h at 4
°C with an anti-PLC2 polyclonal Ab (4 µg) in the
presence of 30 µl of 50% protein A-Sepharose beads. The beads were washed
and subjected to 7.5% SDS-PAGE under a reducing condition, immunoblotted with
an anti-phosphotyrosine mAb PY-20 or an anti-PLC2 Ab, and
developed with Enhanced Chemiluminescence (Dupont) as previously described
(29).
Quantitation of IP3 Levels—Washed platelets (2 x 109/ml) were treated with cFab 7E3 (20 µg/ml) for 10 min, then aggregated with vWf (20 µg/ml) in the presence of ristocetin (1 mg/ml) for the indicated period of time. Platelets were then lysed, and IP3 in the total cell lysates quantitated using a commercial IP3 assay kit (Amersham Biosciences, UK) according to the manufacturer's instruction. The IP3 levels were determined using a standard curve established using known amounts of IP3.
Analysis of Cytosolic Ca2+
Flux—The platelet intracellular Ca2+
changes were monitored according to the previously detailed method
(30). Washed human platelets
were loaded with two membrane-permeable Ca2+ indicator
dyes, Oregon Green 488 BAPTA-AM-1 (1.25 µM) and Fura Red AM (1
µM), for 20 min at 37 °C. The Ca2+
dye-loaded platelets were treated with c7E3 Fab (20 µg/ml) and then allowed
to adhere to a vWf (10 µM) matrix in the presence of ristocetin
(1 mg/ml), under static conditions. The changes in fluorescence ratio of
Oregon Green to Fura Red was then measured after 10 min of adhesion using
confocal microscopy (1 frame/s) and converted to intracellular
Ca2+ concentrations as previously described
(31). The
Ca2+ concentrations in resting platelets were also
determined by measuring the fluorescence ratio (mean ratio = 0.6, n =
390) in platelets applied onto a non-reactive (10% human serum-coated)
surface. A transient Ca2+ signal was defined as a change
in fluorescence ratio greater than two standard deviations (2
= 0.38,
n = 390) above the mean fluorescence ratio (the mean fluorescence
ratio in resting platelets). Where indicated, platelets were preincubated with
the inhibitors of PLC, U73122
[GenBank]
(1 µM); of IP3
receptor, 2-APB (20 µM); of Src kinases, PP2 (5
µM); or of FcRIIA, IV.3 mAb (5 µg/ml), prior to
adhesion to vWf.
When these studies were performed on mouse platelets, adhesion assays were
carried out in the presence of botrocetin (1 µg/ml). EDTA (1 mM)
was also present to prevent the integrin
IIb3-vWf interaction and extracellular
Ca2+ influx.
Statistical Analysis—The statistical significance of differences between means was evaluated using Student's t test for paired samples, and p values of less than 0.05 were considered to be significant.
|
RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The vWf-GPIb Interaction Induces IP3 Formation—We have previously demonstrated that vWf engagement of GPIb induces transient Ca2+ spikes that initiate cytoskeletal remodeling (5). In platelets, release of Ca2+ from intracellular stores is primarily mediated by inositol 1,4,5-trisphosphate (IP3), generated through the hydrolysis of the membrane phospholipid, phosphatidylinositol 4,5-diphosphate, by one or more phospholipase C (PLC) isoforms. To date, there is limited evidence that the vWf-GPIb interaction can induce PLC activation and IP3 generation in platelets (16). To examine this possibility, we initially quantified IP3 levels in platelets aggregated by vWf (10 µg/ml) and ristocetin (1 mg/ml), in the presence of the anti-integrin
IIb3 antibody c7E3 Fab.
IP3 levels were determined on platelet aggregates rather than from
adhesion assays, because we were unable to harvest sufficient quantities of
platelet lysates from the latter assays to reliably detect changes in
IP3. Stimulation of platelets in suspension with vWf/ristocetin
resulted in a transient increase in IP3 levels from a resting
concentration (1.1 ± 1.4 pmol/109 platelets) up to a peak of
5.2 pmol/109 platelets after 10 s of stimulation. By 30 s,
IP3 levels had returned to basal levels
(Fig. 1A). A similar
transient increase in IP3 was also observed in Glanzmann
thrombasthenic platelets, confirming the lack of involvement of integrin
IIb3
(Fig. 1A). In all
experiments, the increase in IP3 induced by the vWf-GPIb
interaction was weak relative to other agonists, with thrombin inducing peak
IP3 levels as high as 90 pmol/109 platelets
(Fig. 1B and data not
shown).
|
To investigate the relationship between IP3 generation and
calcium mobilization during platelet adhesion on vWf, platelet
Ca2+ levels were monitored using a quantitative dual-dye
ratiometric assay, as detailed under "Experimental Procedures." As
demonstrated in Fig.
1C, the Ca2+ concentration in
resting platelets was low. Following adhesion to vWf, 
60% of adherent
platelets elicited transient Ca2+ spikes ranging from
50–200 nM (Fig.
1C). Consistent with the low IP3 levels,
GPIb-dependent Ca2+ signals were small relative to those
induced by thrombin (up to 2000 nM).
Platelet adhesion on vWf, in the presence of integrin
IIb3 antagonists, results in morphological
alterations characterized by sphere forming of the cell body and extension of
multiple filopodia (5). To
further establish the relationship between IP3 generation,
Ca2+ flux, and platelet shape change on vWf, the effects
of the IP3 receptor antagonist, 2-aminoethoxydiphenyl borate
(APB-2), were examined. As demonstrated in
Fig. 2, APB-2 decreased the
proportion of platelets undergoing shape change from 85% in control platelets
to less than 20% in treated platelets (Fig.
2, A and B). Pretreating platelets with APB-2
(20 µM) completely abolished GPIb-induced
Ca2+ spikes (Fig.
2C). In control studies, we confirmed that APB-2 was not
having nonspecific inhibitory effects on platelets, because stimulating
APB-2-treated platelets with low dose Ca2+ ionophore
A23187
[GenBank]
(40 nM) restored platelet shape change
(Fig. 2, A and
B). This effect of ABP-2 was not caused by its blockade
of extracellular Ca2+ entry, as GPIb-induced
Ca2+ mobilization occurred in the presence of the
extracellular Ca2+ chelator EDTA
(5) and similar inhibitory
effects of APB-2 were observed under these conditions (data not shown).
|
Involvement of PLC in Platelet Cytoskeletal Reorganization Triggered by
the vWf-GPIb-V-IX Interaction—To investigate further the potential
involvement of PLC in GPIb signaling, we examined the effects of the PLC
antagonist, U73122
[GenBank]
(32), on
GPIb-induced Ca2+ mobilization and platelet shape
change. As shown in Fig.
3A, U73122
[GenBank]
reduced the proportion of platelets undergoing
filopodial extension from 95% down to less than 5% of platelets
(Fig. 3B). The effect
on filopodial formation was observed at similar concentrations to those
required to inhibit shape change induced by collagen or serotonin, two
agonists known to activate the PLC and PLC isoforms, respectively
(Fig. 3C). U73122
[GenBank]
(1
µM) totally prevented GPIb-induced intracellular
Ca2+ flux (Fig.
3D). In further control studies, we demonstrated that the
structurally related inactive U73122
[GenBank]
analogue, U73343
[GenBank]
, had no effect on
vWf-induced platelet shape change, and furthermore, the inhibitory effects of
U73122
[GenBank]
on platelet shape change could be completely reversed with low dose
ionophore A23187
[GenBank]
(40 nM) (data not shown).
|
CHO cells transfected with the GPIb-IX complex are able to adhere to a vWf matrix and extend filopodia in a manner similar to platelets (5). Incubation with U73122 [GenBank] prevented cytoskeletal reorganization in GPIb-IX-transfected CHO cells, whereas control U73343 [GenBank] had no effect (Fig. 4, A and B). Such comparable inhibition in different cellular systems supports the involvement of PLC activation in GPIb-IX signaling leading to cytoskeletal reorganization.
|
Cytoskeletal Reorganization Induced by vWf-GPIb Interaction Does Not
Require the PLC Isotype—To investigate the potential
PLC isotypes involved in GPIb-dependent cytoskeletal reorganization, adhesion
assays were performed on mouse platelets deficient in the subunit of
the Gq heterotrimeric protein
(23). Platelets express
Gq but not its structural homologue G11, and as a result
Gq-deficient platelets do not activate PLC
(23).
Gq-deficient and wild type platelets displayed no difference
in their capacity to adhere to a vWf matrix and undergo shape change,
indicating that the PLC-dependent pathway was not required for vWf-GPIb
signaling (Fig. 5,
A–C). In control studies, we confirmed that
vWf-induced cytoskeletal reorganization in mouse platelets was PLC-dependent,
because it was abolished by the PLC inhibitor U73122
[GenBank]
(data not shown).
|
The vWf-GPIb Interaction Induces PLC2 Tyrosine
Phosphorylation through Src Family Tyrosine Kinases—Previous
studies have reported PLC2 tyrosine phosphorylation in vWf-aggregated
platelets (15,
18), although the functional
significance of this event remains unclear. Moreover, it is not clear whether
PLC2 tyrosine phosphorylation occurs downstream of GPIb or requires vWf
binding to integrin IIb3. In platelets
aggregated by HvWf (10 µg/ml) and ristocetin (1 mg/ml), in the presence of
the anti-integrin IIb3 antibody c7E3 Fab
(Fig. 6A), a rapid
increase in PLC2 tyrosine phosphorylation was observed at 10 s, which
slowly decreased over time with kinetics that resemble the production of
IP3. In comparison to collagen, phosphorylation induced by vWf was
weak (Fig. 6A),
consistent with low level phosphoinositide turnover in these platelets. To
investigate the ability of GPIb to induce tyrosine phosphorylation of
PLC2 during platelet adhesion on immobilized vWf, platelets were
allowed to adhere to a vWf matrix in the presence of the integrin
IIb3 antagonist, c7E3 Fab. Platelet
adhesion to vWf was associated with the tyrosine phosphorylation of
PLC2. Pretreating platelets with the Src family kinase inhibitor, PP2,
completely eliminated GPIb-induced PLC2 phosphorylation
(Fig. 6B), confirming
an important role for Src kinases in this process.
|
To investigate the functional significance of Src kinases for GPIb-induced
cytoskeletal reorganization, platelet adhesion experiments were performed in
the presence of PP1 and PP2. Although PP1 and PP2 had no significant effect on
the number of platelets adhering to vWf, they resulted in a >75% reduction
in the proportion of platelets undergoing shape change
(Fig. 7, A and
B), and Ca2+ responses
(Fig. 7D).
Dose-response studies revealed that PP1 and PP2 inhibited shape change induced
by vWf and collagen over a concentration range previously demonstrated to be
Src kinase-selective (Fig. 7
and data not shown). Taken together, these studies are consistent with a
potential role for Src kinase-mediated activation of PLC isoforms in
GPIb-induced cytoskeletal remodeling.
|
Cytoskeletal Reorganization Induced by vWf-GPIb Interaction Is
Regulated by PLC2—PLC1 and PLC2
isotypes have been detected in platelets, and the latter is clearly implicated
in signaling by ITAM-bearing receptors (GPVI and FcRIIA). Indeed,
PLC2-deficient platelets have a major defect in collagen signaling
(25,
33,
34). To investigate the
potential role of PLC2 in GPIb signaling, adhesion studies were
performed on PLC2-deficient platelets. As demonstrated in
Fig. 8B, these
platelets adhered as efficiently to vWf as PLC2+/+ controls, however,
PLC2-deficient platelets exhibited a significant reduction in their
capacity to extend filopodia (Fig. 8,
A and C). Studies examining
Ca2+ flux demonstrated 35% of adherent wild-type
mouse platelets displaying GPIb-induced Ca2+ transients
similar to those observed for human platelets, with peak
Ca2+ concentrations ranging from 175 to 500
nM (Fig.
8D). In PLC2-deficient mouse, a decreased number
of adherent platelets were able to sustain Ca2+
oscillations. Interestingly, this defect in platelet shape change and
Ca2+ mobilization
(Fig. 8, D and
E) was not as profound as those observed in platelets
treated with Src kinase, PLC, or IP3 receptor antagonists,
suggesting the possible involvement of other PLC isoforms in GPIb
signaling.
|
The FcRIIA Receptor and FcR Chain Are
Not Required for GPIb-induced Platelet Shape Change—The
demonstration that GPIb-induced cytoskeletal reorganization is regulated by
Src kinases and PLC2, raises the possibility that GPIb utilizes
ITAM-containing signaling receptors to induce platelet activation. In support
of this hypothesis, the two major ITAM-containing receptors in platelets,
FcRIIA receptor and FcR chain, have previously been demonstrated
to be physically associated with GPIb
(15,
17). In addition, recent
studies utilizing the FcRIIA function-blocking mAb antibody, IV.3, have
demonstrated an important role for this receptor in regulating PLC2
tyrosine phosphorylation and platelet secretion in vWf-aggregated platelets
(18,
35). To examine the
involvement of FcRIIA in GPIb-induced shape change, human platelets
were pretreated with IV.3. As demonstrated in
Fig. 9, IV.3 had no inhibitory
effect on GPIb-induced platelet shape change (A and B) or
Ca2+ mobilization (C). In control studies, we
confirmed that the IV.3 antibody effectively blocks FcRIIA by its
ability to completely abolish platelet aggregation induced by heat-aggregated
immunoglobulin (IgG) (data not shown).
|
A recent study has suggested a potentially important role for the
FcR chain in promoting vWf-induced PLC2 tyrosine phosphorylation
and platelet aggregation (16).
To examine the necessity of FcR chain for GPIb-induced platelet shape
change and Ca2+ mobilization, studies were performed on
platelets derived from FcR chain-deficient mice
(FcR–/–)
(24). As demonstrated in
Fig. 10, platelets from
wild-type (FcR+/+) and FcR–/– mice changed shape and
extended filopodia in an identical manner
(Fig. 10A). Analysis
of the adherent platelets indicated that the percentage of adherent platelets
undergoing shape change and the number of adherent platelets were
indistinguishable between FcR+/+ and FcR–/– mouse
platelets (Fig. 10, B and
C). A similar pattern of Ca2+
transients was detected in FcR+/+ and FcR–/– mouse
platelets (Fig. 10D).
In contrast to FcR+/+ platelets, on a type 1 fibrillar collagen matrix,
FcR-deficient platelets failed to undergo any
Ca2+ responses (Fig.
10D) and morphological changes following adhesion to
collagen (data not shown). These findings do not support an important role for
the FcR chain for vWf-induced cytoskeletal reorganization.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The GPIb-V-IX receptor complex is unique among adhesion receptors in that it not only regulates the cytoskeletal architecture of resting platelets but can also induce cytoskeletal remodeling following engagement of its adhesive ligand (5). Recent evidence suggests that vWf-induced cytoskeletal reorganization may play a potentially important role in regulating the adhesive function of GPIb, relevant to shear-induced platelet activation (6). Despite the potential functional importance of vWf-induced cytoskeletal changes, the signaling mechanisms regulating this process have remained ill-defined. The studies presented here demonstrate an important role for PLC-dependent phosphoinositide turnover, leading to IP3 generation and subsequent calcium mobilization, for GPIb-dependent cytoskeletal remodeling. More specifically, through the analysis of PLC
2-deficient platelets and
pharmacological inhibition of Src kinases, our studies have demonstrated the
involvement of PLC2, and potentially a second Src kinase-regulated form
of PLC, in GPIb signaling. Finally, our studies do not support a major role
for the ITAM-bearing receptors, FcRIIA or FcR chain, in
GPIb-dependent cytoskeletal remodeling.
Several lines of experimental evidence support a functionally important
role for PLC-mediated phosphoinositide turnover for GPIb-induced cytoskeletal
change. First, we have demonstrated that the vWf-GPIb interaction is
sufficient to induce a small increase in IP3, independent of
integrin IIb3. Second, that pharmacological
blockade of the IP3 receptor completely blocks platelet shape
change. Third, inhibition of PLC with U73122
[GenBank]
, but not its inactive structural
analogue U73343
[GenBank]
, abolishes GPIb-dependent cytoskeletal changes. Finally,
studies on mouse platelets lacking PLC2 demonstrated the involvement of
this enzyme in GPIb-dependent cytoskeletal remodeling. These findings,
combined with recent reports demonstrating that the vWf-GPIb interaction is
sufficient to mobilize calcium from internal stores
(5), provide strong evidence
that GPIb can induce phosphoinositide turnover in platelets.
The ability of the vWf-GPIb interaction to induce a cytosolic calcium response has remained controversial, with some studies demonstrating transmembrane calcium influx (36, 37), other studies suggesting calcium release from internal stores (38, 39), whereas other studies have failed to detect a cytosolic calcium signal (16, 42). The reason for these discrepancies is likely to reflect technical differences between the studies. From previous studies (5, 38) and the present results it appears that GPIb is a weak activator eliciting weak transient Ca2+ response in platelets. Furthermore, in contrast to soluble agonists, vWf does not induce a synchronized calcium response throughout the platelet population, with a low percentage of primary adherent platelets undergoing a calcium response at any one time. The low level, transient IP3 generation in our studies is consistent with the calcium dynamics observed in primary adherent platelets. These findings may partly explain why other studies have failed to demonstrate IP3 generation or a detectable Ca2+ signal following vWf engagement of GPIb.
Our studies define an important, albeit not absolute requirement for
PLC2 for GPIb-dependent cytoskeletal remodeling and
Ca2+ mobilization. The partial defect in filopodia
formation in PLC2–/– platelets was accompanied by a partial
decrease in Ca2+ signaling suggesting the involvement of
another PLC isotype. Gq-deficient mouse platelets have
impaired receptor-coupled PLC activation and do not aggregate in
response to ADP, U46619
[GenBank]
, or thrombin nor change shape in response to ADP
stimulation (23). Although
platelets primarily express two PLC isotypes (PLC2 and
PLC3), the demonstration that Gq-deficient platelets
undergo shape change indistinguishable from normal platelets rules out an
important role for these enzymes in GPIb-shape change. Platelets also contain
the PLC1 and PLC
1 isotypes, however, their levels of expression
are much lower than other PLC isoforms and their roles in platelets and
mechanisms of activation remain unclear. Platelets express both members of the
PLC family: PLC1 and PLC2; however the latter is
expressed at higher levels than PLC1
(40). Nonetheless, it remains
conceivable that this enzyme may also contribute to GPIb signaling,
particularly in light of our observations that inhibition of Src kinases or
PLCs has a more profound effect on platelet shape change and
Ca2+ mobilization than that observed in
PLC2-deficient platelets. The recent observation of a residual
activation to collagen in PLC2-deficient mice
(33,
34) involving the PLC1
isotype raises the possibility of a similar involvement of PLC1 in
GPIb/vWf-triggered activation
(34). This possibility will
require further investigation.
Our studies do not support an important role for the ITAM-bearing
receptors, FcRIIa and the FcR chain, for GPIb signaling, at
least in the context of cytoskeletal remodeling. Evidence for an important
role for FcRIIA in GPIb signaling has been derived from studies
demonstrating physical association between the receptors
(15,
17) and from functional
studies demonstrating that the anti-FcRIIA-blocking antibody, IV.3,
prevents vWf-induced granule release and FcRIIA tyrosine
phosphorylation (18). However,
our finding that IV.3 did not inhibit GPIb-dependent cytoskeletal change is
not altogether surprising given previous findings that GPIb-V-IX-transfected
CHO cells, which do not express Fc receptors
(41), undergo cytoskeletal
reorganization following adhesion to vWf. Furthermore, in contrast to human
platelets, mouse platelets do not naturally express FcRIIA, yet undergo
normal shape change in response to vWf. Given the important role of Src
kinases, and in particular the involvement of PLC2 in GPIb signaling,
we sought evidence for the involvement of FcR chain in vWf-induced
cytoskeletal changes. It has previously been demonstrated that the FcR
chain becomes tyrosine-phosphorylated following vWf stimulation of platelets
and that it co-immunoprecipitates with GPIb in GST-Syk pull-downs
(16). Furthermore,
FcR-deficient mouse platelets have decreased phosphorylation of Syk,
PLC2, and linker for activation of T-cell (LAT) and defective platelet
aggregation in response to vWf. This has lead to a model in which vWf
engagement of GPIb promotes Src-dependent FcR chain phosphorylation and
assembly of a multicomponent signaling complex involving p72Syk,
SLP76, and LAT. However, recent studies
(42) have questioned the
functional significance of this pathway with respect to GPIb signaling, a
finding consistent with our inability to detect differences in the shape
change and calcium responses of FcR-deficient mouse platelets.
The exact signaling pathway utilized by GPIb to induce PLC2
activation remains unclear but undoubtedly involves one or more members of the
Src kinase family. The mode of activation of Src kinases by GPIb is not
obvious, because the intracellular domains of the GPIb-V-IX complex are devoid
of Tyr residues and SH2 domains. A recent study has demonstrated an indirect
association between GPIb and Src through a complex involving the p85 subunit
of PI 3-kinase (13). Other
studies have reported co-precipitation of a non-receptor tyrosine kinase,
possibly Src, with GPIb, although the molecular basis for this association has
not been defined (43). There
is also evidence that GPIb may signal through lipid rafts where GPIb has been
found to co-localize with signaling enzymes and adaptor molecules
(44), although others have not
confirmed these findings (36).
GPIb may also signal through the assembly of cytoskeletal signaling complexes,
because it has previously been demonstrated that the vWf-GPIb interaction can
induce the cytoskeletal association of Src and a range of other signaling
enzymes (13). Resolution of
this important issue will require more detailed analysis of the key structural
domains of the GPIb/V/IX complex involved in signal transduction.
|
FOOTNOTES |
|---|
* This work was supported in part by a grant from Association de Recherche et de Développement en Médecine et Santé Publique and from the Australia National Health & Medical Research Council and National Heart Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
¶ Supported by the Association de Recherche et de Développement en
Médecine et Santé Publique.
** To whom correspondence should be addressed. Tel.: 33-388-21-25-25; Fax: 33-388-21-25-21; E-mail: francois.lanza{at}efs-alsace.fr.
1 The abbreviations used are: vWf, von Willebrand factor; HvWf, human vWf;
GP, glycoprotein; ABP, actin-binding protein; PI, phosphoinositide; ITAM,
immunoreceptor tyrosine-based activation motif; PLC, phospholipase C;
IP3, inositol 1,4,5-triphosphate; CHO, Chinese hamster ovary;
2-APB, 2-aminoethoxydiphenyl borate; TRITC, tetramethylrhodamine
isothiocyanate; mAb, monoclonal antibody; GST, glutathione
S-transferase.
|
ACKNOWLEDGMENTS |
|---|
We thank Prof. J. Ihle for providing the PLC
2-deficient mice, Prof.
S. Offermanns for the Gq-deficient mice, and Prof. Takashi
Saito for the FcR-deficient mice. |
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