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PLATELETS AND THROMBOPOIESIS
S6K1 and mTOR regulate Rac1-driven platelet activation and aggregation
Joseph E. Aslan,1,2 Garth W. Tormoen,1 Cassandra P. Loren,1 Jiaqing Pang,1 and Owen J. T. McCarty1,2
Departments of 1Biomedical Engineering and 2Cell and Developmental Biology, School of Medicine, Oregon Health & Science University,
Portland, OR
Platelet activation and thrombus formation are under the control of signaling
systems that integrate cellular homeostasis with cytoskeletal dynamics. Here,
we identify a role for the ribosome protein S6 kinase (S6K1) and its upstream
regulator mTOR in the control of platelet
activation and aggregate formation under shear flow. Platelet engagement of
fibrinogen initiated a signaling cascade
that triggered the activation of S6K1 and
Rac1. Fibrinogen-induced S6K1 activa-
tion was abolished by inhibitors of Src
kinases, but not Rac1 inhibitors, demonstrating that S6K1 acts upstream of
Rac1. S6K1 and Rac1 interacted in a protein complex with the Rac1 GEF TIAM1
and colocalized with actin at the platelet lamellipodial edge, suggesting that
S6K1 and Rac1 work together to drive
platelet spreading. Pharmacologic inhibitors of mTOR and S6K1 blocked Rac1
activation and prevented platelet spreading on fibrinogen, but had no effect on
Src or FAK kinase activation. mTOR inhibitors dramatically reduced collageninduced platelet aggregation and promoted the destabilization of platelet
aggregates formed under shear flow
conditions. Together, these results reveal
novel roles for S6K1 and mTOR in the
regulation of Rac1 activity and provide
insights into the relationship between the
pharmacology of the mTOR system and
the molecular mechanisms of platelet activation. (Blood. 2011;118(11):3129-3136)
Introduction
Platelets represent a specialized set of peripheral blood cells that
are optimally configured for adhesion, secretion and aggregation
at sites of vascular injury.1,2 The exposure of platelets to extracellular matrix proteins such as collagen or laminin, or endogenous
agonists such as ADP or thromboxanes, mediates hemostasis by
activating signaling pathways that ultimately result in platelet
adhesion and aggregation.3 On the engagement of the adhesive
proteins fibrinogen and fibronectin, platelet tyrosine kinases such
as Src, Syk and FAK are recruited to the platelet cytosolic cell
surface to initiate signaling pathways to drive platelet cytoskeletal
reorganization through the Rho family small GTPase Rac1.3-5 Rac1
regulates actin polymerization at the cell membrane to drive the
growth and extension of platelet lamellipodiae that form the basis
for platelet spreading.4 The molecular mechanisms by which
tyrosine kinases ultimately activate Rac1 remain ill-defined.
The 70 kDa ribosome S6 protein kinase (S6K1) regulates the
ribosome S6 protein to integrate processes of protein translation
with cell growth and cell proliferation.6 In cultured cells as well
as in vivo, mitogenic signals triggered by nutrients and growth
factors initiate a complex sequence of signaling events to activate
the mammalian target of rapamycin (mTOR), a serine/threonine
kinase which regulates S6K1 phosphorylation and activation.7
Treatment of cells with rapamycin (Sirolimus) or other inhibitors
of mTOR blocks S6K1 Thr389 phosphorylation and inhibits
S6K1 activation.8 The ability of mTOR inhibitors to arrest the
growth of transformed tumor cells with dysregulated mTOR
signaling has led to their development as antineoplastic agents that
are currently used to treat several malignancies.9 Imbalances in the
mTOR pathway are also involved in obesity, diabetes, inflammatory diseases and cardiac hypertrophy, and pharmacologic inter-
vention of mTOR has been proposed as a potential treatment for
these conditions.6
In addition to controlling protein translation and cell growth,
S6K1 and mTOR have roles in chemotaxis, cell migration, and
tumor cell invasion.10-12 Inhibition of mTOR and S6K1 with
rapamycin blocks the growth factor and nutrient mediated migration of intestinal cells,13 smooth muscle cells,14 and carcinoma
cells on surface substrates such as fibronectin.15-17 As these cells
migrate, integrin-mediated signals trigger an activation of mTOR
and S6K1, which in turn regulate the remodeling of the actin
cytoskeleton to control cell motility. The manner in which mTOR
pathways direct actin remodeling and cell movement are not
understood but may involve a colocalization of S6K1 with actin
stress fibers18 as well as actin remodeling proteins such as Rac1.
For instance, S6K1 interacts with Rac1 in transfected HEK
293 cells,19 and rapamycin can prevent cell migration through
inhibition of the small GTPases RhoA, Cdc42, and Rac1.20 Furthermore, S6K1 and mTOR work with Rac1 to reorganize the actin
cytoskeleton and direct the migration of ovarian cancer21 and
colorectal cancer cells.22 Rac1 activity is also regulated by the
tuberous sclerosis protein TSC2, a downstream target of Akt that
suppresses mTOR and S6K1 activity to control tumor cell polarity
and migration.23
Despite known functions of S6K1 and mTOR in cell migration
and chemotaxis, the roles of these signaling kinases in motilityrelated platelet processes in hemostasis and thrombosis remain
unexplored. This is of clinical relevance as mTOR inhibitors are
used as chemotherapy drugs for a growing number of malignancies.24 mTOR inhibitors such as rapamycin are also potent immunosuppressive and antiproliferative agents which prevent the rejection
Submitted January 31, 2011; accepted June 24, 2011. Prepublished online as
Blood First Edition paper, July 14, 2011; DOI 10.1182/blood-2011-02-331579.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2011 by The American Society of Hematology
BLOOD, 15 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 11
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3130
ASLAN et al
of transplanted organs as well the restenosis of intravascular
stents.25-27 The exact mechanisms by which mTOR inhibitors act as
immunosuppressants are not completely understood but may
involve inhibition of peripheral blood cells with immunologic roles
such as macrophages28 and neutrophils29 as well as platelets.30 In
this study, we investigate the role of mTOR/S6K1-mediated Rac1
activation in platelet spreading and platelet aggregation. We find
that platelets use an mTOR/S6K1 pathway to activate Rac1 and
regulate platelet spreading on fibrinogen. Moreover, mTOR has a
role in glycoprotein GPVI-mediated platelet aggregation as well
as maintaining the integrity of platelet aggregates formed under
physiologic shear conditions. Our findings provide novel insights
into the role of the mTOR signaling system in platelet biology and
contribute to an understanding of how mTOR and S6K1 function in
regulating Rac1 activation.
Methods
Reagents
EHT 1864, PP2, BAY 61-3606, apyrase, and fatty-acid–free BSA and
all other reagents were from Sigma-Aldrich except for as noted. WYE354 and Ku-0063794 were from Chemdea. Human fibrinogen was from
Enzyme Research. Collagen was from Chrono-Log. Rapamycin and
RAD001 were from LC Labs. MCF10A cells were from ATCC and
provided by the National Cancer Institute Physics-Oncology Program.
Cells were cultured in DMEM supplemented with 5% horse serum
(Invitrogen) and hydrocortisone, EGF, insulin, and cholera toxin.
D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone (PPACK) anticoagulant was from Calbiochem. Plasmids encoding GST and PAK-CRIB-GST were
provided by L. Machesky (Beatson Institute for Cancer Research, Glasgow,
United Kingdom). pGEX-Rac1 was from Addgene. Anti–Syk-pTyr323
(2715), Syk (2712), LAT-pTyr171 (3581), LAT (9166), FAK-pTyr576/577
(3281), FAK (3285), mTOR (2983), Raptor (2280), Rictor (2114), G␤L (3274),
GSK3␤-pSer9 (9336), and S6K1-pThr389 (9234) were from Cell Signaling.
Anti-Rac1 (23A8) and Raptor (1H6.2) were from Millipore. Anti-S6K1 (sc-230),
GSK3␤ (sc-9166), Rictor (sc-271081), TIAM1 (sc-872), mouse (sc-2025),
and rabbit (sc-2345) IgGs, and secondary goat anti–rabbit and anti–mouse
HRP secondary antibodies were from Santa Cruz Biotechnology.
Preparation of human washed platelets
Human venous blood was drawn in accordance with an Oregon Health &
Science University IRB–approved protocol from healthy donors into
sodium citrate and acid/citrate/dextrose as previously described.4 Plateletrich plasma (PRP) was prepared by centrifugation of anticoagulated
blood at 200g for 10 minutes. Platelets were further purified from PRP
by centrifugation at 1000g in the presence of prostacyclin (0.1 ␮g/mL).
Purified platelets were resuspended in modified HEPES/Tyrode buffer
(129mM NaCl, 0.34mM Na2HPO4, 2.9mM KCl, 12mM NaHCO3, 20mM
HEPES, 5mM glucose, 1mM MgCl2; pH 7.3) containing 0.1 ␮g/mL prostacyclin. Platelets were washed once by centrifugation and resuspended in
modified HEPES/Tyrode buffer at indicated concentrations.
Static adhesion assays
Glass coverslips were coated with human fibrinogen (100 ␮g/mL) followed
by surface blocking with BSA (5 mg/mL). Inhibitors or vehicle were added
to platelets in solution (2 ⫻ 107/mL) with 2 U/mL apyrase for 10 minutes
before exposure to immobilized fibrinogen at 37°C. After 45 minutes,
nonadherent platelets were discarded and surface-bound platelets were
washed 3 times with PBS. Coverslips were fixed in 4% paraformaldehyde
and washed with PBS. Platelets were imaged using Kohler illuminated
Nomarski differential interference contrast (DIC) optics with a Zeiss
63⫻ oil immersion 1.40 NA plan-apochromat lens on a Zeiss Axiovert
200M microscope as previously described.4
BLOOD, 15 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 11
Western blotting and immunoprecipitations
For platelet lysate experiments, 24-well tissue culture treated plates
(Corning) were coated with human fibrinogen (50 ␮g/mL) followed by
surface blocking with BSA (5 mg/mL). Platelets (300 ␮L, 5 ⫻ 108/mL in
modified HEPES/Tyrode buffer with 2 U/mL apyrase) were incubated in
fibrinogen-coated dishes for 45 minutes at 22°C. Nonadherent platelets
were discarded and surface-bound platelets were washed 3 times in 1 mL
PBS before collection into 100 ␮L lysis buffer (1% Nonidet P-40, 1%
N-octyl glucoside, 150mM NaCl, 10mM Tris/HCl, 1mM EGTA, 10mM
MgCl2, 1mM phenylmethylsulfonyl fluoride, 10 ␮g/mL leupeptin, 2mM
orthovanadate, and Sigma Phosphatase Inhibitor Cocktail, pH 7.4). Lysates
were denatured in an equal volume of Laemmli sample buffer (Bio-Rad)
with 0.5M dithiothreitol (100°C, 5 minutes), separated by SDS-PAGE,
transferred to polyvinylidene difluoride membranes, and immunoblotted
with indicated antibodies and HRP-conjugated secondary antibodies as
previously described.31 Protein was detected using ECL (Amersham
Biosciences). Blot films were digitally scanned and analyzed for band
intensity with ImageJ Volume 1.44 software. Band densitometry statistical
analysis was performed using a paired t test between vehicle and each test
condition for the antigen specified (supplemental Figure 2, available on the
Blood Web site; see the Supplemental Materials link at the top of the online
article). For coimmunoprecipitation studies, platelets treated as above were
lysed in 1 mL of M-PER Mammalian Protein Extraction Reagent (Pierce)
supplemented with 1mM DTT, 1mM phenylmethylsulfonyl fluoride,
10 ␮g/mL leupeptin, and Phosphatase Inhibitor Cocktail. Immunoprecipitation lysates were precleared with 25 ␮L Protein A/G PLUS-Agarose (Santa
Cruz Biotechnology) before incubation with 10 ␮g of indicated antisera or
control IgGs overnight at 4°C, followed by the addition of 25 ␮L Protein
A/G agarose. Captured protein complexes were washed 3 times in 1 mL of
M-PER and eluted into 50 ␮L of Laemmli sample buffer. To capture S6K1
from platelet lysates, M-PER lysates as above were incubated with 1 ␮g of
Rac1-GST or GST alone conjugated to glutathione sepharose for 1 hour,
then washed and eluted into Laemmli sample buffer with 0.5M DTT.
Rac1 activity assays
Rac1 activation was measured through capture of Rac1-GTP using the
CRIB domain of PAK1 as previously described.4 Platelets (5 ⫻ 107/mL in
modified HEPES/Tyrode buffer) were incubated at 37°C for 45 minutes in
35-mm dishes coated with human fibrinogen (100 ␮g/mL) or BSA in the
presence of apyrase (2 U/mL) and inhibitors as indicated. Nonadherent
platelets were discarded and surface-bound platelets were washed 3 times
in 1 mL of PBS and collected into 1 mL of lysis buffer (as in “Western
blotting and immunoprecipitations”). Lysates were incubated with 50 ␮L of
a 50% slurry of glutathione-sepharose beads (GE Healthcare Life Sciences)
conjugated to GST-PAK CRIB (60 minutes, 4°C). Beads were washed in
lysis buffer and bound protein was eluted by the addition of 50 ␮L of
Laemmli sample buffer with 0.5M DTT.
Confocal microscopy
Purified human platelets (1 ⫻ 107/mL) were spread on coverglass coated
with 25 ␮g/mL fibrinogen for 45 minutes at 37°C. Adherent platelets were
washed 3 times with PBS before fixation with 4% paraformaldehyde,
then washed in PBS and permeabilized with blocking buffer (PBS,
0.1% SDS ⫹ 1% BSA) for 1 hour. Platelets were stained with anti-S6K1
(1:50) or anti-TIAM1 (1:50) and anti-Rac1 (1:100) in blocking buffer
overnight at 4°C. Invitrogen Alexa Fluor secondary antibodies (1:200) and
Alexa Fluor 647 phalloidin (1:100) were added in blocking buffer for
1 hour. Coverslips were mounted with Fluoromount G (Southern Biotech)
on glass slides and visualized with a 60⫻ oil immersion objective on an
Olympus Fluo-View FV300 laser scanning confocal microscope.
Platelet aggregation and shape change
For platelet aggregation studies, 300 ␮L of purified human platelets
(2 ⫻ 108/mL) were treated with inhibitors as indicated in the presence of
2 U/mL apyrase. Platelet aggregation was triggered by 10 ␮g/mL collagen
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BLOOD, 15 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 11
mTOR REGULATES PLATELET ACTIVATION
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and monitored under continuous stirring at 1200 rpm at 37°C by measuring
changes in light transmission using a PAP-4 aggregometer as previously
described.4
Platelet aggregate stability under flow
Glass capillary tubes (Vitrocom) were coated with collagen (100 ␮g/mL)
and surface blocked with denatured BSA. PPACK-anticoagulated blood
was perfused at 37°C to form platelet aggregates, which were then chased
with modified HEPES/Tyrodes buffer containing 3 mg/mL fibrinogen
containing indicated treatments. Aggregate dissociation was imaged in real
time using Kohler illuminated Nomarski DIC optics with a Zeiss 40⫻
0.75 NA EC Plan Neofluar lens on a Zeiss Axiovert 200M microscope.
Time-lapse images were recorded with a Zeiss Axiocam MRm camera
using Slidebook 5.0 (Intelligent Imaging Innovations Inc). To compute
dissociation, images were manually outlined and quantified by determining
the number of pixels within each outline using a Java plug-in for ImageJ as
described previously.4
Results
S6K1 is activated upstream of Rac1 and platelet
lamellipodia formation
Previously, we determined that the small GTPase Rac1 is required
for actin remodeling events that drive platelet lamellipodia formation and thrombus stability under shear.4 As platelets adhere
to an immobilized surface of fibrinogen, the platelet integrin
␣IIb␤3 recruits and activates tyrosine kinases such as Src and
Syk at its intracellular domains to ultimately lead to the activation of Rac1.4,32 To better understand how these tyrosine kinase
pathways activate Rac1 to mediate platelet spreading, we first
examined the effects of Src and Syk kinase inhibition on platelet lamellipodia formation. Human platelets were purified from
freshly drawn blood and allowed to bind to an immobilized
surface of fibrinogen. Surface-bound platelets were washed, fixed,
and then analyzed by Kohler-illuminated Normarski DIC microscopy. As seen in Figure 1A, pretreatment of platelets with PP2 or
BAY 61-3606, which inhibit Src or Syk kinases, respectively,
blocked the formation of platelet lamellipodia and prevented
the spreading of platelets on fibrinogen. Platelet lamellipodia
formation and spreading was similarly blocked by treatment with
the Rac inhibitors EHT 1864 (Figure 1A) and NSC23766 (supplemental Figure 1). Western blot analyses of platelet lysates
confirmed that PP2 blocked Src kinases as evidenced by loss of
the Src-dependent phosphorylation of Syk Tyr323 (Figure 1B).
Moreover, inhibition of Src kinases with PP2 abrogated the phosphorylation of the downstream effector FAK at Tyr576/577 (Figure
1B). The Syk inhibitor BAY61-3606 led to the inhibition Syk
activation as seen by blockade of LAT Tyr171 phosphorylation.
Rac1 appeared to have no role in these tyrosine kinase pathways
as the Rac1 inhibitor EHT 1864 did not affect Syk, FAK or LAT
phosphorylation.
To study the pathways by which platelet tyrosine kinases trigger
Rac1 activation, we measured the activation state of this small
GTPase through capture of GTP-Rac1 by the CRIB domain of
the p21-activated kinase PAK. Platelet spreading on fibrinogen
was marked by an increase in Rac1 activation as evidenced by
the precipitation of GTP-Rac1 from lysates of activated platelets
versus resting platelets (Figure 1C). Pretreatment of platelets
with PP2 or BAY61-3606 blocked Rac1 activation, indicating that
Src or Syk kinases are needed for Rac1 activation in response to
Figure 1. S6K1 is activated upstream of Rac1 in platelets. (A) Representative
DIC images of purified human platelets (2 ⫻ 107/mL) treated with vehicle (DMSO),
the Src inhibitor PP2 (20␮M), the Syk inhibitor BAY 61-3606 (1␮M) or the
Rac1 inhibitor EHT 1864 (50␮M), on a surface of fibrinogen (FG). Scale bar ⫽ 10 ␮m.
(B) Lysates from quiescent platelets in solution (basal) or FG-surface-attached
platelets were analyzed for Src, Syk and FAK activation by Western blotting (WB)
for Syk-pTyr323, LAT-pTyr171 and FAK-pTyr576/577. (C) Lysates were incubated
with glutathione-sepharose conjugated to GST-PAK-CRIB to capture activated
GTP-bound Rac1. Captured GTP-Rac1 and total Rac1 inputs were analyzed by
Western blotting. (D) Platelets were treated with inhibitors as above and analyzed
for S6K1 activation by Western blotting for S6K1-pThr389. PP2 and BAY 61-3606
decreased S6K1 phosphorylation by 86.9% and 66.9%, respectively (n ⫽ 3, P ⬍ .05).
EHT 1864 increased pS6K1 levels by 121% relative to vehicle (n ⫽ 3, P ⬍ .05).
fibrinogen. Rac1 activation was also inhibited by EHT 1864. To
test the hypothesis that S6K1 plays a role in Rac1-based platelet
lamellipodia formation we next looked at S6K1 phosphorylation
in fibrinogen-activated platelets. Quiescent platelets in solution
had no detectable S6K1 activation as demonstrated by Western
blotting for S6K1 phosphorylation at Thr389 (Figure 1D). Spreading on a surface of fibrinogen increased the phosphorylation of
S6K1 Thr389. Src and Syk inhibitors, which prevented platelet
spreading (Figure 1A), blocked S6K1 activation. However, EHT
1864, which also inhibits platelet spreading, did not block S6K1
activation, suggesting that S6K1 is activated upstream of Rac1 in
spreading platelets (Figure 1D).
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ASLAN et al
BLOOD, 15 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 11
established that Rac1 activity in tumor cells is regulated in part by
TIAM1,33 a Rac guanine nucleotide exchange factor (GEF) that is
known to interact with S6K1.34 To determine whether S6K1 and
Rac1 are associated with TIAM1 in platelets, we next immunoprecipitated TIAM1 from platelet lysates with sc-872, an antibody
that specifically captures TIAM1 from platelet lysates (supplemental Figure 3). TIAM1 was precipitated at similar levels from
quiescent platelets in solution as well as platelets spread on
fibrinogen (Figure 2D). As seen in Figure 2D, platelet activation
by a surface of fibrinogen increased the association of Rac1 with
TIAM1. Moreover, platelet activation was associated with an
increase in phosphorylation of S6K1 associated with TIAM1. Like
S6K1, TIAM1 also colocalized with Rac1 at the platelet lamellipodial edge (Figure 2E). Together, these data suggest that TIAM1
may associate with an S6K1-containing Rac1 signaling complex
which localizes to the lamellipodial edge of platelets to regulate the
spreading of platelets on fibrinogen.
mTOR regulates S6K1 phosphorylation, Rac1 activation,
and platelet spreading
Figure 2. S6K1 and Rac1 localize to the lamellipodial edge of spreading
platelets. (A) Purified human platelets were spread on coverglass coated with
25 ␮g/mL fibrinogen. After 45 minutes, platelets were fixed, stained for S6K1, Rac1,
and actin, and visualized by confocal microscopy. Scale bar ⫽ 2 ␮m. (B) S6K1 was
immunoprecipitated (IP) from lysates of quiescent platelets in solution or platelets
spread on fibrinogen and analyzed for coprecipitating Rac1 by Western blot.
Nonspecific rabbit immunoglobulins (IgG) were used as negative control for immunoprecipitations. Total S6K1 and Rac1 levels in whole-platelet lysates serve as input
controls. (C) Platelet lysates were incubated with GST or Rac1-GST glutathione
sepharose and captured S6K1 was analyzed by Western blot. Coomassie-stained
GST and Rac1-GST inputs are shown. (D) TIAM1 was immunoprecipitated from
platelet lysates as above and examined for coprecipitating S6K1 and Rac1 by
Western blot. (E) Localization of TIAM1, Rac1 and actin in fibrinogen-activated
human platelets visualized by confocal microscopy. Scale bar ⫽ 2 ␮m. Western
blot, IP, protein capture, and imaging results are representative of 3 independent
experiments.
S6K1 and Rac1 colocalize at the leading edge of
activated platelets
To better understand how S6K1 coordinates the activation of
Rac1 in platelets, we examined the localization of these proteins
in fibrinogen-activated platelets. Purified human platelets were
spread on fibrinogen-coated coverglass and stained for S6K1, Rac1
and actin. Laser scanning confocal immunofluorescence microscopy showed that S6K1 localized to the actin-rich leading edge
of activated platelets (Figure 2A). S6K1 colocalized with Rac1 and
lamellipodial actin at the leading edge of platelets, suggesting
that S6K1 may interact with Rac1 in a lamellipodial signaling
complex. To test this hypothesis, we immunoprecipitated S6K1
from human platelets and detected coimmunoprecipitating Rac1 by
Western blot. Anti-S6K1 precipitated S6K1 from both quiescent
and activated platelets (Figure 2B). S6K1 immunoprecipitates from
spread platelets showed more S6K1 phosphorylation than those
from quiescent platelets. Immunoprecipitation of S6K1 supported
the cocapture of Rac1 from platelets in solution as well as
fibrinogen-activated platelets. To determine whether Rac1 itself
can support an interaction with S6K1, we incubated platelet lysates
with glutathione sepharose conjugated to Rac1-GST. As seen in
Figure 2C, Rac1-GST captured S6K1 from platelet lysates while
GST alone failed to interact with S6K1. Previous work has
mTOR is a serine/threonine protein kinase that mediates cellular
responses to growth factors, cell cycle transitions, cell growth and
cell migration through 2 separate protein complexes, the mTOR
complex 1 (mTORC1) and the mTOR complex 2 (mTORC2).7
These complexes are distinguished by their uniquely associated
regulatory proteins, Raptor and Rictor, respectively.7 To first determine whether mTORC1 and mTORC2 protein complexes are
present in platelets, we prepared lysates from human platelets as
well as MCF10A cells. Lysates were normalized for total protein
content and separated by gel electrophoresis and blotted for the
presence of the mTOR complex components mTOR, Raptor,
Rictor and G␤L. As seen in Figure 3A, mTOR, Raptor and Rictor
were found in comparable levels in cytosolic lysates from both
platelets and MCF10A cells. G␤L, a G-protein ␤-subunit-like
protein component of both mTORC1 and mTORC2 was also
present in both cell types, though in slightly lower amounts in
platelets. To next determine whether mTORC1 and mTORC2 complexes are intact in human platelets, we performed coimmunoprecipitation experiments. Platelet lysates were incubated with antibodies against Raptor, Rictor, or control IgGs and analyzed for
coprecipitating mTOR by Western blot. As seen in Figures 3B and
C, Raptor and Rictor both coprecipitated mTOR, indicating that
mTORC1 and mTORC2 complexes are present in platelets.
Figure 3. mTORC1 and mTORC2 complexes are present in human platelets.
(A) Centrifugally cleared lysates-(50 ␮g) from human platelets or MCF10A breast
epithelial cells were analyzed by Western blot for levels of mTOR, Raptor, Rictor, or
G␤L. (B) mTORC1 complexes were identified by immunoprecipitating Raptor from
platelet lysates and Western blotting for associated mTOR. (C) mTORC2 complexes
were identified by immunoprecipitation of Rictor followed by Western blotting for
coprecipitating mTOR. Nonspecific immunoglobulins (IgG) served as a negative
control for immunoprecipitations. Immunoprecipitation results are representative of
3 independent experiments.
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To determine whether S6K1 and mTOR have roles in platelet
function, we next studied the effects of mTOR and S6K1 inhibition
on platelet activation. Several agents block S6K1 activity through
inhibition of mTORC1, most notably rapamycin and the rapalogue
compounds such as RAD001 (Everolimus). These agents bind to
FK-binding protein 12 (FKBP12) to form a complex that allosterically inhibits mTORC1 and subsequently S6K1 activation.6
mTORC1 and S6K1 are also inhibited by mTOR substrate inhibitors that engage the active site of mTOR directly.8 These
next generation mTOR inhibitors include compounds such as
WYE-35435 and Ku-0063794.8,36 As seen in Figure 4A, pretreatment of platelets with any of these agents inhibited platelet
S6K1 phosphorylation in response to spreading on immobilized
fibrinogen. mTOR inhibitors abrogated S6K1 activation but had
no effect on early signaling pathways of platelet activation;
rapamycin, RAD001, WYE-354 and Ku-0063794 had no significant effect on Src or FAK activation or the PI3K/Akt-mediated
phosphorylation GSK3␤ (Figure 4B).37 However, mTOR/S6K1 inhibitors blocked Rac1 activation (Figure 4C). To confirm that this
Rac1 inhibition had a consequence to platelet function, we used
DIC microscopy to analyze platelet spreading on fibrinogen after
pretreatment with mTOR inhibitors. As seen in Figure 4D, mTOR/
S6K1 inhibitors blocked lamellipodia formation and platelet spreading on a surface of fibrinogen.
mTOR controls platelet aggregation and aggregate stability
under flow
We next examined the role of S6K1 in the process of platelet
shape change and aggregation using a Born aggregometer. This
apparatus measures light transmission through a platelet suspension, with an increase in optical density correlating to platelet
sphericity and shape change and a decrease indicative of platelet
aggregation. The Rac1 inhibitor EHT 1864 abrogated the shape
change and aggregation of platelets stimulated with collagen in
the presence of the ADP scavenger, apyrase (Figure 5). mTOR/
S6K1 inhibitors also significantly decreased the magnitude and
time course of both the shape change and aggregation in response
to 10 ␮g/mL collagen (Figure 5).
The role of S6K1 and mTOR in platelet activation and
aggregation prompted us to investigate the function of these signaling proteins in maintaining platelet spreading. Purified human
platelets were first spread on an immobilized surface of fibrinogen. After 30 minutes, nonadherent platelets were washed away
and spread platelets were exposed to vehicle (DMSO), the mTOR/
S6K1 inhibitor WYE-354 or the Rac1 inhibitor EHT 1864. As
seen in Figure 6A, DMSO treatment did not reverse the spreading
of individual platelets on fibrinogen. Treatment with WYE-354 or
EHT 1864, however, caused a lamellipodial withdrawal of individual platelets previously spread on fibrinogen. We next aimed
to determine whether mTOR/S6K1 plays a role in platelet aggregate stability under shear flow. Platelet aggregates were formed
after perfusion of PPACK-anticoagulated blood over a surface of
collagen. After a secondary perfusion with buffer containing
fibrinogen and vehicle alone, platelet aggregates remained stable.
Aggregate stability was assessed during secondary perfusion
of buffer containing physiologic concentrations of fibrinogen.
Our data show that while platelet aggregates remained stable in
the presence of vehicle, the mTOR/S6K1 inhibitor WYE-354 led
to aggregate instability at 150 seconds, with further instability
at 300 seconds (Figure 6C). Quantification of the surface area
changes of these aggregates revealed an increase in coverage of
Figure 4. mTOR and S6K1 inhibition blocks Rac1 activation and platelet spreading.
(A) Purified human platelets were treated with apyrase (2 units/mL) and vehicle
(DMSO), Rapamycin (1␮M), RAD001 (1 ␮M), WYE-354 (10␮M), or Ku-0063794
(10␮M) before spreading on a surface of fibrinogen. Lysates from surface-bound
platelets were analyzed for S6K1 activation by Western blotting for S6K1-pThr389.
(B) Western blot analysis of Syk-pTyr323, FAK-pTyr576/577, and GSK3␤-pSer9
phosphorylation of fibrinogen-activated platelets pretreated with Rapamycin,
RAD001, WYE-354, or Ku-0063794. (C) Lysates from surface-bound platelets were
incubated with PAK-CRIB-GST to capture activated Rac1. Captured GTP-Rac1 and
total Rac1 levels were determined by Western blot. (D) Representative DIC images
of human platelets spread on coverglass coated with 100 ␮g/mL fibrinogen after
treatment with apyrase and vehicle (DMSO), Rapamycin, RAD001, or WYE-354.
Scale bar ⫽ 10 ␮m. Results are representative of 3 independent experiments.
28.8% after 150 seconds (P ⬍ .005 vs vehicle) extending to 37.0%
at 300 seconds (P ⬍ .05 vs vehicle). EHT 1864 similarly destabilized platelet aggregates within 150 seconds under shear flow
conditions (Figure 6C).
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3134
ASLAN et al
Figure 5. Inhibitors of mTOR and S6K1 block platelet aggregation. Washed
human platelets (2 ⫻ 108/mL) were stimulated with 10 ␮g/mL collagen in the
presence of 2 U/mL apyrase and the change in optical density indicative of platelet
aggregation was recorded after 10 minutes of preincubation with EHT 1864 (50␮M),
WYE-354 (10␮M), Ku-0063794 (10␮M), Rapamycin (1␮M), RAD001 (1␮M), or
vehicle (DMSO). One experiment representative of 3 separate experiments is shown.
BLOOD, 15 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 11
fected NIH 3T3 cells.19 In platelets, however, Rac1 is not required
for S6K1 activation, but rather the contrary. These disparities may
be explained in part by the roles of Rac1 and S6K1 in cell cycle
transitions in nucleated, dividing cells. In this study, the anucleate
and nontransformed nature of platelets facilitates the examination
of the roles of S6K1/mTOR in fundamental cell biologic processes
such as cytoskeletal reorganization free from nuclear events.
The formation of platelet plugs at sites of vascular injury
consists of 3 sequential phases of thrombus initiation, propagation
and perpetuation.1,2,39 Intriguingly, these processes are interdependent, as many of the signaling systems at the core of thrombus
initiation must remain active to maintain thrombus integrity and to
remodel thrombi in processes of wound healing. For instance,
continuous PI3K signaling is required for thrombus stabilization.39
Discussion
Here we identify mTOR and its effector S6K1 as mediators of
Rac1 signaling in platelet activation, platelet spreading and the
maintenance of platelet aggregate stability under flow. As platelets
engaged a surface of immobilized fibrinogen, a signaling cascade
triggered through tyrosine kinases activated S6K1 to switch on
Rac1 to drive platelet spreading. Inhibition of mTOR, S6K1 or
Rac1 had no effect on early, upstream tyrosine kinase activation of
Src, Syk and FAK, demonstrating that the activation of S6K1 and
Rac1 occur after the initiation of these tyrosine kinase pathways
in platelets. mTOR/S6K1 inhibitors, however, blocked Rac1 activation in response to the binding of platelets to immobilized
fibrinogen. In accordance with the hypothesis that mTOR and
S6K1 activate Rac1, the Rac1 inhibitor EHT 1864 did not block
S6K1 activation by fibrinogen. Rather, Rac1 inhibition robustly
increased S6K1 Thr389 phosphorylation (Figure 1E), raising
the possibility that Rac1 may also regulate a negative feedback
loop to dephosphorylate S6K1 during platelet activation. Our
model of platelet cytoskeletal reorganization in which S6K1 drives
Rac1 activation is further evidenced by imaging analyses showing
that S6K1 colocalized with Rac1 and actin at the leading lamellipodial edge of activated platelets. Moreover, Rac1 coprecipitated with
S6K1 in activated platelets, suggesting that S6K1 acts proximal to
Rac1 to control platelet activation. S6K1 may work together with
the Rac1 GEF TIAM1,34 as TIAM1 coprecipitated both Rac1 and
S6K1 from platelet lysates. Current models of TIAM function
hypothesize that TIAM1 recruits and activates Rac1 at the cytosolic surface of the plasma membrane.33 As platelet activation
increases the association of TIAM1 with Rac1 and phosphorylated
S6K1 (Figure 2D), TIAM1 may recruit a Rac1-S6K1 complex to
the plasma membrane to help drive lamellipodia formation. In
support of this model, confocal microscopy revealed that TIAM1
colocalized with Rac1 at the lamellipodial edge of activated
platelets (Figure 2E). Importantly, the effects observed in this study
occurred with platelets in presence of the ADP scavenger apyrase,
an agent known to reduce the fibrinogen-mediated activation of
platelet tyrosine kinase pathways.38 The extent to which mTOR
influences tyrosine kinase, S6K1 and Rac activation and platelet
function in the absence of ADP inhibitors remains to be determined. Interestingly, earlier studies of the S6K1-Rac1 axis revealed
that Cdc42 and Rac1 are required for S6K1 activation in trans-
Figure 6. mTOR is required for platelet aggregate stability under flow. (A) Purified human platelets (2 ⫻ 107/mL) were spread on glass coverslips coated with
100 ␮g/mL fibrinogen. After 30 minutes, unbound platelets were removed and activated
platelets were treated with vehicle (DMSO), WYE-354 (10␮M), or EHT 1864 (50␮M)
for an additional 30 minutes. Platelet spreading and lamellipodial withdrawal were
evaluated by DIC microscopy. Scale bar ⫽ 10 ␮m. (B) PPACK-anticoagulated blood
was perfused over collagen for 4 minutes to produce platelet aggregates that were
subsequently perfused with buffer containing fibrinogen for an additional 4 minutes
before perfusion with buffer, fibrinogen, and vehicle (DMSO), WYE-354 (10␮M), or
EHT 1864 (50␮M). Representative DIC images of platelet aggregates are shown.
Arrow indicates direction of flow. Scale bar ⫽ 50 ␮m. (C) Quantification of platelet
disaggregation promoted by WYE-354 or EHT 1864 under flow (n ⫽ 3). Data are
represented as mean ⫾ SEM.
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BLOOD, 15 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 11
During thrombus perpetuation, fibrin formation and other postaggregation phenomena maintain the activation of signaling programs such as the PI3K system through integrins to keep thrombi
stable. Previously, we showed that Rac1 plays a critical role in
thrombus formation in vivo.4 Here, we show that like PI3K, continuous Rac1 activation is also required to maintain the stability of
platelet aggregates under physiologic conditions of shear, as EHT
1864 disaggregates platelet clusters within seconds of administration (Figure 6B). Our hypothesis that mTOR and S6K1 control
platelet Rac1 activity to mediate platelet aggregate stability is
similarly supported through these assays using the mTOR inhibitor
WYE-354. Like Rac1, continuous mTOR activation was also
required to maintain the integrity of platelet aggregates formed on a
collagen matrix under shear. mTOR activity was also required for
collagen-stimulated platelet aggregation in solution (Figure 5). It is
noteworthy that our aggregation experiments were performed in
the presence of inhibitors of ADP, as previous studies have linked
the inhibition of mTOR with ADP secretion and ADP-triggered
platelet aggregation.40
mTOR and S6K1 prototypically control cell size and cell
proliferation by regulating mRNA translation through substrates
such as the ribosome S6 protein and the 4E binding protein
4E-BP1.6 Intriguingly, platelets synthesize proteins in an mTORdependent manner on binding to fibrinogen.41,42 Platelet engagement of fibrinogen promotes the synthesis of several proteins
including Bcl-3,41 a B cell lymphoma oncoprotein that can interact with the tyrosine kinase Fyn.42 Here, however, we find that
translation of Bcl-3 mRNA is not likely to have any instantaneous
effects on platelet Rac1 activity as puromycin, which also inhibits
Bcl-3 translation in platelets,41,42 had no effect on platelet spreading
or aggregation (supplemental Figures 4-5). Previous work has also
established that rapamycin can inhibit the retraction of platelet
fibrin clots by blocking the translation of Bcl-3 mRNA in platelets over a time course of minutes to several hours in the process
of thrombus consolidation and wound healing.43 Here, our data
suggests that mTOR and S6K1 also have more immediate effects
in thrombus formation and in the maintenance of platelet aggregate stability. Like Rac1 inhibitors, mTOR inhibitors also disrupt
platelet lamellipodia formation, platelet aggregate formation and
platelet aggregate stability within seconds to minutes through a
mechanism that may not be dependent on mRNA translation.
mTORC1 inhibitors such as rapamycin are used as chemotherapeutic agents for the treatment of renal cell44 and other
carcinomas.24 Rapamycins selectively block cancer cell growth and
induce apoptosis of transformed tumor cells with dysregulated
mTOR signaling with minimal effects on healthy cells and only
mild physiologic complications.45 The majority of studies investigating the antineoplastic effects of mTOR inhibition have focused
on the antiproliferative actions of the rapamycins. However,
rapamycins also inhibit cell migration and have been proposed to
block tumor cell growth by halting tumor cell chemotactic responses to nutrients and growth factors such as IGF-I.15,17,46 In
tumor cells, the effects of rapamycin on chemotaxis are in part
mediated by an inhibition of FAK through mTORC1, as rapamycin
inhibits IGF-I–induced F-actin reorganization and phosphorylation
of focal adhesion proteins in an mTORC1-dependent manner.16
Here, we find that mTOR inhibitors have no effect on platelet
FAK activation, suggesting that an mTORC1 activation of FAK
may be specific to tumor cells and other transformed cells with
irregular mTOR signaling. mTOR also has roles in the migration
of intestinal cells13 and smooth muscle cells14 as well as tumor
cells. Such migratory processes are driven by an interaction of
mTOR REGULATES PLATELET ACTIVATION
3135
cell surface integrins with immobilized substrate proteins such as
fibronectin and fibrinogen. In cultured cells, the engagement of
integrins ␣5␤1 and ␣v␤3 by fibronectin activates an mTOR/S6K1
signal that is required to drive cell migration.14,47 The mechanisms by which mTOR and S6K1 are involved in directing the
motility of these cells, however, have not been described. Here, we
demonstrate that mTOR and S6K1 are required for Rac1 signaling
which prompts cytoskeletal remodeling events that form the basis
for cell motility of platelets triggered by fibrinogen binding to
integrin ␣IIb␤3. As an integrin-mediated activation of S6K1 drives
Rac1 processes in platelets, S6K1/mTOR may similarly have
Rac1-related roles in migratory processes of other cell types such
as tumor cells and other peripheral blood cells with immune
functions such as macrophages and neutrophils.
In addition to their use as chemotherapeutic agents, rapamycins
are also potent immunomodulators used to prevent organ rejection in transplant recipients.48 The mechanisms by which rapamycins suppress the immune system are not fully understood, but
may involve immunoregulators such as B cells, neutrophils,29 and
perhaps platelets.30 While our work ascribes a role for mTOR/
S6K1 in mediating Rac1-based cytoskeletal reorganization in
platelets, similar mechanisms may be present in other peripheral
blood cells. Our results may in part explain how rapamycins block
immune responses by preventing a Rac1-mediated activation of
chemotaxis in peripheral blood cells involved in immunity. The
antiproliferative actions of rapamycins are further exploited in
drug-eluting intravascular stents to prevent restenosis.25,27 As
thrombosis is a common long-term complication of stent implantation, antiplatelet regimens are often prescribed to patients after
stent insertion.26 Intriguingly, rapamycin-eluting stents show reduced platelet binding and activation.49 Our data suggests that
rapamycins and other mTORC1 inhibitors may play a role in
inhibiting platelet function in regions proximal to drug-eluting
stents and may provide insights into future treatments for stent
thrombosis.
In conclusion, our results uncover a novel mechanism by which
tyrosine kinase systems work through S6K1/mTOR to control
Rac1 activity and platelet action. While we have determined that
S6K1 and Rac1 activation occur downstream of platelet tyrosine
kinases, fundamental questions remain about how integrintriggered tyrosine kinase pathways can activate S6K1 in platelets.
Future work will aim to identify the precise signaling network
that leads to and from tyrosine kinases to mTOR/S6K1 and then
to Rac1 to drive platelet activation. While Rac1 associates
with S6K1 in platelet lysates, the nature of how S6K1, Rac1 and
TIAM1 may all come together remains an open question. Furthermore, exactly how S6K1 may help to promote the exchange
of GDP for GTP by Rac1 is also mysterious. It is possible that
undetermined partners or substrates of mTOR or S6K1 may be
present in a complex with Rac1 to control platelet action. The
hypothesis that platelet S6K1/mTOR can work directly with
Rac1 or a Rac-associated guanine nucleotide exchange factor such
as TIAM1 is currently under investigation.
Acknowledgments
The authors thank M. Larson (Augustana College), M. Berny-Lang
(Harvard), and D. Greenberg, M. Hinds, and A. Gruber (OHSU) for
helpful discussions.
This work was supported by National Institutes of Health grants
T32HL007781 (J.E.A.), U54CA143906 (O.J.T.M.), and R01HL101972
(O.J.T.M.). C.P.L. is an Oregon State University Johnson Scholar.
From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
3136
BLOOD, 15 SEPTEMBER 2011 䡠 VOLUME 118, NUMBER 11
ASLAN et al
The content is solely the responsibility of the authors and does
not necessarily represent the official view of the National Cancer
Institute or the National Institutes of Health.
Authorship
Contribution: J.E.A. designed the project, performed research,
analyzed data, and wrote the manuscript; G.W.T. designed and
performed the aggregation experiments under flow, analyzed data,
and assisted in writing the manuscript; C.P.L. conducted DIC
microscopy experiments; J.P. carried out Western blotting and
platelet aggregation experiments; and O.J.T.M. supervised the
research and assisted in writing the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Dr Joseph E. Aslan, 3303 SW Bond Ave,
Center for Health and Healing CH13B, Oregon Health & Science
University, Portland, OR 97239; e-mail: [email protected].
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From www.bloodjournal.org by guest on June 18, 2017. For personal use only.
2011 118: 3129-3136
doi:10.1182/blood-2011-02-331579 originally published
online July 14, 2011
S6K1 and mTOR regulate Rac1-driven platelet activation and
aggregation
Joseph E. Aslan, Garth W. Tormoen, Cassandra P. Loren, Jiaqing Pang and Owen J. T. McCarty
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