 |
INTRODUCTION |
As the suspension medium temperature falls below 15 °C
platelets abruptly change their shape from smooth discs into spiny spheres with irregular projections and aggregate (1-5). These events
resemble platelet responses to thrombin, ADP, collagen, and other
stimuli that operate optimally at 37 °C through
energy-dependent signaling reactions resulting in actin
remodeling. However, cooling slows these reactions and causes most cell
types to round up. The response of human blood platelets to chilling
has profound medical consequences. It limits the storage of over 90 milion platelet units collected worldwide per year for transfusion to 5 days at room temperature, because longer storage leads to unacceptable amounts of microbial growth. Even 5-day storage of platelets without refrigeration results in occasional septic complications following transfusion (6, 7).
Unactivated discoid platelets have a unique submembrane coil of
microtubules. Some microtubules depolymerize at low temperatures, and
the microtubule coils of platelets dissolve in the cold (1, 8). At
reduced temperatures, cells also become less able to maintain
energy-dependent low cytosolic calcium levels, and chilled platelets have increased cytosolic calcium concentrations (4). Since
calcium-dependent severing of the actin scaffolding that maintains the discoid shape of the resting platelet is an early step in
normal platelet activation, this calcium rise is presumably a mediator
of cold-induced platelet activation. In addition to these clues to
possible mechanisms underlying cold activation of platelets, recent
work has implicated phosphoinositide-mediated actin assembly (9, 10).
Platelet stimulation through the PAR-1 receptor activates the Rho
GTPase Rac leading to the synthesis of polyphosphoinositides
(ppIs).1 Studies in
permeabilized platelets indicated that these lipids induce actin
assembly by producing actin nucleation sites equivalent to actin
filament fast growing barbed ends. Since these ppIs release barbed end
capping proteins such as gelsolin from actin filaments in permeabilized
platelets, uncapping of preexisting actin filaments is one pathway
proposed from ppIs to actin assembly and is amplifiable by actin
filament severing and capping, which increase the number of ends. A
second pathway involves ppIs and Cdc42-activated unfolding of
Wiskott-Aldrich syndrome protein (WASp) family proteins, which then
bind the Arp2/3 complex, resulting in branching barbed end nucleation
at the cell cortex that leads to cell movement (11-16). Receptor
tyrosine kinases, the Rho family GTPase Cdc42, and probably G-protein-coupled receptors transmit the signals to WASp-Arp2/3 (17-20) and link signaling pathways to cell motility.
Although ppI turnover would predictably diminish in the cold, if
degradation declined more than synthesis, net ppI levels might
increase, leading to actin assembly. A more plausible explanation is
that temperature influences the structure of these lipids. The physical
chemistry of lipid presentation affects lipid-protein interactions in
general and gelsolin actin binding in particular (21). Tablin et
al. (22) have proposed evidence for lipid phase changes at the
critical temperature for cold-induced platelet activation.
We previously showed that chilled platelets assemble actin from barbed
end nuclei (4). In this paper, we document that chilling activates
ppI-induced barbed end assembly independently of phospholipid synthesis
and GTPase activation. We show that ppIs work through both actin
filament barbed end uncapping and Arp2/3-mediated nucleation in chilled
platelets. We also provide direct evidence that chilling of platelets
activates gelsolin to amplify the actin remodeling response.
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EXPERIMENTAL PROCEDURES |
Materials--
Chemical reagents were purchased from Sigma.
Rabbit skeletal muscle actin was isolated and labeled with pyrene as
previously described (23). The QRLFQVKGRR 10-mer
polyphosphoinositide-binding peptide was synthesized based on residues
160-169 of gelsolin (24). The Arp3 component of the Arp2/3 complex was
detected using affinity-purified anti-Arp3 antibody provided by Dr.
Matt Welch (University of California, Berkeley, CA). Rabbit polyclonal anti-adducin antibody was kindly provided by Dr. Vann Bennett (Duke
University Medical Center, Durham, NC). The monoclonal anti-gelsolin antibody (2C4) was previously described (25).
GST Fusion Proteins--
Vectors encoding GST-CA (aa 450-505 of
N-WASp) and GST-VCA (aa 392-505 of N-WASp) constructs, derived from
the C-terminal end of N-WASp, were kindly provided by Drs. Rajat
Rohatgi and Marc Kirschner (Department of Cell Biology, Harvard Medical
School, Boston, MA). The GST-RacN17 and -Cdc42N17 expression constructs were kindly provided by Dr. G. Bokoch (Scripps Research Institute, La
Jolla, CA). GST proteins were expressed in Escherichia coli, purified on glutathione-agarose beads, and stored at
80 °C in concentrations of
1 mg/ml. Proteins were thawed at 37 °C and diluted immediately before use. Purity was checked following
SDS-polyacrylamide gel electrophoresis through 15% gels and
staining with Coomassie Brilliant Blue.
Preparation of Human and Mouse Platelets--
Blood was drawn
from normal human volunteers by venipuncture into 0.1 volume of
Aster-Jandl citrate-based anticoagulant as previously described (26).
Platelet-rich plasma was prepared by centrifugation of anticoagulated
blood at 100 × g for 20 min, and platelets were
separated from the plasma proteins by gel filtration (26) through a
small Sepharose 2B column. Blood was obtained from wild-type and
gelsolin
/
(27) mice by cardiac puncture into 0.1 volume of
Aster-Jandl anticoagulant. Mouse platelet-rich plasma was separated
from the red blood cells by centrifugation of the blood at 100 × g for 6 min, followed by centrifugation of the supernatant
and the buffy coat again at 100 × g for 6 min. Mouse
platelets were isolated from platelet-rich plasma using a metrizamide
gradient (28). Briefly, platelets were concentrated between 25 and 10%
metrizamide in 140 mM NaCl, 5 mM KCl, 12 mM trisodium citrate, 10 mM glucose, 12.5 mM sucrose, pH 6, by centrifugation at 1,100 × g for 12 min. This washing procedure was repeated, and the
platelets were resuspended in 140 mM NaCl, 3 mM
KCl, 0.5 mM MgCl2, 5 mM
NaHCO3, 10 mM glucose, and 10 mM
Hepes, pH 7.4. The concentration of human and mouse platelets was
adjusted to 3 × 108/ml, and platelets were allowed to
rest for 30 min at 37 °C before use.
Permeabilization of Platelets with
n-Octyl-
-D-glucopyranoside (OG)--
Resting platelets
in suspension (90 µl containing 1.5 × 108
platelets) were permeabilized at 37 °C by the addition of one-ninth volume (10 µl) of 2.5% OG in 60 mM Pipes, 25 mM HEPES, 10 mM EGTA, 2 mM
MgCl2, 2 µM phallacidin, and protease
inhibitors (PHEM buffer) (29). The detergent-platelet suspension was
mixed, and the platelets were allowed to extract for 30 s at
37 °C. TRAP, GTP
S, GDP
S, GST-RacN17, GST-Cdc42N17, GST-VCA, or
GST-CA were added to the OG-permeabilized cells just before cooling.
Permeabilized cells were then cooled for 5 min at ice bath temperatures
and rewarmed for 2 min at 37 °C. This procedure resulted in 70-80%
of the platelet permeabilized as judged by the incorporation of
FITC-phalloidin into the platelets (data not shown).
Phospholipids--
Phosphatidylserine
(L-
-phosphatidyl-L-serine dipalmitoyl
(C16:0)) and PtdIns-4,5-P2 were obtained from Sigma.
Phosphatidylserine and PtdIns-4,5-P2 were dissolved by
sonication in water as described by Janmey and Stossel (21) and were
added to the chilled and rewarmed OG permeabilized platelets.
Morphological Studies--
25-mm round coverslips were attached
to the bottom of 35-mm plastic Petri dishes, each having a 10-mm hole
punched in its bottom. The coverslips were coated with 1 mg/ml bovine
serum albumin in Dulbecco's phosphate-buffered saline (Life
Technologies, Inc.) for 10 min. For experiments using OG, the platelets
were permeabilized for 30 s using a final concentration of 0.25%
OG. OG suspensions were chilled in plastic tubes for 5 min in an ice
bath. In experiments on rewarmed platelets, the tubes were rewarmed to
37 °C for 2 min in a water bath. Platelets were fixed for
morphological examination with a final concentration of 3.4%
formaldehyde in Dulbecco's phosphate-buffered saline. The fixed
platelets were viewed in a Zeiss IM-35 inverted microscope using
differential interference contrast optics and a 100× oil immersion
objective. Images were captured with a Hamamatsu C2400 CCD camera
(Hamamatsu, Japan), processed for background subtraction and frame
averaging with a Hamamatsu ARGUS image processor, and digitally stored
with a Macintosh computer equipped with a SCION frame grabber LG-3
(SCION, Frederick, MD).
Phospholipid Labeling and Extraction--
Human platelets were
pelleted and washed from platelet-rich plasma by two sequential
centrifugations at 800 × g for 10 min in the presence
of 1 µM prostaglandin E1 (Sigma). The
purified platelets (~109/ml) were incubated for 1 h
at 37 °C with 2 mCi/ml of [32P]orthophosphoric acid.
32P in the medium was separated from the platelets
by gel filtration over a Sepharose 2B column as described above. The
platelets were incubated at ice bath temperatures for 10, 20, 30, or 40 min, and total phospholipids were extracted using two combined washes of chloroform/methanol/HCl. ppIs were deacylated and analyzed by HPLC
as described (9, 30, 31).
Measurement of Filament End Numbers--
Intact platelets (90 µl containing 1.5 × 108 platelets), incubated at
ice bath temperatures for 5 min or treated with 1 unit/ml thrombin for
60 s, were permeabilized with one-ninth volume of 1% Triton X-100
in PHEM buffer. To assess filament end numbers in a
temperature-dependent manner, intact platelets (90 µl
containing 1.5 × 108 platelets) were incubated for 5 min at 37, 25, 20, 15, 10, 5, or 0 °C with or without the addition
of 25 µM TRAP for 60 s and were permeabilized as
described above. To assay filament barbed ends, 185 µl of a solution
containing 100 mM KCl, 2 mM MgCl2, 0.5 mM ATP, 0.1 mM EGTA, 0.5 mM
dithiothreitol, and 10 mM Tris, pH 7.0, was added to 100 µl of the Triton X-100 or OG-lysate. The actin polymerization rate
assay was started by the addition of 15 µl of 20 µM
monomeric pyrene-labeled rabbit skeletal muscle actin to a final
concentration of 1 µM. In this assay, the fluorescence increase is proportional to actin assembled into filaments.
Fluorescence was recorded in a LS50 spectrofluorimeter (PerkinElmer
Life Sciences) using excitation and emission wavelengths of 366 and 386 nm, respectively. Actin assembly inhibited by 2 µM
cytochalasin B is defined as occurring at the barbed end of the actin
filament. Activity not inhibited by cytochalasin B is defined as
pointed end assembly. The number of free actin filament barbed ends was
calculated from the known assembly rates of actin and the cytochalasin
sensitivity of this assembly as previously described (23). The barbed
and pointed end addition rates used are 10 and 1 monomer
s
1, respectively (32).
Immunoblot Analysis of Platelet Cytoskeletal
Proteins--
Resting and cold-activated platelets (1, 2, 5, and 30 min at ice bath temperatures) or platelets loaded with 40 µM EGTA-AM for 30 min at 37 °C and then chilled (1, 2, 5, and 30 min at ice bath temperatures) were lysed using a final Triton
X-100 concentration of 0.1% in PHEM buffer. The lysates were
centrifuged at 450,000 × g for 30 min at 4 °C, and
the resultant pellets and supernatants were separated. The pellet,
corresponding to the cytoskeleton, was resuspended to 133% of original
volume in 1× SDS-polyacrylamide gel electrophoresis loading buffer
(33) containing 5%
-mercaptoethanol. The supernatant was dissolved
by the addition of 33% of the original volume of 4×
SDS-polyacrylamide gel electrophoresis buffer. The samples were boiled
for 5 min. Proteins were displayed by SDS-polyacrylamide gel
electrophoresis and transferred onto Immobilon-P membrane (Millipore
Corp.). Membranes were blocked using 1% bovine serum albumin 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).
Measurement of F-actin Content--
Resting or cold-activated
platelets (1, 2, 5, and 30 min at ice bath temperatures) were fixed in
3.4% formaldehyde and permeabilized with 0.1% Triton X-100 in the
presence of 10 µM FITC-phalloidin. Bound FITC-phalloidin
was quantified by fluorescence-activated cell sorting analysis using a
Beckton Dickinson FACSCalibur flow cytometer (Franklin Lakes, NJ). A
total of 10,000 events were analyzed for each sample.
 |
RESULTS |
Actin Assembly in OG-permeabilized Platelets Induced by
Chilling--
Fig. 1 shows that most
platelets remain discoid at 37 °C after permeabilization with OG
(Fig. 1a) and that these permeabilized platelets change
shape when chilled (Fig. 1b). Chilled OG-permeabilized platelets have blebs on their surfaces and elaborate filopodial-like structures that remain after rewarming (Fig. 1c). Rewarming
is, however, required to detect actin filament barbed ends that
nucleate the assembly of pyrene-actin, suggesting that OG-treated
platelets reseal upon cooling but become permeable again when rewarmed
(Fig. 1d). We verified this idea by extracting
OG-permeabilized cells with Triton X-100 after cooling, followed
or not by rewarming. After Triton X-100 treatment, 199 ± 30 barbed end nucleation sites were detectable in chilled platelets, an
increase of 3-fold over cells maintained at 37 °C. Chilling of
OG-permeabilized platelets for
5 min at 4 °C followed by rewarming
led to the production of 170 ± 10 barbed ends/cell, demonstrating
that the permeabilized cells retain ~90% of their response to cold.
Exposure of filament ends was restricted to barbed ends in
OG-permeabilized platelets, since 2 µM cytochalasin B, an
agent that binds to the barbed end and inhibits monomer assembly onto
this end, abolished all detectable actin assembly from OG-permeabilized
platelets (data not shown). In summary, cooling and rewarming of
permeabilized platelets reports barbed end exposure and allows
dissection of this process through the addition of inhibitory reagents
normally unable to penetrate into platelets.

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Fig. 1.
Morphology and barbed end actin nucleation
capacity of OG-permeabilized platelets before and after chilling.
This gallery of DIC micrographs shows that OG-permeabilized platelets
retain their capacity to change shape in the cold by blebbing and
forming protrusions at their surfaces and that rewarming does not
reverse these changes. a, OG-permeabilized platelets remain
discoid when maintained at 37 °C. b, chilling of
OG-permeabilized platelets to ice bath temperatures for 5 min causes
the elaboration of membrane protrusions similar to pseudopodia extended
by activated cells. c, chilled OG-permeabilized cells retain
their spiny appearances after rewarming. d, chilling of
OG-permeabilized platelets causes the exposure of barbed end-directed
actin nucleation sites. Shown are barbed end actin filament nucleation
site counts in OG-permeabilized platelets maintained at 37 °C
platelets (Rest), chilled for 5 min in an ice-bath
(Cold), or chilled for 5 min and then rewarmed to 37 °C
for 2 min (Cold + RW). All platelets were
permeabilized with OG. Some OG-permeabilized platelets were further
treated with 0.1% Triton X-100 after the temperature shifts to
guarantee complete permeabilization and access of the pyrene-actin
probe. The bars show the mean ± S.D. for five separate
experiments.
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A ppI-binding Peptide Inhibits Actin Filament Barbed End Exposure
in Response to Cold--
The addition of a 10-mer peptide derived from
the ppI-binding site of gelsolin to OG-permeabilized platelets
diminished barbed actin filament end exposure when permeabilized
platelets were chilled for 5 min. 2 µM peptide decreased
detectable barbed ends by ~65%, and 20-30 µM
completely inhibited barbed end exposure following chilling (Fig.
2a). Resting platelets have
44 ± 16 exposed actin filaments barbed ends. This result is
consistent with previous studies showing that 25 µM of
the 10-mer peptide inhibits all barbed end nucleation in permeabilized
PAR-1-activated platelets and
formyl-methionine-leucine-phenylalanine-activated neutrophils (9, 34).
Incubation with 25 µM of the 10-mer peptide prevented the
growth of protrusions from the surface of chilled permeabilized platelets (Fig. 2b). The addition of
PtdIns-4,5-P2 to OG-permeabilized, peptide-treated
platelets rescued actin nucleation in a dose-dependent manner, whereas phosphatidylserine (PS) was without effect
(Fig. 2c). These data show that ppIs are intimately involved
in the actin assembly reaction that distorts the shape of the platelet in the cold. To determine whether the ppI content changes during the
chilling process, platelets, loaded with 32P to label the
phospholipid pool, were incubated at ice bath temperatures for 5-40
min. Fig. 3 shows that cooling did not
alter the content of D3- and D4-containing ppIs in platelet membranes
at time points when actin assembly and shape change are maximal.

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Fig. 2.
A peptide that binds and sequesters ppIs
greatly diminishes the exposure of barbed end directed actin nucleation
sites induced by cold. a, effect of the
gelsolin-derived ppI-binding peptide on the number of free barbed ends
exposed in OG-permeabilized and chilled platelets. The peptide was
added after OG-permeabilization and before chilling and rewarming of
the platelets. b, chilling of OG-permeabilized platelets in
the presence of 25 µM of the gelsolin-derived ppI-binding
peptide prevents the bulk of the shape change induced by cooling.
Peptide-treated platelets lack cellular protrusions although some cells
still convert from discs into more spherical shapes. c,
PtdIns-4,5-P2 reverses the inhibition of nucleation induced
by chilling. PtdIns-4.5-P4 or phosphatidylserine (PS)
micelles were added to OG-permeabilized platelets treated with a 25 µM concentration of the gelsolin-derived ppI-binding
peptide. Actin nucleation was recovered only in the presence of
PtdIns-4,5-P2. The bars show the mean ± S.D. for three separate experiments.
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Fig. 3.
Chilling of platelets to 4 °C does not
result in the synthesis or degradation of membrane ppIs. Platelet
ppIs, labeled to equilibrium with 32P, were isolated from
resting and chilled platelets. The relative distribution of each ppI
type was quantified by HPLC analysis. Profiles are shown of
phosphatidylinositol 3-trisphosphate
(PI3P) and phosphatidylinositol
3,4-bisphosphate (PI34P2)
(top) and of phosphatidylinositol 4-phosphate
(PI4P) and PtdIns-4,5-P2
(PI45P2) (bottom) in
platelets incubated at ice bath temperatures for 5-40 min.
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Formation of Barbed End Nucleation Sites is
Temperature-dependent--
To determine the temperature
dependence of cold-induced actin assembly, platelets were incubated at
decreasing temperatures for 5 min. As the temperature decreased, the
barbed end number per platelet increased from 55 ± 9 at 37 °C
to maximal values at or below 10 °C of 254 ± 21 barbed ends
per cell. The platelet F-actin content increased maximally by ~25%
at temperatures of
15 °C. Both barbed end exposure and F-actin
content begin to increase when the temperature decreases to
20 °C.
Tablin et al. (22) have shown that membrane phase
transitions and platelet shape changes begin at this temperature.
Platelets lose their responsiveness as their temperatures decrease and
do not respond to TRAP at
5 °C (Fig.
4a).

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Fig. 4.
Temperature dependence of the formation of
barbed end nucleation sites and actin assembly. Platelets were
incubated at various temperatures (37, 25, 20, 15, 10, 5, or 0 °C)
for 5 min. a, barbed ends were counted before
(closed circles) or after the addition of 25 µM TRAP for 60 s (open
circles). b, F-actin content was measured before
(closed circles) or after the addition of 25 µM TRAP for 60 s (open
circles). The values of both experiments are the means ± S.D. of six individual points.
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GDP
S and Dominant Negative GTPases Do Not Inhibit the
Cold-mediated Actin Filament Barbed End Exposure--
When platelets
are activated at 37 °C by ligation of the PAR-1 receptor, GTPases
are upstream of ppI production (9, 10). As reported, GTPase
antagonists, GDP
S or dominant negative GTPases, are potent
inhibitors of PAR-1-mediated signaling to actin in platelets (9). Fig.
5 shows that actin assembly is not
coupled to GTPases in chilled OG-permeabilized platelets. The
nonhydrolyzable GDP
S and GTP
S analogs fail to affect the number
of barbed ends exposed after chilling of OG-permeabilized platelets. In
accordance with this finding, the dominant negative GTPases, 1.5 µM GST-N17Rac1 or 3 µM GST-N17Cdc42, do not
affect the number of barbed filament ends produced in chilled
OG-permeabilized platelets (Fig. 5). These data, combined with a lack
of new ppI synthesis in the cold and the temperature dependence of
barbed end exposure, support the notion that lipid rearrangements,
uncoupled from receptors and GTPases, induce actin assembly and shape
change in cooled platelets.

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Fig. 5.
The production of barbed end directed
nucleation sites is uncoupled from GTPases. Effect of treating
OG-permeabilized platelets with the guanine nucleotide analogs GDP S
or GTP S on the number of free actin filament barbed end nucleation
sites in resting or chilled platelets for 5 min (Cold).
OG-permeabilized platelets were also treated with 25 µM
TRAP for 1 min at 37 °C in the absence and presence of GDP S.
GDP S has been shown previously to inhibit barbed end exposure
mediated by TRAP. Negative dominant GTPases do not block the exposure
of actin filament nucleation sites induced by cooling. Effects of
bacterially expressed GST-N17Rac1 (1.5 µM) or
GST-N17Cdc42 (3 µM) on the number of barbed filament ends
in OG-permeabilized chilled platelets. Values are mean ± S.D. for
five individual experiments.
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Barbed End Capping Proteins--
Barbed end capping proteins such
as gelsolin or adducin regulate platelet actin assembly. In the resting
platelet, gelsolin is inactive, and most of it is not bound to actin
and therefore extractable by detergents (35). Binding to actin by
gelsolin is rapidly induced by the intracellular free calcium increase following PAR-1 ligation. A large fraction of the bound gelsolin subsequently dissociates from actin. Fig.
6a shows that gelsolin reversibly associates with the actin cytoskeleton fraction of chilled
platelets. Gelsolin is ~80-90% detergent-extractable in resting
platelets at 37 °C, but 20-30% becomes inextractable
5 min after
chilling. In contrast, the small amount of gelsolin in the resting
cytoskeleton (10%) dissociates from the cytoskeletal fraction of
EGTA-AM-loaded and cooled platelets (Fig. 6a). As we
previously reported, platelet F-actin content increased following cooling (4). The F-actin content increases from 40 to ~60% after
chilling for 30 min, as determined by FITC-phalloidin binding by flow
cytometry (data not shown) (4). The inset in Fig.
6a shows the gelsolin/F-actin ratio in cooled untreated
platelets and in platelets preloaded with 40 µM EGTA-AM.
The ratio increases from 1:300 in resting cells to 1:180 after 5 min of
cooling and then decreases to 1:250 after 30 min of chilling. In
contrast, the ratio decreased to 1:1000 within the first minute of
cooling in platelets loaded with EGTA-AM. Direct evidence that gelsolin is involved in the actin response induced by chilling comes from experiments on platelets from gelsolin
/
mice. Fig. 6b
shows that gelsolin
/
platelets undergo only small distortions from their discoid shape when cooled compared with wild-type mouse platelets. The most prominent shape change is the elongation of the
discs into barbell shapes, although a few filopodia and blebs are
observable. Wild-type mouse platelets lose their discoid shapes and
protrude filopodia and blebs in similar fashion to human platelets when
chilled. Gelsolin
/
platelets expose 50% less barbed ends upon
chilling compared with gelsolin +/+ platelets. Fig. 6c
reports that cold activation stimulates a ~3-fold increase of barbed
end exposure in wild-type mouse platelets, whereas gelsolin
/
platelets have only a ~1.5-fold increase in barbed ends. Exposure of
filament ends is restricted to the barbed ends in the Triton X-100
permeabilized platelets, since 2 µM cytochalasin B
abolished all detectable pyrene actin assembly (data not shown). Murine
platelets activated with 1 unit/ml thrombin for 1 min at 37 °C
increased the number of actin nuclei by ~5-fold in wild-type and
~2.8-fold in the gelsolin null platelets.

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Fig. 6.
Role of gelsolin in barbed end exposure
mediated by cooling. A, interaction of gelsolin with
the actin cytoskeleton of untreated chilled platelets
(closed circles) or platelets first incubated
with 40 µM EGTA-AM for 30 min (open
circles). Soluble gelsolin was separated from cytoskeletal
bound gelsolin in Triton X-100 platelet lysates by centrifugation at
450,000 × g for 30 min. Gelsolin was detected by
immunoblotting. The graphs quantify the movement of gelsolin into the
platelet cytoskeleton. The values are the means ± S.D. for three
individual experiments. The inset shows the gelsolin/F-actin
ratio in the cytoskeleton of chilled platelets (closed
squares) and platelets preloaded with EGTA-AM and then
chilled (open squares). B, gelsolin
/ platelets have a diminished shape change response to chilling
compared with wild-type mouse platelets. The panel compares
the morphology of resting (Rest) and chilled
(Cold) platelets of wild-type (Gsn +/+) and gelsolin /
mice. C, gelsolin / platelets expose fewer barbed end
nucleation sites when chilled compared with normal mouse platelets.
Actin nucleation sites when quantified in Triton X-100 permeabilized
gelsolin +/+ and gelsolin / platelets chilled for 5 min or
stimulated with 1 unit/ml thrombin for 1 min. The resting barbed end
numbers were 65 ± 4 in wild-type platelets and 83 ± 16 in
gelsolin / platelets.
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Chilling also dissociates the barbed end capping protein adducin from
the platelet actin cytoskeleton (Fig. 7).
In resting platelets, ~70-80% of the total adducin is bound to the
cytoskeletal fraction. Adducin begins to dissociate from the platelet
actin cytoskeleton 2 min after chilling, and the dissociation becomes maximal after 30 min (Fig. 7). The inset in Fig. 7 shows the
adducin/F-actin ratio in the cytoskeleton of cold-activated platelets.
The amount of adducin bound to the cytoskeletal fraction decreases
during the cold activation 1:100 in resting cells to 1:333 after 30 min. The distribution of CapZ, a barbed end capping protein that
terminates actin filament assembly by capping the ends of elongating
filaments (36-38), did not change following cooling of platelets (data
not shown).

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Fig. 7.
Chilling dissociates adducin from the
platelet cytoskeleton. Time course of adducin release from the
cytoskeleton as detected by immunoblotting of soluble and insoluble
fractions of lysates of chilled platelets (circles). The
inset shows the adducin/F-actin ratio during cooling
(squares). The data are the mean ± S.D. for three
experiments.
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Role of the Arp2/3 Complex in the Platelet Actin Assembly Induced
by Chilling--
To determine whether the Arp2/3 complex participates
in cold-induced actin assembly of OG-permeabilized platelets, we used constructs derived from N-WASp that inhibit Arp2/3 complex-mediated actin nucleation. The C-terminal CA domain of N-WASp (amino acids 450-505) binds to the Arp2/3 complex, inhibiting its function. In
contrast, N-WASp VCA domain (amino acids 392-505) binds to actin
monomers and the Arp2/3 complex and leads to an enhancement of actin
nucleation (20). GST-CA added to the OG-permeabilized platelets before
chilling inhibits the barbed end number induced by chilling in a
concentration-dependent manner (Fig.
8). The addition of >0.1
µM of GST-CA to the OG-permeabilized platelets diminished
the number of barbed ends measurable after chilling by ~40% (Fig.
8), indicating that Arp2/3 activation contributes about half the nuclei
used in the actin assembly reaction of chilled platelets.

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Fig. 8.
Role of the Arp2/3 complex in the formation
of barbed end nucleation sites induced by chilling to 4°C. Shown
is dose response for the inhibition of barbed end nuclei by the
dominant negative N-WASp C terminus (GST-CA) construct in
response to the activation of OG-permeabilized platelets through
chilling. The addition of saturating concentrations of GST-CA reduced
by ~40% the production/exposure of barbed end-directed nucleation
sites in OG-permeabilized chilled platelets. The resting barbed end
numbers were 57 ± 19. The values are the means ± S.D. for
three individual experiments.
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 |
DISCUSSION |
Our results show that actin assembly observed during cold
activation of platelets results from membrane lipid rearrangements without activation of GTPases and synthesis of ppIs. The mechanics of
cold activation involve known regulatory processes of actin assembly. First is the activation of gelsolin to sever actin filaments through increased intracellular calcium (23, 35). Cooling of platelets
leads to a slow rise in free cytosolic calcium to levels of 200-300
nM (4, 39), which is not surprising, because cold decreases
the activity of calcium pumps that function to extrude calcium (40). In
chilled platelets, gelsolin, the major actin filament-severing protein
of platelets, transiently associates with the actin cytoskeleton and
becomes maximally bound after 5 min. Translocation into the
cytoskeleton is prevented if platelets are loaded with EGTA. We
previously demonstrated barbed end exposure to be inhibited by ~50%
in Quin2-loaded platelets, consistent with the loss of
calcium-activated gelsolin severing activity (4). Second is the
exposure of actin filament barbed ends that induce new actin assembly
by ppIs (9). ppIs are known to be intimately involved in the
barbed end-based nucleation and actin assembly of PAR-1-activated
platelets (9). Unlike PAR-1-mediated activation, barbed end
exposure/nucleation is uncoupled in the cold from GTPases and ppI
synthesis. The addition of GDP
S or the negative dominant Rho family
GTPases N17Cdc42 or N17Rac did not effect barbed end exposure in
permeabilized and chilled platelets.
To investigate whether the cold-induced actin assembly observed in
platelets involves ppI-mediated uncapping of filaments, we applied a
permeabilization scheme used to define the mechanisms of the
PAR-1-mediated actin assembly in platelets and
formyl-methionine-leucine-phenylalanine-induced actin assembly in
polymorphonuclear leukocytes (9, 34). Platelets, briefly permeabilized
with OG, were chilled to 4 °C, and shape change and actin assembly
were monitored. After chilling, blebs and protrusions developed on the
surfaces of OG-permeabilized platelets in similar fashion to those
extruded by chilled intact platelets (43). These findings demonstrate
that OG-treated platelets retain their response to cold in terms of
actin-driven shape change. Cold induced the formation of ~200 new
barbed filament ends per platelet within 5 min, consistent with our
previous study showing barbed end exposure to increase within minutes
when platelets were shifted to ice bath temperature (4). This number of
barbed ends represents ~40% of the total barbed end number exposed
following PAR-1 activation (9). Treatment of OG-permeabilized platelets with a peptide that binds and sequesters ppIs prevents cold-induced protrusive activity and blocks all barbed end exposure. The inhibitory effect of the ppI binding peptide on actin assembly was rescued by the
addition of PtdIns-4,5-P2 micelles to the OG-permeabilized and chilled platelets. This demonstrates that ppIs are involved in the
reactions that lead to barbed end exposure/nucleation in the cold.
Since net ppI synthesis or degradation was not observed following
chilling, we propose that a structural change of ppIs in the membrane
induces the exposure of actin filament barbed ends and the induction of
actin assembly. The temperature dependence of barbed end exposure
correlates with that of the membrane phase transition and would change
lipid packing, causing ppI clustering (22). Aggregation of ppIs would
potentiate the activity of the phosphoinositol-lipid head groups in the
platelet plasma membrane as has been shown for ppIs in mixed lipid
vesicles in vitro (41). The aggregation process would also
be predicted to occur independently of the activity of GTPases, as observed.
Targets of ppIs--
Targets of these phospholipid clusters
include barbed end capping proteins and the Arp2/3 complex. The
uncapping of actin filament barbed ends was the first pathway defined
to be involved in ppI-induced actin assembly, and evidence has been
provided by us that barbed end capping proteins are released from actin filaments during cell activation by the addition of ppIs to
permeabilized platelets (36). Our data are consistent with the idea
that 50-60% of the barbed end-based nucleation activity in chilled
platelets derives from actin filaments severed by gelsolin and
subsequently uncapped. The small amount of gelsolin associated with the
resting cytoskeleton dissociates when EGTA-AM-loaded platelets are
chilled for 5 min, indicating that signals (ppIs) that
dissociate gelsolin from actin remain intact in these cells. A similar
dissociation from the cytoskeletal fraction was observed after 30 min
in untreated cooled platelets. In addition gelsolin-deficient platelets
were found to be less responsive to chilling and to produce only 50% of barbed end nucleation sites of normal platelets.
Adducin is a second platelet protein that may contribute to the actin
nucleation induced by ppIs. Adducin was first identified as a barbed
end capping protein in the membrane cytoskeleton of red blood cells
(42, 43). Adducin possesses a myristoylated alanine-rich protein kinase
C substrate-related domain that is required for its actin filament
barbed end capping activity (44). This 25-amino acid basic domain is
phosphorylated at multiple serines by protein kinase C and also binds
to calmodulin or PtdIns-4,5-P2 to regulate its interaction
with actin (45, 46). In the resting platelet, 70-80% of the total
adducin is associated with the actin cytoskeleton. Chilling dissociates
adducin from the cytoskeleton. Adducin's dissociation could be due to
the calcium activation of calmodulin in chilled platelets or, since the
gelsolin-derived ppI-binding peptide completely inhibits cold-induced
barbed end exposure, by phospholipid binding within this myristoylated
alanine-rich protein kinase C substrate domain. It is not clear yet
which mechanism is involved, but dissociation of adducin from actin
filaments is expected to contribute to barbed end exposure.
A more recently discovered mechanism leading to cytoplasmic actin
assembly is the de novo actin nucleation by the Arp2/3
complex (19, 20, 47-51). We investigated whether the Arp2/3 complex participates in cold-induced actin assembly by adding a negative dominant inhibitor of its function (GST-CA) to the permeabilized platelets. GST-CA inhibits the barbed end production induced through chilling by ~40% when added to OG-permeabilized platelets prior to
chilling. The Arp2/3 complex nucleates actin downstream of WASp family
proteins (WASp, N-WASp, Scar/WAVE), which require GTPases and ppIs to
become active (20, 52, 53). PtdIns-4,5-P2 and the small
GTPase Cdc42 co-activate N-WASp (20). N-WASp can be activated by
PtdIns-4,5-P2 micelles alone, whereas GTPCdc42 is a poor
activator of N-WASp in the absence of PtdIns-4,5-P2 (53).
Temperature Dependence of the Cold-induced Response--
It is
well established that platelets begin to change shape at temperatures
of <25 °C and that the number of activated platelets increases with
decreasing temperatures until 5 °C (22). We investigated the
relation of temperature to the barbed end nucleation activity in
platelets and found that free barbed end nucleation begins at
temperatures of <25 °C, as has been reported for the membrane phase
transitions that change the packing of membrane lipids (22). As
platelets are activated by cold, they lose their ability to respond to
receptor-mediated stimuli. Below 5 °C, platelets lack responsiveness
to TRAP. Although we expected barbed end nucleation in platelets
induced by TRAP to be maximal at 37 °C, higher barbed end nucleation
and actin assembly were found in platelets activated at 20-25 °C.
These results imply that at 20-25 °C nucleation activity derives
from the combined effects of temperature-induced nucleation and the
normal receptor-coupled signaling events. It also suggests that the
exquisite sensitivity of platelets to temperature may lead to a
coupling of membrane dynamics and signaling events mediating a maximal
physiological response at temperatures below 37 °C, such as those
that exist in dermis and at wound sites.
Our data show that platelet actin filament assembly induced by cooling
requires membrane ppIs to mediate the uncapping of actin filaments by
gelsolin and adducin and de novo actin nucleation by the
Arp2/3 complex. However, the signaling pathways are uncoupled from
receptors and small GTPases. The data lead to a better understanding of
how cooling leads to platelet shape changes and actin assembly and
shows their sensitivity to temperature changes. This knowledge may
contribute to a better storage of platelets.