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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
CalDAG-GEFI and protein kinase C represent alternative pathways leading to
activation of integrin ␣IIb␤3 in platelets
Stephen M. Cifuni,1 Denisa D. Wagner,1,2 and Wolfgang Bergmeier1-3
1Immune
Disease Institute and 2Department of Pathology, Harvard Medical School, Boston, MA; and 3Cardeza Foundation and Department of Medicine,
Thomas Jefferson University, Philadelphia, PA
Second messenger-mediated inside-out
activation of integrin ␣IIb␤3 is a key step
in platelet aggregation. We recently
showed strongly impaired but not absent
␣IIb␤3-mediated aggregation of CalDAGGEFI–deficient platelets activated with
various agonists. Here we further evaluated the roles of CalDAG-GEFI and protein kinase C (PKC) for ␣IIb␤3 activation
in platelets activated with a PAR4 receptor–
specific agonist, GYPGKF (PAR4p).
Compared with wild-type controls, platelets treated with the PKC inhibitor Ro31-
8220 or CalDAG-GEFI–deficient platelets
showed a marked defect in aggregation at
low (< 1mM PAR4p) but not high PAR4p
concentrations. Blocking of PKC function
in CalDAG-GEFI–deficient platelets, however, strongly decreased aggregation at
all PAR4p concentrations, demonstrating
that CalDAG-GEFI and PKC represent
separate, but synergizing, pathways important for ␣IIb␤3 activation. PAR4pinduced aggregation in the absence of
CalDAG-GEFI required cosignaling
through the G␣i-coupled receptor for ADP,
P2Y12. Independent roles for CalDAGGEFI and PKC/G␣i signaling were also
observed for PAR4p-induced activation
of the small GTPase Rap1, with CalDAGGEFI mediating the rapid but reversible
activation of this small GTPase. In summary, our study identifies CalDAG-GEFI
and PKC as independent pathways leading to Rap1 and ␣IIb␤3 activation in mouse
platelets activated through the PAR4 receptor. (Blood. 2008;112:1696-1703)
Introduction
Integrins are heterodimeric cell-surface receptors that mediate cell
adhesion to the extracellular matrix and cell-cell interactions.
Probably the best-studied integrin is ␣IIb␤3, the platelet receptor
for fibrinogen. Activation of ␣IIb␤3 in platelets is largely dependent on the generation of the second messengers Ca2⫹ and
diacylglycerol (DAG), and the concomitant activation of members
of the G␣i family of heterotrimeric G proteins. Ca2⫹ and DAG are
generated by phospholipase C␤ (PLC␤) downstream of G proteincoupled receptors (GPCRs), such as PAR receptors (thrombin
receptors) or P2Y1 (ADP receptor), or by PLC␥ downstream of the
activating collagen receptor, GPVI.1,2 Signaling via G␣i proteins in
platelets is selectively coupled to various agonist receptors: G␣z
preferentially couples to the ␣2A adrenergic receptor for epinephrine,3 whereas G␣i2 is preferred by the ADP receptor P2Y12.4 G␣i
signaling leads to the activation of phosphoinositide 3-kinase
(PI3K), Akt, Rap1B, and the inhibition of adenylyl cyclase but has
little, if any, ability to activate PLC.5 Studies with PKC inhibitors
and calcium chelators identified separate PKC-dependent and
Ca2⫹-dependent signaling pathways that synergize with G␣i signaling in the activation of ␣IIb␤3.6,7 In addition, signaling by
both PKC and Ca2⫹ plays a key role in granule secretion from
activated platelets.8,9
The Rap family of small GTPases has recently gained much
attention as a central player in integrin activation downstream of
second messengers.10 In mammals, the Rap family consists of
2 rap1 genes and 2 rap2 genes encoding proteins that are
approximately 65% homologous. Platelets express significant
amounts of Rap1B and Rap2B, with Rap1B accounting for
approximately 90% of the total Rap protein.11 Rap proteins
cycle between an inactive GDP-bound and an active GTP-bound
form. The GDP-GTP cycle is regulated by guanine nucleotide
exchange factors (GEFs), which facilitate the release of GDP
and allow GTP to bind. GTPase-activating proteins (GAPs)
facilitate the hydrolysis of bound GTP to complete the cycle. On
platelet stimulation, Rap1B activation is mediated via Ca2⫹dependent and -independent mechanisms, the latter being dependent on G␣i signaling and the subsequent activation of PI3K.12-15
The importance of Rap1B for ␣IIb␤3 activation has recently
been demonstrated in Rap1B-deficient mice.16
Ca2⫹ and diacylglycerol regulated guanine nucleotide exchange
factor I (CalDAG-GEFI, RasGRP2) is a member of the CalDAGGEF/RasGRP family of intracellular signaling molecules involved
in the activation of small G proteins of the Ras superfamily.17,18
CalDAG-GEFI contains binding sites for Ca2⫹ and DAG and a
GEF domain that predominantly activates Rap1.18 We have previously shown that CalDAG-GEFI plays a key role in the activation
of Rap1, ␣IIb␤3 integrin, and ␤1 integrins in platelets19,20 as well
as Rap1, ␤1, and ␤2 integrins in neutrophils.20 Platelets from mice
deficient in CalDAG-GEFI did not aggregate when stimulated with
ADP, thromboxane A2 (TxA2), or Ca2⫹ ionophore. In contrast,
aggregation in response to collagen and, especially, thrombin was
only slightly affected. CalDAG-GEFI⫺/⫺ mice were characterized
by a prolonged bleeding time and protection against experimental
thrombosis, demonstrating the importance of this molecule for
thrombus formation in vivo.19,20
In the present study, we have evaluated how CalDAG-GEFI
signaling cooperates with PKC and G␣i signaling in platelet
Submitted February 15, 2008; accepted May 28, 2008. Prepublished online as
Blood First Edition paper, June 10, 2008; DOI 10.1182/blood-2008-02-139733.
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The publication costs of this article were defrayed in part by page charge
© 2008 by The American Society of Hematology
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BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5
aggregation. We show that CalDAG-GEFI and PKC can independently promote the activation of Rap1 and integrin ␣IIb␤3 in
mouse platelets specifically activated through the major thrombin
receptor, PAR4. Whereas CalDAG-GEFI mediates the rapid but
reversible activation of Rap1, PKC ensures sustained Rap1 activation through its central role in the release of ADP. In platelets
lacking CalDAG-GEFI, integrin activation required cosignaling by
PKC and G␣i.
Methods
Reagents and antibodies
Lovenox (enoxaparin sodium; Sanofi-Aventis, Bridgewater, NJ), heparincoated microcapillaries (VWR, West Chester, PA), bovine serum albumin
(BSA), prostacyclin (PGI2), apyrase, 2⬘-deoxy-N6-methyl adenosine 3⬘,5⬘diphosphate diammonium salt (MRS2179, P2Y1 inhibitor), 2-methylthioAMP triethylammonium salt hydrate (2-MesAMP, P2Y12 inhibitor), epinephrine, serotonin (all from Sigma-Aldrich, St Louis, MO), protein kinase
C (PKC) inhibitor Ro31-8220 (EMD Chemicals, Gibbstown, NJ), ADP
(Bio/Data, Horsham, PA), 14C-serotonin (GE Healthcare, Little Chalfont,
Buckinghamshire, United Kingdom), PAR4 receptor-activating peptide
H-GYPGKF-NH2 (Advanced Chemtech, Louisville, KY), calcium-sensing
dye Fluo-3 (Invitrogen, Carlsbad, CA), RalGDS-RBD coupled to agarose
beads (Upstate Biotechnology, Charlottesville, VA), were purchased. Monoclonal antibody directed against the activated form of murine ␣IIb␤3
integrin, JON/A-PE, was purchased from emfret Analytics (Wuerzburg,
Germany). Polyclonal antibodies to CalDAG-GEFI were described recently19 and antibodies to Rap1 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
CalDAG-GEFI AND PKC IN ␣IIb␤3 ACTIVATION
1697
Rap1 activation
Amounts of activated Rap1 in platelets were measured using a protocol
similar to the one previously described.19 Platelets were activated for
various times under static or stirring conditions in the presence or absence
of inhibitors and immediately lysed with ice-cold lysis buffer (25 mM
Tris-HCl at pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 5 mM MgCl2,
5% glycerol, and complete protease inhibitor cocktail lacking ethylenediaminetetraacetic acid; Roche Applied Science, Indianapolis, IN). A total of
30 ␮L of sample was immediately withdrawn from the lysates for the
determination of total Rap1 levels. Detection of activated Rap1 (Rap1GTP) in platelet lysates was done according to the instructions of the
manufacturer. Briefly, Rap1-GTP was precipitated from lysates using
RalGDS-RBD beads. Precipitated proteins were separated on a 15%
SDS-PAGE gel and transferred to polyvinylidene fluoride membranes
(Millipore, Billerica, MA). Rap1 was detected with rabbit polyclonal
antibodies followed by antirabbit antibodies conjugated to horseradish
peroxidase (Vector Laboratories, Burlingame, CA). Immunoreactivity was
detected by Western Lightning enhanced chemiluminescence (PerkinElmer
Life and Analytical Sciences, Waltham, MA).
Serotonin release
PRP was incubated in a 37°C water bath for 15 minutes with
14C-serotonin (57 mCi/mmol per mL of PRP), and diluted with modified
Tyrode buffer containing 1 mM of CaCl2. Platelets were activated under
stirring conditions (standard aggregometry), and activation was terminated by adding 1 volume of 4% ice-cold paraformaldehyde solution.
The samples were then centrifuged at 2500 g for 5 minutes, and the
supernatants were used for scintillation counting of 14C-serotonin. Total
or 100% 14C-serotonin secretion was defined as the 14C-serotonin in
samples lysed with 0.5% Triton X-100.
Mice
CalDAG-GEFI⫺/⫺19 and littermate control (wild-type, WT) mice were
received from the mouse facility at the Massachusetts Institute of Technology and were bred in the mouse facilities of the Immune Disease Institute
and Thomas Jefferson University. Experimental procedures were approved
by the Animal Care and Use Committees of the Immune Disease Institute
and of Thomas Jefferson University.
Aggregometry
Platelet-rich plasma (PRP) was obtained from heparinized whole blood by
centrifugation at 100g for 10 minutes. Light transmission was measured by
using PRP adjusted to 3 ⫻ 108 platelets per milliliter with modified
Tyrode’s buffer (137 mM NaCl, 0.3 mM Na2HPO4, 2 mM KCl,
12 mM NaHCO3, 5 mM N-2-hydroxyethylpiperazine-N⬘-2-ethanesulfonic
acid, 5 mM glucose, pH 7.3) containing 0.35% BSA and 1 mM CaCl2.
Inhibitors and agonists were added at the indicated concentrations, and
transmission was recorded over 12 minutes on a Chrono-log 4-channel
optical aggregation system (Chrono-log, Havertown, PA).
Flow cytometry
PRP was centrifuged at 700g in the presence of PGI2 (2 ␮g/mL) for
7 minutes at room temperature. After 2 washing steps, pelleted platelets
were resuspended in modified Tyrode’s buffer.
Calcium flux measurement. Washed platelets were incubated with
5 ␮M of the calcium-sensing dye Fluo-3 for 15 minutes, activated with the
indicated concentrations of PAR4p, and immediately analyzed on a
FACScalibur. Data were analyzed with FlowJo software (TreeStar,
Ashland, OR).
Integrin activation. Platelets were diluted in Tyrode buffer containing 1 mM CaCl2, activated with PAR4p, epinephrine, or serotonin in the
presence of JON/A-PE21 for 10 minutes, and studied immediately by
flow cytometry. Inhibitors to PKC (Ro31-8220), P2Y12 (2-MesAMP),
or P2Y1 (MRS2179) were added before stimulation to block the
respective signaling pathways.
Results
CalDAG-GEFI and PKC represent independent pathways
leading to activation of ␣IIb␤3
We have previously shown that CalDAG-GEFI⫺/⫺ mouse platelets
show impaired aggregation in response to stimulation by low-dose
thrombin, whereas no such defect was observed at higher concentrations of the agonist.19 These results indicated the existence of a
CalDAG-GEFI-independent pathway downstream of PLC␤ activation that is sufficient to activate ␣IIb␤3. To evaluate whether PKC
is involved in this signaling pathway, PKC function was inhibited
in WT and CalDAG-GEFI–deficient platelets with Ro31-8220, an
inhibitor of conventional and novel PKC isoforms22 (Figure 1).
Activation of platelets through the major activating thrombin
receptor on mouse platelets, PAR4,23,24 was induced with the
peptide GYPGKF (PAR4p). Both WT platelets pretreated with
Ro31-8220 and CalDAG-GEFI–deficient platelets showed a significantly impaired aggregation response when stimulated with
0.5 mM PAR4p (P ⬍ .001 and .001, respectively), a concentration
sufficient to induce full aggregation of WT platelets (Figure 1A).
When stimulated with 1 mM of PAR4p, aggregation of CalDAGGEFI-deficient (P ⬍ .01) but not WT/Ro31-8220 platelets was
significantly reduced. No significant difference in maximum aggregation was observed between WT platelets and WT/Ro31-8220 or
CalDAG-GEFI–deficient platelets when the cells were activated
with PAR4p concentrations equal or higher than 1.5 mM. In
contrast, compared with WT platelets, CalDAG-GEFI–deficient
platelets pretreated with Ro31-8220 showed significantly reduced
aggregation at all tested PAR4p concentrations (P ⬍ .001). Figure
1B (top panel) shows representative aggregation traces for platelets
activated with 1.25 mM of PAR4p, a concentration that allows
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CIFUNI et al
BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5
Figure 1. CalDAG-GEFI and PKC synergize in ␣IIb␤3
activation in platelets activated through the PAR4
receptor. (A) Dose response for PAR4p-induced aggregation of wild-type (WT) and CalDAG-GEFI–deficient
(KO) platelets. The data shown represent the percentage
aggregation measured 5 minutes after addition of the
agonist; n ⫽ 6 (P values given in “Results”). (B) WT or
CalDAG-GEFI–deficient platelets were stimulated with
1.25 mM PAR4p in the presence or absence of the
broad-range PKC inhibitor Ro31-8220 (5 ␮g/mL). (Top
panel) Representative aggregation traces. (Bottom panel)
Representative histograms for the activation of integrin
␣IIb␤3 as measured by flow cytometry. Gray curve
represents untreated; black curve, treated with PAR4p.
The results are representative of 5 individual experi) or CalDAG-GEFI–deficient (
)
ments. (C) WT (
platelets were stimulated with 1.25 mM of PAR4p in the
presence or absence of the broad-range PKC inhibitor
Ro31–8220. Binding of JON/A-PE was measured to
determine the level of ␣IIb␤3 activation by flow cytometry;
n ⫽ 6 (***P ⬍ .001). (D) Calcium flux was measured over
time in Fluo-3–loaded WT or CalDAG-GEFI–deficient
platelets stimulated with the indicated concentrations of
PAR4p. The bar graph shows the maximum fluorescence
) and CalDAGintensity (Fluo-3) measured in WT (
) platelets within 1 minute after addiGEFI–deficient (
tion of PAR4p; n ⫽ 6. No significant difference in Fluo-3
fluorescence signals between WT and KO platelets was
observed.
aggregation of platelets lacking either functional PKC or CalDAGGEFI but that does not cause aggregation in CalDAG-GEFIdeficient platelets treated with Ro31-8220. Shape change as
indicated by the initial decrease in light transmission was observed
in all samples. Direct assessment of ␣IIb␤3 activation in platelets
stimulated with 1.25 mM of PAR4p, using an antibody against the
activated form of the receptor (JON/A-PE21), showed significantly
reduced activation of ␣IIb␤3 in both Ro31-8220–treated and
CalDAG-GEFI–deficient platelets (Figure 1B bottom panel; Figure
1C). Almost complete inhibition of ␣IIb␤3 activation
(and platelet aggregation), however, was only observed in CalDAGGEFI–deficient platelets treated with Ro31-8220. Similar results
were observed when integrin activation was studied using
FITC-labeled fibrinogen (not shown). These data demonstrate
a synergistic role of PKC and CalDAG-GEFI in ␣IIb␤3 activation
in platelets.
Figure 2. PAR4p-induced activation of ␣IIb␤3 in
CalDAG-GEFI–deficient platelets requires signaling
by PKC and G␣i-coupled receptors. (A) WT platelets
pretreated with 5 ␮g/mL Ro31-8220 or CalDAG-GEFI–
deficient (CalDAG-GEFI⫺/⫺) platelets were activated with
1.25 mM of PAR4p in the presence of (1) the P2Y1
inhibitor MRS2179 (100 ␮M), (2) the P2Y12 inhibitor
2-MesAMP (50 ␮M), or (3) the ADP degrading enzyme
apyrase (8 U/mL). Aggregation of platelets was recorded
) or CalDAG-GEFI–deficient
for 10 minutes. (B) WT (
) platelets were stimulated with 1.25 mM of PAR4p in
(
the presence or absence of 2-MesAMP or MRS2179.
Binding of JON/A-PE was measured to determine the
level of ␣IIb␤3 activation by flow cytometry; n ⫽ 6
(***P ⬍ .001). (C) CalDAG-GEFI–deficient platelets pretreated with apyrase (8 U/mL) were activated with
1.25 mM of PAR4p, followed by 10 ␮M of epinephrine or
10 ␮M of serotonin. Platelet aggregation was recorded for
10 minutes. (D) CalDAG-GEFI–deficient platelets were
preincubated with 2-MesAMP, followed by stimulation
with 1.25 mM of PAR4p in combination with epinephrine
or serotonin. Binding of JON/A-PE was measured to
determine the level of ␣IIb␤3 activation by flow cytometry;
n ⫽ 6 (***P ⬍ .001; ns indicates not significant).
To rule out the possibility that impaired aggregation of CalDAGGEFI–deficient platelets at low dose of the agonist was because of
a defect in signaling events upstream of CalDAG-GEFI, calcium
flux was studied in PAR4p-stimulated platelets. As shown in Figure
1D, a similar calcium flux response of WT and CalDAG-GEFI–
deficient platelets to various doses of PAR4p was observed.
␣IIb␤3 activation in the absence of functional PKC or
CalDAG-GEFI depends on G␣i signaling provided by
secreted ADP
In a next step, the dependency of ␣IIb␤3 activation in the absence
of functional PKC or CalDAG-GEFI on the second wave mediators
ADP and/or TxA2 was investigated. Whereas inhibition of TxA2
formation by aspirin did not affect aggregation of WT/Ro31-8220
or CalDAG-GEFI–deficient platelets activated with 1.25 mM of
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BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5
CalDAG-GEFI AND PKC IN ␣IIb␤3 ACTIVATION
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PAR4p (not shown), preincubation of platelets with the ADP
scavenger apyrase inhibited the aggregation of CalDAG-GEFI–
deficient but not WT/ Ro31-8220 platelets (Figure 2A). The same
inhibitory effect was observed when platelets were preincubated
with 2-MesAMP, a specific inhibitor of G␣i-coupled P2Y12
receptors. In contrast, preincubation with MRS2179, an inhibitor of
Gq-coupled P2Y1 receptors, did not affect the aggregation of
PAR4p-stimulated CalDAG-GEFI–deficient platelets. Using JON/
A-PE as a probe for activated ␣IIb␤3, we observed a significant
inhibitory effect of 2-MesAMP but not MRS2179 on PAR4pinduced ␣IIb␤3 activation in WT and CalDAG-GEFI–deficient
platelets (Figure 2B). Almost complete inhibition of ␣IIb␤3
activation was observed in 2-MesAMP-treated CalDAG-GEFI–
deficient platelets.
To confirm that ␣IIb␤3 activation in the absence of CalDAGGEFI depends on G␣i signaling, epinephrine was used to specifically stimulate this signaling pathway.3 Addition of epinephrine
restored aggregation and significantly increased ␣IIb␤3 activation
in CalDAG-GEFI–deficient platelets pretreated with apyrase (Figure 2C) or 2-MesAMP (Figure 2D), respectively. In contrast,
aggregation or ␣IIb␤3 aggregation could not be restored by
addition of serotonin, a specific stimulator of G␣q signaling.25
Impaired dense granule release in CalDAG-GEFI–deficient
platelets activated with low- but not high-dose PAR4p
To test whether impaired integrin activation at low PAR4p concentrations could be the result of defective release/cosignaling by ADP,
we next studied dense granule release in CalDAG-GEFI–deficient
platelets. Release of 14C-labeled serotonin from platelets activated
under stirring conditions (standard aggregometry) was measured.
Pretreatment of platelets with Ro31-8220 completely inhibited
dense granule release in platelets activated with various doses of
PAR4p (Figure 3A). In contrast, platelets lacking CalDAG-GEFI
showed significantly reduced dense granule release only at low but
not high concentrations of the agonist (P ⬍ .001 at 0.5 mM
PAR4p). No significant differences in the kinetics of granule
release were observed between WT and CalDAG-GEFI–deficient
platelets activated with 1.25 mM of PAR4p (Figure 3B).
To more directly address whether impaired ADP release accounts for the defect in integrin activation in CalDAG-GEFI–
deficient platelets, mutant platelets were costimulated with 0.5 mM
of PAR4p and 5 ␮M of ADP. As shown in Figure 3C, aggregation
was restored in CalDAG-GEFI–deficient platelets costimulated
with PAR4p and ADP, whereas the individual agonists did not
cause aggregation of the mutant cells. Preincubation of CalDAGGEFI–deficient platelets with 2-MesAMP blocked the costimulatory effect of ADP, demonstrating a key role of G␣i-coupled P2Y12
receptors in this process.
CalDAG-GEFI mediates the rapid but reversible activation of
Rap1 in PAR4p-stimulated platelets
Agonist-induced activation of ␣IIb␤3 strongly depends on the
activation of the small GTPase Rap1.16 CalDAG-GEFI is a key
regulator of Rap1 activation in mouse platelets.19 However,
CalDAG-GEFI is not the sole activator of Rap1 as significant
activation of Rap1 was observed in CalDAG-GEFI–deficient
platelets stimulated with thrombin or ADP. In platelets activated
with 1.25 mM of PAR4p under nonstirring conditions, CalDAGGEFI mediated the rapid activation of Rap1 (Figure 4A). Whereas
maximum activation of Rap1 in WT platelets was seen already
10 seconds after addition of the agonist, Rap1 activation was
Figure 3. Impaired dense granule release from CalDAG-GEFI–deficient platelets stimulated with low-dose PAR4p. (A) 14C-serotonin-loaded WT or CalDAGGEFI–deficient (KO) platelets were activated with various concentrations of PAR4p in
the presence or absence of Ro31-8220 (5 ␮g/mL) under stirring conditions. Activation
was terminated 10 minutes after addition of the agonist, and 14C-serotonin levels in
the supernatant (SN) of activated platelets were determined. 0% indicates 14Cserotonin in SN of resting platelets; 100%, 14C-serotonin levels in platelet samples
after lysis; n ⫽ 6. (B) Time course of PAR4p-induced serotonin release. The
activation process was terminated at the indicated time points after addition of
1.25 mM of PAR4p; n ⫽ 6. (C) WT (black line) or CalDAG-GEFI–deficient (gray lines)
platelets were activated with 0.5 mM of PAR4p and/or 5 ␮M of ADP in the presence or
absence of 2-MesAMP (50 ␮M). Results are representative of 4 experiments.
completely impaired in CalDAG-GEFI–deficient platelets for more
than 1 minute. Weak activation of Rap1 was observed 3 minutes
after stimulation with PAR4p, and activation continued to reach
almost maximum strength at 10 minutes after stimulation. In
contrast, WT platelets treated with Ro31-8220 showed reduced
levels of Rap1 activation but no change in the overall kinetics of
this process. Pretreatment of CalDAG-GEFI–deficient platelets
with Ro31-8220, however, completely abolished Rap1 activation
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BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5
CIFUNI et al
deficient platelets 15 seconds after addition of PAR4p. Aggregation
in the mutant platelets started approximately 45 seconds after
stimulation, a time point when weak Rap1 activation was observed.
Full aggregation and strong Rap1 activation were observed at
t ⫽ 3 minutes. Using the same experimental approach, we also
compared the kinetics of Rap1 activation and aggregation in
2-MesAMP–treated CalDAG-GEFI–deficient platelets activated
with PAR4p and/or epinephrine (Figure 5B). At all time points
tested, Rap1 activation and aggregation were strongly inhibited in
the absence of epinephrine. In contrast, platelets activated with the
combination of PAR4p and epinephrine showed a strong aggregation response and a marked Rap1 activation, both occurring with
similar kinetics.
Figure 4. Rap1 activation in platelets stimulated with PAR4p requires CalDAGGEFI and P2Y12-mediated signaling. (A) Time course of Rap1 activation in WT
( ⫾ 5 ␮g/mL Ro31-8220) or CalDAG-GEFI⫺/⫺ platelets stimulated with 1.25 mM of
PAR4p. (B) Rap1-GTP detected in lysates from CalDAG-GEFI–deficient platelets at
t ⫽ 10 minutes after addition of PAR4p. Rap1 activation was completely inhibited in
the presence of Ro31-8220. (C) Effect of 2-MesAMP on Rap1 activation in WT or
CalDAG-GEFI-deficient platelets. Platelets were pretreated with 50 ␮M 2-MesAMP
for 5 minutes followed by stimulation with 1.25 mM of PAR4p.
induced by PAR4p (Figure 4B). To determine whether the delayed
activation of Rap1 seen in CalDAG-GEFI–deficient platelets is a
result of P2Y12-mediated signaling, we tested the effect of
2-MesAMP on this process. Rapid but reversible Rap1 activation
was observed in 2-MesAMP-pretreated WT platelets stimulated
with PAR4p, whereas no Rap1 activation was observed in 2-MesAMP-pretreated CalDAG-GEFI–deficient platelets (Figure 4C).
These data suggest that CalDAG-GEFI mediates the rapid but
reversible activation of Rap1 in platelets whereas CalDAG-GEFI–
independent stimulation through P2Y12 leads to a delayed but
sustained formation of Rap1-GTP. PKC participates in Rap1
activation through its central role in the release of ADP from
activated platelets.
To directly compare the kinetics of Rap1 activation and
aggregation/␣IIb␤3 activation in CalDAG-GEFI–deficient platelets, we studied the generation of Rap1-GTP in aggregating
platelets activated with 1 mM PAR4p, a threshold concentration for
the activation of the mutant platelets (Figure 5A). In WT platelets,
both aggregation and Rap1 activation occurred within seconds after
the addition of PAR4p. In contrast, both aggregation and Rap1
activation were almost completely inhibited in CalDAG-GEFI–
Discussion
Based on the results of this study, a new model for ␣IIb␤3
activation in platelets can be proposed (Figure 6): CalDAG-GEFI
and PKC represent independent, synergistic signaling pathways
leading to ␣IIb␤3 activation downstream of agonist-induced activation of PLC-␤2. In WT platelets, generation of the second
messengers calcium and DAG led to the concurrent activation of
CalDAG-GEFI and PKC, allowing for ␣IIb␤3 activation independent of the generation of the second wave mediators TxA2 and
ADP. In the absence of CalDAG-GEFI, PKC-mediated activation
of ␣IIb␤3 requires cosignaling by G␣i, provided by plateletderived ADP, most probably because of its role in the activation of
the small GTPase Rap1. In the absence of functional PKC,
activation of Rap1 and ␣IIb␤3 is mediated solely by CalDAGGEFI, as ADP release is inhibited under such experimental
conditions.
This study was designed to further clarify the role of CalDAGGEFI in ␣IIb␤3 activation in platelets stimulated through the
activating thrombin receptor PAR4. A specific PAR4 agonist
(GYPGKF, PAR4p) was used for these studies because (1) PAR4 is
the major activating receptor for thrombin in mouse platelets,
whereas PAR3 facilitates the activation through PAR424;
(2) stimulation of platelets with thrombin would also cause the
activation of signaling cascades downstream of GPIb␣,26 thus
leading to unwanted complexities in the interpretation of the
results; and (3) in contrast to thrombin, PAR4p does not activate the
coagulation system and can thus be used in PRP.
Figure 5. Similar kinetics for Rap1 activation and
aggregation in platelets stimulated with PAR4p.
(A) Time course for the aggregation response (top panel)
and for Rap1 activation (bottom panel) in WT or CalDAGGEFI–deficient (CalDAG-GEFI⫺/⫺) platelets stimulated
with 1.0 mM of PAR4p. Samples for the determination of
Rap1-GTP and total Rap1 levels were withdrawn from the
aggregation tubes at t ⫽ 15, 45, or 180 minutes after
addition of the agonist (indicated by black arrows). Results are representative of 3 independent experiments.
(B) Time course for the aggregation response (top panel)
and for Rap1 activation (bottom panel) in 2-MesAMPpretreated CalDAG-GEFI⫺/⫺ platelets stimulated with
1.5 mM of PAR4p in the presence or absence of 10 ␮M
of epinephrine. Samples for the determination of Rap1GTP and total Rap1 levels were withdrawn from the
aggregation tubes at the indicated time points. Results
are representative of 3 independent experiments.
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BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5
Figure 6. Schematic representation of the CalDAG-GEFI–dependent and PKCdependent signaling pathways leading to ␣IIb␤3 activation in mouse platelets.
PLC-␤2 indicates phospholipase C-␤2; PAR4, protease activated receptor 4; G␣q/
G␣i, heterotrimeric G protein; PKC, protein kinase C.
Synergistic signaling between CalDAG-GEFI and PKC was
evident in 2 ways: (1) at high concentrations of PAR4p, where full
aggregation was observed in platelets lacking functional PKC or
CalDAG-GEFI, preincubation of CalDAG-GEFI–deficient platelets with Ro31-8220 blocked integrin activation/aggregation; and
(2) at a low dose of the agonist, where successful activation of
␣IIb␤3 was observed in WT platelets but not platelets lacking
either functional PKC or CalDAG-GEFI (Figure 1).
In addition to PKC, ␣IIb␤3 activation/aggregation induced by
high PAR4p concentrations in the absence of CalDAG-GEFI
required signaling by the G␣i-coupled ADP receptor, P2Y12
(Figure 2). Interestingly, our studies also suggest that the aggregation defect of CalDAG-GEFI–deficient platelets stimulated with
low-dose PAR4p is the result of impaired ADP/dense granule
release in these platelets (Figure 3). These studies with low-dose
PAR4p-stimulated mutant platelets cannot rule out the possibility
that addition of ADP simply pushes the platelets above the
threshold concentration of stimulus required for ␣IIb␤3 activation.
However, aggregation was again dependent on P2Y12 signaling,
demonstrating the importance of this signaling pathway as an
alternative to CalDAG-GEFI signaling in integrin activation. It is
not clear how CalDAG-GEFI-Rap1 signaling affects granule
release in platelets stimulated with low-dose PAR4p. Studies in
other cell types demonstrated that Rap1 is localized to the surface
of secretory granules and that it translocates to the plasma
membrane on cellular stimulation, suggesting that it may play a
role in granule secretion.27-29 Furthermore, CalDAG-GEFII and
CalDAG-GEFIII have been implicated in granule release in mast
cells and endocrine tissue, respectively.30,31 CalDAG-GEF–
mediated granule release in these cells involved activation of the
small GTPase RhoA, and microtubule formation and was, at least
in part, independent of PKC function. The latter observation is in
contrast to our own findings, which show completely inhibited
granule release in PAR4p-activated platelets lacking PKC signaling. The mechanisms by which CalDAG-GEFI affects granule
release in platelets will be investigated in further studies.
Our results on the role of CalDAG-GEFI and PKC signaling in
platelet integrin activation are well in line with previous studies.
Inhibition of PKC signaling was shown to strongly impair dense
granule release in thrombin-activated human platelets, whereas it
CalDAG-GEFI AND PKC IN ␣IIb␤3 ACTIVATION
1701
only minimally affects the aggregation of these cells.32-34 Using
calcium chelators to remove intracellular calcium, Quinton et al
suggested a calcium-regulated pathway that synergizes with PKC
signaling in fibrinogen receptor activation in human platelets
stimulated through PAR1.6 Our studies suggest that CalDAG-GEFI
is the primary signaling molecule mediating calcium-dependent
integrin activation in PAR4p-stimulated platelets. Like platelets
treated with a calcium chelator, platelets lacking CalDAG-GEFI
require PKC signaling and costimulation through G␣i-coupled
receptors for successful ␣IIb␤3 activation (Figures 1,2). Furthermore, as shown previously,19 CalDAG-GEFI–deficient platelets
show aggregation defects to agonists other than thrombin, which
are very similar to those published for BAPTA-AM–treated
platelets. This involves defective aggregation in response to ADP
and the TxA2 mimetic U46619 and partially inhibited aggregation
in response to collagen. And lastly, calcium ionophore A23187
failed to aggregate CalDAG-GEFI–deficient platelets, whereas the
DAG mimetic PMA led to full platelet aggregation.19
The key role of CalDAG-GEFI in the rapid activation of Rap1
further confirms that this molecule is the main signaling molecule
involved in calcium-induced activation of integrins. In 1997,
Franke et al showed that Rap1 activation in platelets is mediated by
a rapid calcium-dependent mechanism.12 In a subsequent study, the
same group suggested a model for the sequential activation of Rap1
in thrombin-stimulated platelets.35 In this model, Rap1 activation is
initiated by intracellular calcium generated through PLC activity,
followed by a second phase of activation requiring PKC. In later
years, several groups demonstrated a role for ADP-initiated G␣i
signaling in the PKC-mediated pathway of Rap1 activation.13-15 In
CalDAG-GEFI–deficient platelets activated with PAR4p under
static conditions, Rap1 activation was completely inhibited in the
presence of inhibitors of P2Y12 or PKC (Figure 4). Trace amounts
of Rap1-GTP were observed in 2-MesAMP-treated mutant platelets activated under stirring conditions (Figure 5B). These data
show that CalDAG-GEFI is the dominant molecule in PAR4pactivated platelets allowing for the activation of Rap1 in the
absence of G␣i signaling, and that it represents the previously
described calcium-dependent pathway involved in the rapid activation of Rap1. So far, CalDAG-GEFI is the only Rap1-GEF with
documented activity in the regulation of platelet Rap1. Other
identified Rap1-GEFs in platelets include CalDAG-GEFIII,36 PDZGEF1,36 and Epac1 (cAMP-GEFI).37 In addition to GEFs, Rap1
activation is regulated by GAPs. In platelets, Rap1GAP2 was
recently identified.36 In other cell types, G␣i/o signaling was shown
to promote ubiquitination and proteosomal degradation of
Rap1GAP2, leading to enhanced Rap1 activity.38 The role of
Rap1GAP2 in G␣i-dependent Rap1 activation in platelets has not
been addressed.
In addition to identifying CalDAG-GEFI as the central molecule mediating the rapid activation of Rap1, we could demonstrate
that Rap1 activation and aggregation follow very similar kinetics in
activated platelets (Figure 5). In CalDAG-GEFI–deficient platelets
activated with a threshold dose of PAR4p, both aggregation and
Rap1 activation were delayed by approximately 30 seconds.
Whereas Rap1 activation was weak at the beginning of the
aggregation phase, marked Rap1 activation was observed when
maximum aggregation was reached. Similarly, the kinetic of Rap1
activation mirrored that of the aggregation response in 2-MesAMPpretreated CalDAG-GEFI-deficient platelets costimulated with
PAR4p and epinephrine. It cannot be concluded, however, that
impaired ␣IIb␤3 activation/aggregation observed in CalDAGGEFI–deficient platelets pretreated with MesAMP or Ro31-8220 is
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1702
BLOOD, 1 SEPTEMBER 2008 䡠 VOLUME 112, NUMBER 5
CIFUNI et al
the direct result of defective Rap1 signaling. Indeed, studies with
platelets lacking Rap1b make this assumption rather doubtful as
they showed significant ␣IIb␤3 activation in response to various
agonists (including thrombin).16 However, platelets also express
other small GTPases shown to be involved in integrin activation,
such as Rap239 and R-ras.40,41 These proteins may be regulated by
CalDAG-GEFI–dependent and -independent mechanisms and thus
facilitate ␣IIb␤3 activation in the absence of Rap1.
It is important to note that there are significant species-specific
differences in platelet responses to thrombin between human and
mouse platelets, with mice expressing PAR3 to support cellular
activation through PAR4,23 whereas ␣IIb␤3 activation induced by
thrombin in human platelets depends on signaling provided by both
PAR1 and PAR4. Recent studies by Holinstat et al demonstrated
impaired activation of integrin ␣IIb␤3 in PAR4-stimulated human
platelets pretreated with BAPTA-AM and 2-MesAMP.42 In contrast, pretreatment with BAPTA-AM and 2-MesAMP inhibited
␣IIb␤3 activation in human platelets stimulated with a low but not
high dose of PAR1-stimulating peptide. These findings may be
explained by signaling through G␣i/o, which couples to PAR1 but
not PAR4.43
Our findings are also important with regard to the diagnosis of
patients with leukocyte adhesion deficiency type III (LAD-III), a
clinical complication characterized by recurrent infections and a
Glanzmann-like bleeding phenotype.44 Studies in mice20 and
humans45 strongly suggest that impaired expression/function of
CalDAG-GEFI, resulting in defective Rap1 activation and integrinmediated cell adhesion, is a molecular cause for this disease.
Others, however, have not found defects in Rap1 activation in
platelets and leukocytes from patients with a similar inflammatory
and hemostatic phenotype (designated LAD-I/variant).46,47 It is
entirely possible that abnormalities in signaling molecules other
than CalDAG-GEFI can lead to a combined defect in ␤1, ␤2, and
␤3 integrins demonstrated for LAD-III (LAD-I/variant) patients. It
is important to keep in mind, however, that there are alternative
pathways in platelets (and most probably leukocytes), which can
facilitate Rap1 activation in the absence of functional CalDAGGEFI. Based on our studies, we suggest that patients with a
LAD-III phenotype be tested for Rap1 activation in platelets
activated with weak agonists, such as ADP or low-dose thrombin.
If CalDAG-GEFI function is impaired, Rap1 activation will be
strongly reduced in these cells. In contrast, Rap1 activation will
appear normal in platelets activated with high-dose thrombin, as
ADP-induced P2Y12/G␣i signaling will serve as a backup for
CalDAG-GEFI under these experimental conditions.
In conclusion, we show that the activation of Rap1 and ␣IIb␤3
in platelets stimulated through the PAR4 receptor is regulated by
independent CalDAG-GEFI– and PKC/ADP-dependent pathways.
Our data further demonstrate that CalDAG-GEFI is the dominant
signaling molecule allowing for the rapid but reversible activation
of Rap1 in PAR4p-stimulated platelets and that CalDAG-GEFI is
important for dense granule release in platelets stimulated with
low-dose PAR4p.
Acknowledgments
The authors thank Crystal Piffath for her excellent technical
support throughout the study, Janos Polgar for help with the
serotonin release assay and helpful discussions, and Jill Crittenden
and Ann Graybiel for providing the mice and for helpful discussions.
This work was supported by a Scientist Development Grant
0630044N from the American Heart Association (W.B.) and the
National Heart, Lung, and Blood Institute of the National Institutes
of Health (grants R37 HL41002 and P01 HL56949; D.D.W.)
Authorship
Contribution: S.M.C. performed many of the experiments and
helped analyze the data; D.D.W. helped design the study and
interpret the results; and W.B. designed the study, performed many
of the experiments, analyzed the results, and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Wolfgang Bergmeier, Cardeza Foundation
and Department of Medicine, Thomas Jefferson University, 803
Curtis, 1015 Walnut Street, Philadelphia, PA 19107; e-mail:
[email protected].
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2008 112: 1696-1703
doi:10.1182/blood-2008-02-139733 originally published
online June 10, 2008
CalDAG-GEFI and protein kinase C represent alternative pathways
leading to activation of integrin αIIbβ3 in platelets
Stephen M. Cifuni, Denisa D. Wagner and Wolfgang Bergmeier
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