1Division of Hematology, Brigham and Women's Hospital, Department of Medicine, Harvard Medical School, Boston 02115; and 2Beth Israel Deaconess Medical Center, Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115; and 3Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
Submitted 25 April 2003 ; accepted in final form 29 May 2003
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ABSTRACT |
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actin assembly; CD32A
Many receptors are positioned on the platelet surface that lead to actin
assembly and platelet shape change when ligated. Shape change, the signaling
cascade that initiates actin assembly, and proteins that regulate actin
assembly reactions have been examined extensively in platelets activated via
the thrombin receptor protease-activated receptor (PAR)-1 or the collagen
receptor glycoprotein (GP)VI
(4,
12,
18,
19) or after chilling
(22,
39). Signaling from PAR-1
begins with the activation of pertussis toxin-sensitive trimeric G proteins
(6,
7), which couples to
phospholipase C- to cleave phosphatidylinositol 4,5-bisphosphate
(PI4,5P2) into inositol 1,4,5-trisphosphate
(IP3) and diacylglycerol
(25). Subsequently,
IP3 gives rise to elevated intracellular calcium and diacylglycerol
activates protein kinase C (5).
Elevated cytosolic calcium also activates gelsolin, which binds to actin
filaments to sever them and to transiently associate with their barbed ends
(26). PAR-1 also couples to
the small GTPases Rac and Cdc42
(19). Synthesis of
polyphosphoinositides (ppIs) follows, and these new ppIs mediate barbed end
exposure by dissociating actin filament barbed end capping proteins
(4,
19). Barbed ends, exposed when
gelsolin family members dissociate from actin filaments, are subsequently
amplified by Arp2/3 complex
(14), which is also activated
by pathways involving ppIs
(22,
32). This process is
intimately connected to PAR-1-induced actin filament assembly because mice
null for gelsolin have only 25-50% of the wild-type actin filament barbed end
exposure/nucleation activity after stimulation with thrombin
(14,
40).
Early studies indicate that immunoglobulins can activate platelets
(21). Immune thrombocytopenic
purpura and heparin-induced thrombocytopenia have been linked to ligation of
the immunoglobulin receptor FcRIIA (CD32A) on platelets, suggesting
that Fc
RIIA can bind immune complexes in blood and that this leads to
clearance of platelets from the blood by macrophage-mediated phagocytosis
(28). Platelet activation via
Fc
RIIA initiates all the well-defined responses believed to be
important for platelet function
(16,
33). However, the normal
hemostatic function of Fc
RIIA has not been determined.
In this study, we investigated the mechanisms of shape change in
FcRIIA-stimulated platelets. Like ligation of PAR-1
(8,
15,
24,
31), the cross-linking of
Fc
RIIA leads to actin assembly. However, the cytoplasmic signaling
cascades set in motion by these two stimuli differ. Platelet shape change
initiated by Fc
RIIA is particularly dynamic, with readily observed
filopodia, membrane ruffling, and extension of lamellae resulting in platelet
spreading. The underlying structures that support these movements are actin
rich, and actin assembly proceeds in a reproducible and robust response with
exposure of actin filament barbed ends following Fc
RIIA cross-linking.
In addition, proteins that modulate barbed end exposure, such as Arp2/3
complex (14), associate with
actin in Fc
RIIA-activated platelets. Importantly, the data place
phosphoinositide 3-kinase (PI3-kinase) as an essential element in actin
assembly reactions triggered by Fc
RIIA. In particular, PI3-kinase
activity is required for the association of the actin filament barbed end
amplifier Arp2/3 complex with the platelet actin cytoskeleton.
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MATERIALS AND METHODS |
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Videomicroscopy. Coverslips for use in the light microscope were
coated with 50 µg/ml of the anti-FcRIIA antibody IV.3 in PBS for 2 h
at room temperature, blocked with 3% BSA in PBS for 1 h at room temperature,
and washed three times with platelet buffer. Platelets at 2 x
108/ml were diluted with platelet buffer (with or without 30 µM
LY-294002) to 4 x 107/ml, and 100 µl was incubated on the
coverslips for 20 min at 37°C. A Zeiss inverted microscope provided images
of platelets by using differential interference contrast (DIC) optics with a
x 100 oil immersion objective. Images were captured as described
previously (12,
13,
22).
Immunofluorescent staining. Adherent platelets were fixed with 3.7% formaldehyde in platelet buffer for 15 min and permeabilized with 0.5% Triton X-100 in PIPES-HEPESEGTA-MgCl2 (PHEM) buffer. Arp2/3 complex was stained as described previously (13, 14).
Electron microscopy. Platelets were adhered for 30 min to glass
surfaces previously coated for 2 h with 25 µg/ml of the anti-FcRIIA
antibody IV.3. Unattached platelets were washed away, and adherent cells were
extracted in PHEM buffer (in mM: 60 PIPES, 25 HEPES, 10 EGTA, and 2
MgCl2, pH 6.9) containing 1 µM phallacidin, protease inhibitors
(10 µg/ml each of leupeptin, aprotinin, and benzamidine), and 0.75% Triton
X-100 for 2 min. Cytoskeletons were processed for electron microscopy as
previously reported (18). They
were fixed for 10 min with 1% glutaraldehyde in PHEM buffer containing 0.1
µM phallacidin, washed in deionized, filtered water and rapidly frozen,
freeze dried, and coated with 1.4-nm tungsten-tantalum with rotation and
2.5-nm carbon without rotation. Cytoskeletons were examined and photographed
in a JEOL 1200-EX electron microscope at an accelerating voltage of 100
kV.
Actin assembly assay. Platelets were activated with 25 µM thrombin receptor PAR-1 activating peptide (TRAP) or with 3 µg/ml IV.3 (the concentration that gave the maximum response in all quantitative experiments) for 3 min, followed by 30 µg/ml goat anti-mouse F(ab)2 (GAM), and then fixed at the desired time point by the addition of an equal volume of 6.8% formalin. The samples were extracted and labeled in PHEM buffer with 0.1% Triton X-100, and the actin filaments were labeled with 1 µM FITC-phalloidin for 30 min. FACS analysis with a BD Biosciences FACSCalibur (San Jose, CA) used forward and side scattering to identify platelets and measure the mean fluorescence of 10,000 platelets to quantitate filamentous actin content.
Actin filament barbed end exposure in platelets. The average
number of barbed ends exposed per cell was obtained from a nucleation assay
with pyrene-labeled rabbit skeletal muscle actin as previously described
(18,
19). Ninety micro-liters of
platelets at 2 x 108/ml was activated with TRAP or IV.3-GAM
and extracted at various time points as described in Actin assembly
assay. Platelet extracts were diluted to 300 µl with buffer B
(0.1 M KCl, 0.2 mM MgCl2, 0.1 mM EGTA, 0.5 mM ATP, 10 mM Tris, and
0.5 mM -mercaptoethanol pH 7.4). The assay was started by the addition
of 1 µM monomeric pyrene-labeled actin, and fluorescence followed in a
Perkin-Elmer LS-50B fluorimeter with an excitation wavelength of 366 nm and an
emission wavelength of 386 nm. Actin filament barbed ends were calculated as
described previously (18,
19).
When actin nucleation was tested in the presence of recombinant PI3-kinase subunits p85 and p110, resting platelets were permeabilized with 0.25-0.4% octylglucoside (OG) for 30 s and then treated for 2 min with various concentrations of the kinase. Kinase-treated and control platelets were then diluted in buffer B, and the polymerization of 1 µM pyrene-actin was followed as above. Gelsolin and CapZ release from OG-permeabilized platelets was analyzed for sensitivity to added ppI micelles. OG-permeabilized platelets were washed twice to remove soluble proteins by two successive rounds of centrifugation at 10,000 g for 1 min after resuspension in PHEM buffer with 0.1 µM phallacidin. These platelet cytoskeletal "ghosts" were treated with ppIs, in the form of sonicated micelles, and the F-actin-associated pellet and soluble fractions were separated by centrifugation for 15 min at 15,000 g to generate a pellet and soluble fraction. Gelsolin and CapZ were detected in immunoblots of all fractions.
GTP-Rac and -Cdc42 trapping assay. Platelets activated through
FcRIIA in suspension were lysed with 0.1% Triton X-100 in PHEM buffer
containing 1 µM phallacidin and protease inhibitors. Triton releases all of
the Rac and Cdc42 into the soluble phase
(2), allowing platelet lysates
to be clarified by centrifugation at 100,000 g for 1 h. GTP-Rac and
-Cdc42, in the soluble fraction, were selectively adsorbed to glutathione
S-transferase (GST)-GTPase-binding domain (GBD) domain of human
p21-activated kinase 1 (PAK-1) (amino acids 67-150) adsorbed to glutathione
Sepharose beads in an overnight incubation at 4°C. Unbound material was
removed with five successive washes in PBS containing 0.02% Tween 20. Bound
Rac and Cdc42 were released from the glutathione Sepharose beads with SDS-PAGE
sample buffer, displayed by 12% SDS-PAGE, transferred to polyvinylidene
difluoride (PVDF) membrane, and detected by antibodies directed against Rac
and Cdc42 (2,
3). As controls, Rac and Cdc42
in the detergent-soluble lysate from resting platelets were loaded with
guanosine 5'-O-(2-thiodiphosphate) (GDP
S) or guanosine
5'-O-(3-thiotriphosphate) (GTP
S) and incubated with
affinity GBD-domain beads. GDP
S-Rac and -Cdc42 did not bind to PAK-1.
GTP
S-Rac and -Cdc42 bound to the GST-PAK-1 beads
(2).
Immunoblot analysis of platelet cytoskeletal proteins. Resting and
activated platelets were lysed with a final Triton X-100 concentration of 0.1%
in PHEM buffer. The lysates were centrifuged at 100,000 g for 30 min
at 4°C, the resultant pellets and supernatants were separated, and
SDS-PAGE loading buffer containing 5% -mercaptoethanol was added. The
samples were boiled for 5 min. Proteins were displayed by SDS-PAGE and
transferred onto Immobilon-P membrane (Millipore). Membranes were blocked with
5% dry milk in 100 mM NaCl-20 mM Tris · HCl, pH 7.4, and then probed
with specific antibodies and appropriate peroxidase-tagged secondary
antibodies. Detection was performed with an enhanced chemiluminescence system
(Pierce).
Intracellular calcium measurement. Isolated platelets were preincubated in the dark with 2 µM indo 1-AM (Molecular Probes, Eugene, OR) for 45 min at room temperature. Indo 1-labeled platelets were collected by centrifugation at 800 g and resuspended in platelet buffer containing 1 mM CaCl2. Platelets were activated as described in Actin assembly assay, and fluorescence was recorded (excitation 331 nm, emission 410 nm). The intracellular calcium concentration ([Ca2+]i) was calculated as described previously (17): [Ca2+]i = Kd x (F - Fmin)/(Fmax - F) where Kd = 250 nM, F is fluorescence, Fmin is fluorescence after extraction with detergent in the absence of free calcium, and Fmax is fluorescence after extraction with detergent in the presence of millimolar free calcium.
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RESULTS |
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Cross-linking of FcRIIA on platelets in solution with the IV.3
antibody also initiates shape change and actin assembly, which peaks
2
min after addition of the cross-linking secondary antibody
(Fig. 1B). Actin
assembly in response to Fc
RIIA cross-linking is dose dependent and
saturates when the amount of IV.3 antibody is 3 µg/ml (data not shown).
Actin assembly induced through Fc
RIIA shows a lag period compared with
that seen when platelets are activated through PAR-1, although activation
through Fc
RIIA and PAR-1 both lead to similar numbers of exposed actin
filament barbed ends (Fig.
1C). As expected, there is temporal coupling between
actin filament barbed end exposure and actin assembly such that the kinetics
of barbed end exposure parallel those of actin filament assembly.
Whereas light microscopy underscores the dynamic nature of platelet filopodial extension, membrane ruffling, and spreading when platelets adhere to IV.3-coated surfaces, high-resolution electron microscopy reveals the ultrastructure of the underlying actin cytoskeleton in these processes (Fig. 2A). Filamentous actin fills ruffles, lamellae, and filopodia. Actin filaments in filopodia and lamellae organize into bundles and networks comparable to those seen in platelets activated by contact with glass (18). Filopodia are cored by long actin filaments, which originate from the cell center, coalesce into bundles at the base of filopodia, and extend to their tips. Lamellae have a dense three-dimensional network of shorter actin filaments. Ruffles, rare in glass-activated platelets, have densely packed actin filament arrays that appear to form on top of the less dense actin arrays of lamellae.
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Role of PI3-kinase in platelet shape change and actin assembly induced
by FcRIIA cross-linking. The p85 subunit of PI3-kinase
associates with Fc
RIIA in immunoprecipitates, and platelets activated
by IV.3 synthesize ppIs phosphorylated in the D-3 position of the inositol
ring (9,
16). Consistent with these
observations, Fig. 3A
shows that the platelet mass of phosphatidylinositol 3,4-bisphosphate
(PI3,4P2) and phosphatidylinositol 3,4,5-trisphosphate
(PI3,4,5P3) increases after Fc
RIIA activation.
Inhibition of PI3-kinase by 25 nM wortmannin blocked D-3 phosphorylation
induced by Fc
RIIA cross-linking.
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We examined the requirement for D-3 ppI synthesis in the actin assembly reaction by inhibiting PI3-kinase activity (Fig. 3B). Actin filament assembly induced by IV.3, detectable by FACS analysis with FITC-labeled phalloidin, was completely inhibited by pretreatment of platelets with wortmannin concentrations as low as 10 nM, specific for PI3-kinase. Wortmannin, in a dose-dependent manner, also markedly reduced but did not completely inhibit actin filament barbed end exposure; 20% of the normal barbed end nucleation sites remained wortmannin insensitive. The PI3-kinase inhibitor LY-294002 also blocked actin assembly at concentrations consistent with the selectable inhibition of PI3-kinase (data not shown).
Platelet spreading on IV.3 coated surfaces is highly sensitive to
wortmannin (Fig. 2B).
Wortmannin-treated platelets, however, do round up and grow sparse filopodia
after stimulation of FcRIIA. Electron micrographs of cytoskeletons from
platelets treated with 25 nM wortmannin and activated on IV.3-coated surfaces
lack well-developed actin cortices but have many filopodia containing long
actin filaments arranged in bundles. Because filopodial growth in the presence
of wortmannin is inhibited by cytochalasin B (data not shown), some residual
PI3-kinase-insensitive actin filament assembly remained that is necessary to
elaborate the filopodia. The dose response for inhibition of barbed end
exposure by wortmannin paralleled that found for its inhibition of actin
assembly (Fig.
3B).
Inhibition of PI3-kinase also blunts other platelet activation responses,
such as secretion and activation of the fibrinogen receptor, the integrin
IIb
3 (9,
16,
33). We therefore examined
platelets lacking
IIb
3 from a patient with Glanzmann
thrombasthenia and found that
IIb
3 is not required for spreading
on IV.3-coated surfaces or for actin assembly in response to Fc
RIIA
cross-linking (data not shown). Similarly, other reports have suggested that
release of ADP by platelets activated via Fc
RIIA is a costimulus.
However, using apyrase to scavenge ADP did not affect the actin filament
barbed end exposure reaction in response to Fc
RIIA cross-linking (data
not shown). Similarly, inhibition of thromboxane production during
Fc
RIIA-induced spreading does not contribute to the shape change
because indomethacin had no effect on spreading (data not shown).
FcRIIA cross-linking leads to activation of small
GTPases Rac and Cdc42. One potential downstream target of Fc
RIIA
signaling to actin is the small GTP-ase Rac. Because Triton X-100 treatment
releases all of platelet Rac independently of its guanine nucleotide-bound
state (2), we trapped and
segregated GTP-Rac in soluble lysates with the GST-PAK-1 construct that binds
GTP-Rac but not GDP-Rac in Triton-soluble platelet extracts
(Fig. 4A). These and
other earlier experiments (2)
reveal that the majority of Rac in resting platelets is GDP bound. Activation
of platelets through Fc
RIIA increased the amount of GTP-Rac in
platelets to 20 ± 10% of the total at 3 min, the time of maximal actin
assembly and barbed end exposure. Treatment of platelets with 30 µM
LY-294002 completely prevented Rac charging, indicating that activation of Rac
induced by Fc
RIIA cross-linking is dependent on PI3-kinase.
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GTP charging of Cdc42 was evaluated with the same approach
(Fig. 4B). The amount
of active Cdc42 in resting platelets was small, representing <5% of the
total, as reported previously
(2,
34,
36). Cross-linking of
FcRIIA increased the amount of GTP-Cdc42 over the time course of the
experiment. The extent of Cdc42 converted to the GTP state, however, was
small, representing only 10% of the total after 1 min. Treatment of platelets
with 25 nM wortmannin had little effect on the amount of GTP-Cdc42 in resting
and Fc
RIIA-activated platelets.
Incorporation of Arp2/3 complex and CapZ in platelet actin
cytoskeleton. Proteins that regulate actin filament exposure in platelets
were examined for their association with F-actin in platelet cytoskeletons
activated through FcRIIA with or without PI3-kinase inhibitors
(Fig. 4, C and
D). The actin filament barbed end amplifier Arp2/3
complex (Fig. 4C) and
the capping protein CapZ (Fig.
4D) both associated with the cytoskeleton of
Fc
RIIA-activated platelets with kinetics that slightly lagged behind
the formation of actin filament barbed ends. Twenty-five percent of the total
platelet Arp2/3 complex associated with the resting platelet cytoskeleton.
Fc
RIIA activation of platelets increased the amount of Arp2/3 complex
in the cytoskeleton to a maximum of 65% of the total in 2-3 min. In similar
fashion,
35-40% of the actin filament barbed end capping protein CapZ
associated with the Triton-insoluble cytoskeletal actin in resting platelets.
Platelet activation by Fc
RIIA increased the amount of CapZ in the
detergent-insoluble cytoskeleton to 60-65%. PI 3-kinase inhibition by
wortmannin and/or LY-294002 diminished the association of Arp2/3 complex and
CapZ with the active cytoskeleton by 80% and 40%, respectively.
Immunofluorescence localization of Arp2/3 complex in platelets spread on
anti-FcRIIA IgG showed it to move to the platelet cortex
(Fig. 5A), as
described previously (13,
14). A portion of Arp2/3
complex also concentrated in foci in the center of these platelets. Analysis
of Arp2/3 complex distribution in surface-activated platelets pretreated with
LY-294002 revealed that Arp2/3 complex failed to move to the cell periphery
and instead incorporated only into distinct foci within the cell center
(Fig. 5C). Resting
platelets showed diffuse labeling for the Arp2/3 complex throughout the
platelet cytoskeleton (14).
F-actin staining with tetramethylrhodamine isothiocyanate (TRITC)-phalloidin
confirmed that platelets adhered less well to IV.3-coated surfaces after
exposure to LY-294002 (Fig. 5, B
and D). The platelets that did adhere rounded up and
extended some filopodia but were unable to protrude lamellae or make
ruffles.
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Role of calcium mobilization in platelet actin assembly induced by
FcRIIA cross-linking. We examined whether a calcium
transient is required for Fc
RIIA-mediated actin assembly
(Fig. 6).
Figure 6A compares the
free calcium increases in Fc
RIIA- and PAR-1-activated platelets. IV.3
induced a sustained rise in free calcium after a 1-min lag, from a resting
value of
170 nM to a maximum of 500 nM after 3 min. Calcium remained
elevated for the time of the assay (5 min). TRAP, on the other hand, mobilized
calcium within seconds and was maximal by 15 s, after which the calcium
transient decayed. Mobilization of calcium by Fc
RIIA activation
requires the activity of PI3-kinase because it is inhibited by wortmannin
(Fig. 6A) or LY-294002
(data not shown).
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Chelation of intracellular calcium with EGTA-AM markedly reduced the
ability of activated platelets to expose actin filament barbed ends in both
FcRIIA- and PAR-1-stimulated platelets
(Fig. 6B). The
inability to generate actin filament barbed ends translated into reduced actin
assembly in platelets preloaded with EGTA-AM and activated through
Fc
RIIA (Fig.
6C). Furthermore, platelets preloaded with EGTA-AM, at
sufficient levels to buffer intracellular calcium
(20), did not spread on
IV.3-coated surfaces, although filopod-like processes were extended, and
movement of Arp2/3 complex to the cell cortex was impaired in platelets
treated with EGTA-AM (data not shown). Arp2/3 complex once again was clustered
in intracellular foci, as was observed when PI3-kinase was inhibited.
Products of PI3-kinase mediate actin assembly in OG-permeabilized platelets. We further measured the ability of ppIs phosphorylated in positions D-3 and D-4 to release the F-actin capping proteins CapZ and gelsolin from OG-permeabilized platelets (Fig. 7A). Ten percent of platelet CapZ and gelsolin is found in the OG-insoluble fraction (4). PI3,4,5P3 and PI3,4P2 release both CapZ and gelsolin from these preparations. Control experiments using phosphatidylserine showed no release of these proteins (4). These experiments demonstrate that gelsolin and CapZ are two putative downstream effectors of D-3-containing ppIs that can lead to barbed end exposure in platelets.
|
Actin filament barbed end exposure was also measured in OG-permeabilized
platelets after addition of the p85-p110 PI3-kinase complex.
Figure 7B shows the
PI3-kinase dose dependence of barbed end exposure in permeabilized platelets.
Addition of >5 nM PI3-kinase to permeabilized platelets induced a maximal
response of 500 barbed ends per platelet. This implies that PI3-kinase
and the synthesis of D-3 phosphorylated ppIs can lead to actin filament barbed
end exposure in permeabilized platelets.
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DISCUSSION |
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PI3-kinase rapidly and transiently associates with FcRIIA in
platelets on clustering of Fc
RIIA
(9). Consistent with previous
results (9,
16,
33), we find both
PI3,4P2 and PI3,4,5P3 to be
synthesized in platelets activated through Fc
RIIA and posit PI3-kinase
at a critical control point for a broad spectrum of activation-related events
in platelets stimulated through Fc
RIIA. The tyrosine kinase Syk is also
recruited to Fc
RIIA
(10) and is required for
Fc
RIIA-dependent phagocytosis
(11). We have observed that
inhibitors of Syk kinase activity prevent Fc
RIIA-induced actin assembly
(data not shown). PI3-kinase may associate with Fc
RIIA either directly
via its SH2 domain or indirectly by using Syk as an adapter. In the latter
case, Syk would bind first to the immunoreceptor tyrosine-based activation
motif (ITAM) of Fc
RIIA and then PI 3-kinase would dock on to the
phosphorylated tyrosine residues on Syk
(9,
23).
Platelet actin assembly induced by FcRIIA requires the enzymatic
activity of PI3-kinase. Platelets adhere less well to surfaces coated with
anti-Fc
RIIA antibody after exposure to LY-294002, and the platelets
that do adhere round up and extend some filopodia but are unable to protrude
lamellae or make ruffles. Some residual actin assembly and filopodia growth
remain in wortmannin-treated and Fc
RIIA-activated platelets that are
inhibited by cytochalasin B. These findings suggest either that the small
amount of actin assembly required to grow filopodia is not detectable in our
assays or that actin disassembly is coupled to filopodial actin assembly under
these conditions. PI3-kinase-independent activation of Cdc42 occurs after
cross-linking of Fc
RIIA with the IV.3 antibody. Further experimentation
is required to determine the precise role of Cdc42 in filopodia formation in
platelets. Interestingly, platelets lacking the Cdc42 effector WASp normally
assemble actin and change shape
(13).
Our findings couple Rac signaling to actin in a PI3-kinase-dependent and
-independent fashion. GTP-Rac can activate PIP5-kinase I to synthesize
the ppI PI4,5P2 on the cytoplasmic face of the plasma
membrane, which can bind and dissociate capping proteins to uncap actin
filament barbed ends (19,
35) and stimulate activation
of Arp2/3 complex (32). Rac
may activate Arp2/3 complex via cortactin
(37), as cortactin
translocation to the cell cortex and to actin-enriched ruffles is Rac
dependent (36,
38). Arp2/3 complex fails to
migrate to the cell cortex and, instead, aggregates into foci in the cell
interior when PI3-kinase is inhibited. It is therefore possible that Arp2/3
complex activation is initiated in a Rac-dependent pathway
(29) with Rac activation
initiated by PI3-kinase (30,
34). Unusual localization and
activation of Arp2/3 complex have previously been linked to low barbed end
production when the actin filament severing and nucleating protein gelsolin is
either impaired or absent
(14). However, signaling to
Arp2/3 complex appears to be impaired here because Arp2/3 complex association
with actin is normal in gelsolin-deficient platelets
(14). It has been shown that
PI3-kinase is not necessary for actin assembly and Arp2/3 complex recruitment
during Fc
RIIA-mediated phagocytosis
(1,
27). The data suggest that
Fc
RIIA signaling cascades to actin during platelet spreading or
phagocytosis by macrophages may differ. CapZ, a heterodimeric actin filament
barbed end capping protein, also associates with the cytoskeleton in
Fc
RIIA-activated platelets as it does after PAR-1 ligation
(4). CapZ binding to filament
barbed ends both stabilizes newly formed filaments and stops filament
elongation.
Platelet shape change and actin assembly in response to activation via
FcRIIA is calcium dependent. Loading platelets with EGTA-AM, at a
concentration that buffers intracellular calcium rise
(20), dramatically reduces the
exposure of actin filament barbed ends and prevents platelet spreading. The
requirement for calcium ions in actin assembly and shape change supports
previous reports in which calcium-dependent actin filament severing by
gelsolin is required for maximal barbed end exposure and actin assembly during
platelet activation (4,
18,
40). Atypical broad filopodia
elaborated when EGTA-loaded platelets are activated through Fc
RIIA also
resemble those previously described in glass-activated and EGTA-loaded
platelets (18). In addition,
because calcium levels do not increase when PI3-kinase is inhibited in
Fc
RIIA-activated platelets, gelsolin-dependent uncapping of actin
filaments to generate free barbed ends does not occur and Arp2/3 complex
amplification of actin filament barbed ends is therefore minimized.
In conclusion, we have shown that activation of FcRIIA results in
platelet spreading in a manner comparable to other mechanisms of platelet
activation. However, the signaling pathway to actin in Fc
RIIA-activated
platelets differs from that seen in PAR-1-stimulated platelets. We have shown
that activation of PI3-kinase is essential for normal shape change and actin
assembly driven by Fc
RIIA. In particular, Arp2/3 complex requires
PI3-kinase activity for binding to actin, where, in concert with gelsolin, it
amplifies actin filament barbed ends
(14). These findings establish
a framework in which we can further understand platelet activation and
signaling between Fc
receptors and actin and clarify physiological
roles for Fc
RIIA on human platelets.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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