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Coordinated signaling through both G12/13 and Gi pathways is sufficient to activate
GPIIb/IIIa in human platelets
Robert T. Dorsam*§, Soochong Kim#, Jianguo Jin#, and Satya P. Kunapuli*#§
*Department of Pharmacology, #Department of Physiology, and §The Sol Sherry ThrombosisResearch Center, Temple University School of Medicine, Philadelphia, PA
*This work was supported by Research Grants HL60683 and HL64943 from the National
Institutes of Health (S. P. K.). R.T.D. is supported by a training grant T32 HL07777 from the
National Institutes of Health.
Corresponding Author:Satya P. Kunapuli, Ph.D.Department of PhysiologyTemple UniversityDepartment of Physiology- Rm. 224, OMS3420 N. Broad StreetPhiladelphia, Pennsylvania 19140 USAPhone: (215) 707-4615Fax: (215) 707-4003E-mail: [email protected]
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on September 23, 2002 as Manuscript M208778200 by guest on July 12, 2020
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ABSTRACT
Activation of GPIIb/IIIa is known to require agonist-induced inside-out signaling through Gq, Gi,
and Gz. Although activated by several platelet agonists, including thrombin and thromboxane A2,
the contribution of the G12/13 signaling pathway to GPIIb/IIIa activation has not been investigated.
In this study, we used selective stimulation of G protein pathways to investigate the contribution of
G12/13 activation to platelet fibrinogen receptor activation. YFLLRNP is a PAR-1-specific partial
agonist that, at low concentrations (60 µM), selectively activates the G12/13 signaling cascade
resulting in platelet shape change without stimulating the Gq or Gi signaling pathways. YFLLRNP-
mediated shape change was completely inhibited by the p160ROCK inhibitor, Y-27632. At this low
concentration, YFLLRNP-mediated G12/13 signaling caused platelet aggregation and enhanced
PAC-1 binding when combined with selective Gi or Gz signaling, via selective stimulation of the
P2Y12 receptor or a2A adrenergic receptor, respectively. Similar data were obtained when using
low dose U46619 (10 nM), a thromboxane A2 mimetic, to activate G12/13 in the presence of Gi
signaling. These results suggest that selective activation of G12/13 causes platelet GPIIb/IIIa
activation when combined with Gi signaling. Unlike either G12/13 or Gi activation alone, co-
activation of both G12/13 and Gi resulted in a small increase in intracellular calcium. Chelation of
intracellular calcium with dimethyl BAPTA dramatically blocked G12/13 and Gi-mediated platelet
aggregation. No significant effect on aggregation was seen when using selective inhibitors for
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p160ROCK, PKC, or MEKK1. PI-3 kinase inhibition lead to near abolishment of platelet
aggregation induced by co-stimulation of Gq and Gi pathways, but not by G12/13 and Gi pathways.
These data demonstrate that co-stimulation of G12/13 and Gi pathways is sufficient to activate
GPIIb/IIIa in human platelets in a mechanism that involves intracellular calcium, and that PI-3
kinase is an important signaling molecule downstream of Gq, but not downstream of G12/13
pathway.
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Agonists for platelet activation, though having varying efficacies for platelet dense
granule secretion and fibrinogen receptor (GPIIb/IIIa; integrin aIIbb3) activation, often signal
through similar G-protein signaling pathways (1,2). GPIIb/IIIa receptor activation occurs by G
protein-mediated inside-out signaling stimulated by platelet agonists such as ADP, thromboxane
A2, and thrombin (3). These agonists cause GPIIb/IIIa to go from a low affinity state to a high
affinity binding state that results in the binding of fibrinogen and cross-linking of platelets (3).
Epinephrine binds to the a2A adrenergic receptor and causes activation of the Gz pathway that
leads to the inhibition of adenylyl cyclase (4,5). Stimulation of the a2A adrenergic receptor
alone is insufficient to cause either dense granule secretion or GPIIb/IIIa activation in washed
platelets, however epinephrine potentiates both secretion and platelet aggregation caused by
other agonists (6-9). ADP binds to the Gq-coupled P2Y1 and the Gi-coupled P2Y12 receptors,
and signaling through both of these pathways is necessary for ADP-induced GPIIb/IIIa
activation (8,10-12), although ADP does not cause dense granule secretion in aspirin-treated
human platelets (13). Thromboxane A2 binds to the TPa and TPb receptor subtypes that
activate both Gq (14,15) and G12/13 signaling (16). Thromboxane receptor stimulation causes
both platelet aggregation and dense granule secretion, but depends upon secreted contents to
provide Gi signaling. The combined signaling from TP receptor stimulation and the Gi
signaling from the secreted ADP or epinephrine causes GPIIb/IIIa activation (17). Both ADP
and thromboxane A2 require co-stimulation of Gq and Gi pathways to cause platelet aggregation
(8,17). Thrombin cleaves the N-terminus of PAR-1 and PAR-4 on human platelets, uncapping
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a tethered ligand that activates the PAR receptors (18). Both PAR-1 and PAR-4 receptors
couple to Gq and G12/13, and cause fibrinogen receptor activation independently of Gi
stimulation by secreted ADP (19).
The heterotrimeric G proteins G12 and G13 are found in human platelets (20), and are
activated upon thromboxane and thrombin receptor stimulation (16). The first evidence for the
role of G12/13 in platelet shape change came from the studies with Gq knockout mice wherein
thrombin and thromboxane A2 failed to cause platelet aggregation but caused platelet shape
change (21). However, ADP failed to cause shape change in these mouse platelets indicating
that ADP receptors do not couple to G12/13 pathways (21). G12/13 activates Rho/Rho-kinase,
causing the phosphorylation of myosin light chain and calcium-independent shape change (22).
G12/13 signaling mediates calcium-independent platelet shape change, involving RhoA and
p160ROCK activity in human and mouse platelets (22). Y-27632, a specific inhibitor of p160ROCK,
blocks the calcium-independent shape change that occurs due to G12/13-mediated signaling,
suggesting that p160ROCK is a key signaling molecule downstream of G12/13 for the platelet
shape change response (23,24). Though the G12/13 pathway has been implicated in p160ROCK
activation and subsequent shape change, this pathway remains the least characterized of the
known G protein-coupled pathways in platelets.
The Gq pathway stimulates phospholipase C, which cleaves phosphatidylinositol 4,5
bisphosphate and results in cofactors that activate protein kinase C (PKC) (1). The a-subunit of
the heterotrimeric G protein Gi pathway inhibits the activity of adenylyl cyclase while the bg-
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subunit activates PI-3 kinase (25). Together, these pathways lead to the activation of numerous
kinases including protein kinase B (PKB/Akt) (26), protein kinase C (PKC) (4), Map kinase
kinase (MEKK1) (27), Src family tyrosine kinases (28), among many others.
YFLLRNP is a heptapeptide that binds to PAR-1 and causes shape change but no
calcium mobilization when used at low concentrations (29). This YFLLRNP-induced platelet
shape change is mediated by the G12/13 – RhoA- p160ROCK pathway and can be completely
blocked by Y-27632 (24). Similarly, low concentrations of the thromboxane mimetic, U46619,
also cause activation G12/13 pathways without activating the Gq pathways (30,31). In this study
we used these selective agonists of G12/13 pathways, in combination with selective activation of
Gi pathways, to demonstrate the contribution of G12/13 signaling cascades to fibrinogen receptor
activation in human platelets. Previously, Gq and Gi have been recognized as the G proteins that
activate pathways leading to platelet aggregation (8). Our studies demonstrate that the G12/13
pathway, in the presence of Gi signaling, can lead to GPIIb/IIIa activation in human platelets
and that PI-3 kinase is an important signaling molecule downstream of Gq, but not downstream
of G12/13 pathway.
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Experimental Procedures:
Materials: Apyrase grade VII, human fibrinogen, and acetylsalicylic acid were obtained from
Sigma (St. Louis, MO). The heptapeptide YFLLRNP was synthesized by New England Biolabs
(Beverly, MA) and the same peptide was also synthesized by Research Genetics (Huntsville,
AL). Adenosine diphosphate (ADP) and Epinephrine were purchased from Chrono-Log Corp.
(Havertown, PA). FITC-conjugated monoclonal antibody PAC-1 was purchased from Becton
Dickinson (San Jose, CA). FURA-2, AM was purchased from Molecular Probes (Eugene, OR).
Adenine [2,8-3H] was purchased from Perkin Elmer (Boston, MA). The acetoxymethyl ester of
5,5’-dimethyl-bis-(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (dimethyl BAPTA), Y-
27632, LY294002, and Ro 31-8220 were purchased from Biomol (Plymouth Meeting, PA).
U0126 was purchased from Alexis Biochemicals (Lausen, Switzerland). AR-C 69931MX was a
gift from Astra-Zeneca Research Laboratories-Charnwood, Loughborough, UK
Platelet preparation: Whole blood was drawn from healthy, consenting human volunteers into
tubes containing 1/6th volume of ACD (2.5 g of sodium citrate, 1.5 g of citric acid, and 2 g of
glucose in 100 mL of deionized water). Blood was centrifuged (Eppendorf 5810R centrifuge,
Hamburg, Germany) at 230 rcf for 20 minutes at room temperature to obtain platelet rich
plasma (PRP). PRP was incubated with 1 mM acetylsalicylic acid (Sigma) for 30 minutes at
37°C, and for calcium measurement PRP was also incubated with 2 mM Fura-2, AM for 45
minutes at 37°C. The PRP was then centrifuged for 10 minutes at 980 rcf (room temperature) to
pellet the platelets. Platelets were resuspended in Tyrode’s buffer (138 mM NaCl, 2.7 mM
KCl, 1 mM MgCl2, 3 mM NaH2PO4, 5 mM glucose, 10 mM Hepes pH 7.4, 0.2% bovine
serum albumin) containing 0.01 U/mL apyrase. Cells were counted using Z1 Coulter Particle
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Counter and adjusted to 2 x 108 platelets/mL. For flow cytometry studies, cells were adjusted to
a concentration of 4.2 X106 platelets/mL.
Aggregometry: Aggregation of 0.5 mL of washed platelets was analyzed using a P.I.C.A.
lumiaggregometer (Chrono-log Corp., Havertown, PA). Aggregation was measured using light
transmission under stirring conditions (900 rpm) at 37°C. Agonists were added simultaneously
for platelet stimulation, however platelets were pre-incubated each inhibitor as follows: 1 µM
dimethyl BAPTA, 10 mM Ro 31-8220 or 25 µM LY294002 for 3 minutes at 37°C and 10 µM
Y-27362 or 10 mM U0126 for 10 minutes at 37°C . Each sample was allowed to aggregate for
at least 3 minutes. The chart recorder (Kipp and Zonen, Bohemia, NY) was set for 0.2 mm/s.
All samples contained exogeneously added human fibrinogen (1 mg/mL).
Intracellular calcium mobilization: Calcium mobilization was measured in platelets that were
loaded with 2 mM FURA-2, AM in PRP for 45 minutes at 37°C, and washed platelets were
isolated as noted above and brought to a final concentration of 2 X 108 platelets/mL in Tyrode’s
buffer. Samples of Fura-2, AM-loaded platelets (0.5 mL) were placed in a quartz cuvette with
a magnetic stirbar, and incubated for 1 minute at 37°C in a temperature-controlled chamber. An
Aminco Bowman Series 2 Luminescence Spectrometer was used for measurement of
intracellular calcium mobilization. Two wavelengths (340 and 380 nm) were used for excitation
and the emitted light was measured at 510 nm. Samples were stimulated after 1 minute
incubation at 37°C and all concentrations of YFLLRNP were added in a volume of 5 mL to
account for dilution effects. Fmin was obtained by addition of 20 mM Tris and 4 mM EGTA,
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and Fmax was determined by adding 0.25% Triton and saturating levels of CaCl2. Calculation of
the calcium mobilization was performed as outlined previously (32).
Analysis of PAC-1 binding: Activation of GPIIb/IIIa was measured by PAC-1 mAb binding to
washed platelets and subsequent analysis by flow cytometry. Aspirin-treated platelets were
isolated by centrifugation as noted, then counted, and brought to a concentration of 4.2 x 106
platelets/mL. The assay was performed considering that three compounds, each 5 mL in
volume, were added to each to each tube prior to addition of the platelets. PAC-1 mAb (5 µL)
was also added to each tube. Tyrode’s buffer was added in samples where less than three
compounds were necessary to normalize the volume. Considering that there is 20 mL total
volume of agonist/mAb added to each sample, adding 50 mL of platelets to the 20 mL of
agonist/Ab resulted in a final concentration to 3 x 106 platelets/mL. The platelets were added to
each tube in 15 second increments to begin stimulation. The samples were stimulated for a
period of 10 minutes in the dark, and then diluted with 450 mL of Tyrode’s buffer. 450 mL of
each sample was transferred to a 12 X 75 mm cuvette (Fisher Scientific, Pittsburgh, PA) and
analyzed by flow cytometry, using FACSCAN (BD Biosciences, San Jose, CA), to measure an
increase in fluorescence that indicates an increase in GPIIb/IIIa receptor activation. The
experiment was performed three times and data are presented as mean +/- S.E.
Measurement of Cyclic AMP Formation in Intact Platelets: Platelet-rich plasma was incubated
with 2 µCi/ml [3H] adenine and aspirin (1mM) for 1 h at 37°C (33). Platelets were isolated from
plasma by centrifugation at 980 X g for 10 min and resuspended in Tyrode’s buffer. Platelet
preparations were incubated with 20 mM forskolin for 3 minutes to stimulate cAMP formation,
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or forskolin and agonist for measurement of Gi signaling stimulated by the agonist. Reactions
were stopped with 1 M HCl and 4000 dpm of [14C] cAMP as recovery standard. Cyclic AMP
was determined by the method of Salomon (34) and expressed as percentage of total [3H]
adenine nucleotides.
RESULTS AND DISCUSSION
The agonists ADP, thrombin, and thromboxane A2 activate multiple G protein pathways,
including Gq, G12/13, and Gi, to activate platelet shape change, dense granule secretion, and
GPIIb/IIIa receptor activation (1). Each agonist has a distinct mechanism to achieve full platelet
activation and much work has been focused on identifying signaling molecules and determining
the roles of each pathway in platelet activation. Whereas Gq and Gi pathways have been
identified as regulating GPIIb/IIIa activation (8), and G12/13 signaling has been implicated in
platelet shape change (22-24), the contribution of G12/13 stimulation to platelet fibrinogen
receptor activation has not been demonstrated.
Determination of the functional coupling specificity of YFLLRNP : Thrombin-
mediated cleavage of the PAR-1 receptor causes activation of both Gq and G12/13 pathways,
leading to a calcium-dependent and calcium-independent shape change, respectively (16,35).
YFLLRNP is a partial agonist at the PAR-1 receptor that antagonizes both a-thrombin-and
SFLLRNP-mediated platelet aggregation and causes platelet shape change without calcium
mobilization or platelet aggregation (29). We first evaluated the concentration-dependent
activation of G proteins by YFLLRNP ranging from 50 mM to 200 mM to identify the proper
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concentration of peptide that is activating G12/13, but not activating Gq signaling. We noted that
60 mM YFLLRNP caused platelet shape change (Fig. 1A) without aggregation or calcium
mobilization (Fig. 1B). Intracellular calcium mobilization occurred at 100 mM YFLLRNP or
higher, suggesting that the peptide activated both Gq and G12/13 at higher concentrations. The
same peptide synthesized from a different source provided similar results (data not shown).
While other studies used up to 300 mM YFLLRNP without calcium mobilization (29), higher
concentrations of YFLLRNP (100-200 mM) caused small calcium mobilization in our hands,
suggesting that there is an increase in Gq coupling. This difference in potency of the peptide
could be due to different quality/purity of the synthesized peptide.
Thromboxane receptors and protease activated receptors couple to Gq and G12/13
pathways and this coupling is dependent on the concentration of the agonist (16,30,31).
Subsequent studies revealed that G12/13-mediated platelet shape change is slow, occurs in the
absence of calcium mobilization, involves p160ROCK as an important signaling molecule, and
can be completely blocked by the p160ROCK inhibitor, Y-27632 (22-24). Thus, the slow platelet
shape change in the absence of intracellular calcium mobilization that can be blocked by Y-
27632 can be taken as a measure of G12/13 activation.
To ensure that YFLLRNP was activating the G12/13 pathway specifically, we measured
YFLLRNP-mediated platelet shape change in the presence or absence of 10 mM Y-27632. As
expected, 10 mM Y-27632 completely inhibited platelet shape change caused by 60 mM
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YFLLRNP (Fig. 1A), suggesting that low-dose YFLLRNP is causing only G12/13-mediated
shape change without a calcium-dependent shape change component.
PAR-1 can couple to the Gi pathway and cause the inhibition of adenylyl cyclase (35);
however, other data suggest that PAR-1 stimulation relies upon secreted ADP for Gi activation
(19). To investigate whether YFLLRNP can activate the Gi pathway, we measured cAMP
formation in YFLLRNP-stimulated platelets. YFLLRNP (60 mM) did not cause significant
inhibition of forskolin-stimulated adenylyl cyclase (Figure 1C), indicating that at this
concentration YFLLRNP does not activate Gi signaling pathways.
Contribution of G12/13 signaling to Platelet aggregation and GPIIb/IIIa receptor
activation : Selective activation of Gq pathways by ADP results only in shape change, while
supplementing Gq signaling with Gi activation, through P2Y12 receptor activation or a2A
receptor activation, results in platelet aggregation (8,36). As selective activation of G12/13
pathways with YFLLRNP (60 µM) resulted only in shape change (Fig. 1A), we investigated the
effect of supplementing this pathway with Gi signaling cascade on platelet fibrinogen receptor
activation.
ADP causes platelet aggregation by stimulating both the Gq-coupled P2Y1 receptor and
the Gi-coupled P2Y12 receptor (8). We used A3P5P, a P2Y1-selective antagonist to block ADP
signaling through the Gq-coupled P2Y1 receptor. Addition of 10 µM ADP in the presence of 1
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mM A3P5P results in selective stimulation of the Gi-coupled P2Y12 receptor, and is evident by
the loss of ADP-induced shape change and aggregation (8). YFLLRNP (60 µM) in the presence
of P2Y12-selective stimulation caused platelet aggregation (Figure 2). Whereas epinephrine
alone does not cause aggregation, simultaneous addition of epinephrine with YFLLRNP caused
platelet aggregation (Figure 2). We also noted that addition of 10 mM epinephrine immediately
subsequent to the addition of YFLLRNP caused platelet aggregation (data not shown).
Though we have demonstrated that platelet aggregation can occur in the presence of
G12/13 and Gi signaling, we wanted to directly correlate concomitant G12/13 and Gi signaling
with GPIIb/IIIa activation. The GPIIb/IIIa receptor shifts from a low-affinity state to a high-
affinity state upon platelet stimulation with agonists such as thrombin, ADP, or thromboxane A2
(3). The PAC-1 mAb is directed against the active conformation of GPIIb/IIIa receptor (37).
YFLLRNP-stimulated platelets bound similar levels of PAC-1 mAb compared to unstimulated
platelets (Figure 3). Platelets treated with either 10 mM epinephrine or ADP and A3P5P bound
background levels of PAC-1 Ab confirming that Gi signaling alone was insufficient to cause
significant GPIIb/IIIa activation. ADP (10 µM) caused a similar magnitude of PAC-1 mAb
binding compared with YFLLRNP plus epinephrine. Also, platelets stimulated simultaneously
with YFLLRNP and selective P2Y12 stimulation bound levels of PAC-1 mAb similar to ADP-
stimulated cells (Figure 3). These results suggest that while activation of either G12/13 or Gi
signaling alone cannot cause GPIIb/IIIa receptor activation, co-stimulation of G12/13 and Gi
signaling pathways can result in GPIIb/IIIa activation.
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The thromboxane receptor couples to Gq and G12/13 in human platelets (16,38). We
used a stable thromboxane A2 mimetic, U46619, for stimulation of the TP receptor. At low
doses of U46619 (10 nM), the receptor couples only to the G12/13 pathway (30,31). Thus, a low
concentration of U46619 provides an alternative to low dose of YFLLRNP to stimulate G12/13
pathways through TP receptors. Stimulation of the platelets with this concentration of U46619
resulted in platelet shape change, but not in calcium mobilization or in platelet aggregation (Fig.
4). However, higher concentration of U46619 (100 nM) causes calcium mobilization (Fig. 4A)
and calcium-dependent shape change that is not inhibited by Y-27632 (Fig. 4B). Simultaneous
addition of either 10 mM epinephrine or 10 µM ADP in the presence of 1 mM A3P5P to 10 nM
U46619-stimulated platelets lead to both shape change and platelet aggregation (Figure 4C).
This illustrates that either P2Y12 receptor or a2A adrenergic receptor stimulation is capable of
causing platelet aggregation when combined with G12/13 signaling from the TP receptor. When
we were finalizing the manuscript, Nieswandt et al (39) reported that stimulation of G12/13 and
Gi is sufficient to cause fibrinogen receptor activation in mouse platelets using mice-deficient in
Gaq. Their results, obtained by a complementary approach, support our conclusions and extend
the observations to mouse platelets. These results may also explain why ADP is weaker agonist
than thromboxane A2 and thrombin. ADP activates only Gq pathways and does not activate the
G12/13 pathways, whereas both thromboxane A2 and thrombin do activate this pathway. Since
either Gq or G12/13 can synergize with Gi to result in the activation of GPIIb/IIIa, thrombin and
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thromboxane A2, activating both Gq and G12/13, could additionally synergize with Gi and
thereby cause more robust platelet aggregation.
Role of intracellular calcium in G12/13 and Gi-mediated human platelet aggregation:
Calcium plays an important role in the platelet function, including the activation of GPIIb/IIIa
(1,3). Although the bg subunits of Gi are known to increase intracellular calcium by the
activation of phospholipase C in other cells (40), selective activation of Gi in platelets through
either P2Y12 or a2A receptors does not mobilize intracellular calcium (8,36). Although neither
epinephrine nor YFLLRNP (60 µM) caused any increases in intracellular calcium, together they
mobilized a small amount of calcium (15 ± 4 nM) from the intracellular stores (Fig. 5A). As
stimulation of G12/13 or Gi alone does not cause increases in intracellular calcium, it is
surprising to see this small increase with co-stimulation of these two pathways. ADP (300 nM)
caused similar increases in intracellular calcium as YFLLRNP and epinephrine together (Fig.
5A). Hence, we used ADP (300 nM) in the presence of AR-C 69931MX, a selective P2Y12
receptor antagonist, to selectively activate the Gq pathway and increase a small and comparable
intracellular calcium (Fig. 5A), and evaluated the effect of epinephrine on platelet aggregation.
As shown in Fig. 5B, although selective activation of P2Y1 receptor alone did not cause any
aggregation, co-stimulation of P2Y1 and a2A adrenergic receptors led to comparable extent of
aggregation as the combined G12/13 and Gi stimulation (Fig. 5B). These data indicate that co-
stimulation of G12/13 and Gi results in a small increase in intracellular calcium which may play
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an important role in the activation of GPIIb/IIIa. Contrary to our results, Nieswandt et al (39)
did not observe any intracellular calcium mobilization with the combined G12/13 and Gi
signaling in mouse platelets. Hence, we investigated the role of this small amount of
intracellular calcium in the platelet fibrinogen receptor activation using an intracellular calcium
chelator, dimethyl BAPTA. As shown in Fig. 5C, pre-incubation of platelets with dimethyl
BAPTA (1 µM) dramatically blocked the aggregation, but not shape change, induced by
YFLLRNP and epinephrine. These results indicate that the small increases in intracellular
calcium, as a result of combined G12/13 and Gi stimulation, play an important role in the
activation of GPIIb/IIIa in human platelets.
Signaling events downstream of concomitant activation of G proteins in human
platelets: The signaling events that occur downstream of platelet receptor stimulation has been
the subject of intense study in several laboratories. Major signaling molecules lying downstream
of G protein activation include PKC (4), MEKK1 (41), PI-3 kinase (25,26), and p160ROCK
(23,24), among many others (3). We measured the effects of selective inhibitors for these
molecules on platelet aggregation stimulated by combined G12/13 and Gi signaling. We then
compared the effects of these inhibitors on concomitant Gq and Gi- mediated platelet
aggregation (8), using ADP as the agonist.
PKC inhibition with Ro 31-8220, an inhibitor of novel and conventional PKC isoforms
(42), had no effect on the aggregation caused by concomitant G12/13 and Gi signaling or Gq and
Gi signaling (Figure 6A and B). These results are consistent with our previous observations, that
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the PKC pathway is important, but not essential, in the activation of GPIIb/IIIa (43). U0126, a
MEKK1 inhibitor (44), also had no effect on the aggregation induced by co-activation of either
G12/13 and Gi or Gq and Gi signaling. Thus, although Erk2 has been implicated in the GP1b-IX-
mediated platelet fibrinogen receptor activation (27), the MEKK-Erk pathway does not play any
significant role in either G12/13 and Gi- or Gq and Gi-mediated GPIIb/IIIa activation in human
platelets.
PI-3 kinase has been known to be involved in platelet activation (3), and knockout
studies show that PI-3 kinase g-deficient mice have decreased aggregation responses to ADP
and collagen (25). LY294002, a PI-3 kinase inhibitor (45), caused a slight decrease in the
extent of combined G12/13 and Gi mediated aggregation, however aggregation and shape change
were still significant in the presence of PI-3 kinase inhibitor (Fig. 6A). This effect was
comparable to the decrease in ADP-induced platelet aggregation in PI-3 kinase g-deficient mice
versus wild type mice (25). While there was a decrease in aggregation, it is unlikely that PI-3
kinase is a key signaling molecule downstream of G12/13 signaling. Rather, LY 294002 is
mediating its effects through decreasing the P2Y12- or a2A adrenergic-stimulated Gi and PI-3
kinase g signaling pathways (46) (depicted in Fig. 7). Conversely, concomitant Gq and Gi
mediated platelet aggregation was nearly abolished by the PI-3 kinase inhibitor (Fig. 6B). These
results indicate that PI-3 kinase is a key signaling molecule in the combined Gq and Gi pathway.
By comparison, PI-3 kinase appears to be a key molecule in the Gq signaling cascade, but not in
G12/13 mediated signaling pathway, leading to the fibrinogen receptor activation (Fig. 7).
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p160ROCK has been identified as a key signaling molecule downstream of G12/13
activation (23,24). Using the p160ROCK inhibitor Y-27632, we expected that platelet
aggregation caused by concomitant G12/13 and Gi signaling would be inhibited. Interestingly,
Y-27632 did not block aggregation caused by simultaneous G12/13 and Gi signaling (Fig. 6A),
suggesting that there is a divergent pathway downstream of G12/13 stimulation. Thus, G12/13
signals through at least two separate pathways, one of which involves p160ROCK and shape
change, and the other that contributes to GPIIb/IIIa activation. As expected, combined Gq and
Gi– mediated platelet aggregation was also unaffected by the p160ROCK inhibitor (Fig. 6B),
indicating that this signaling molecule does not play any significant role in the activation of
fibrinogen receptor (Fig. 7).
In conclusion, we have demonstrated that coordinated signaling between G12/13 and Gi
pathways is a sufficient and redundant mechanism for the activation of fibrinogen receptor in
human platelets. PI-3 kinase appears to be an important signaling molecule downstream of Gq
but not G12/13-medated activation of GPIIb/IIIa. Co-stimulation of G12/13 and Gi pathways
appears to increase intracellular calcium, independently of Gq activation, which plays an
important role in the fibrinogen receptor activation in human platelets. The mechanisms of
increase in intracellular calcium by G12/13 and Gi pathways are under investigation.
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Acknowledgements: We thank Drs. James L. Daniel, Barrie Ashby, and Todd M. Quinton,
Temple University Medical School, for critically reading the manuscript.
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REFERENCES
1. Brass, L. F., Manning, D. R., Cichowski, K., and Abrams, C. S. (1997) Thromb Haemost
78, 581-589
2. Offermanns, S. (2000) Biol Chem 381, 389-396.
3. Shattil, S. J., Kashiwagi, H., and Pampori, N. (1998) Blood 91, 2645-2657
4. Brass, L. F., Woolkalis, M. J., and Manning, D. R. (1988) J Biol Chem 263, 5348-5355.
5. Yang, J., Wu, J., Kowalska, M. A., Dalvi, A., Prevost, N., O'Brien, P. J., Manning, D.,
Poncz, M., Lucki, I., Blendy, J. A., and Brass, L. F. (2000) Proc Natl Acad Sci U S A 97,
9984-9989.
6. Steen, V. M., Holmsen, H., and Aarbakke, G. (1993) Thromb. Haemost. 70, 506-513.
7. Lanza, F., Beretz, A., Stierle, A., Hanau, D., Kubina, M., and Cazenave, J. P. (1988) Am
J Physiol 255, H1276-1288
8. Jin, J., and Kunapuli, S. P. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 8070-8074
9. Dangelmaier, C., Jin, J., Smith, J. B., and Kunapuli, S. P. (2001) Thromb Haemost 85,
341-348.
10. Daniel, J. L., Dangelmaier, C., Jin, J., Ashby, B., Smith, J. B., and Kunapuli, S. P.
(1998) J. Biol. Chem. 273, 2024-2029
11. Hollopeter, J., Jantzen, H.-M., Vincent, D., Li, G., England, L., Ramakrishnan, V.,
Yang, R.-B., Nurden, P., Nurden, A., Julius, D. J., and Conley, P. B. (2001) Nature 409,
202-207
12. Foster, C. J., Prosser, D. M., Agans, J. M., Zhai, Y., Smith, M. D., Lachowicz, J. E.,
Zhang, F. L., Gustafson, E., Monsma, F. J., Jr., Wiekowski, M. T., Abbondanzo, S. J.,
by guest on July 12, 2020http://w
ww
.jbc.org/D
ownloaded from
21
Cook, D. N., Bayne, M. L., Lira, S. A., and Chintala, M. S. (2001) J Clin Invest 107,
1591-1598.
13. Mills, D. C. B. (1996) Thromb. Haemost. 76, 835-856
14. Raychowdhury, M. K., Yukawa, M., Collins, L. J., McGrail, S. H., Kent, K. C., and
Ware, J. A. (1995) J Biol Chem 270, 7011
15. Raychowdhury, M. K., Yukawa, M., Collins, L. J., McGrail, S. H., Kent, K. C., and
Ware, J. A. (1994) J Biol Chem 269, 19256-19261
16. Offermanns, S., Laugwitz, K.-L., Spicher, K., and Schulz, G. (1994) PNAS 91, 504-508
17. Paul, B. Z. S., Jin, J., and Kunapuli, S. P. (1999) J. Biol. Chem. 274, 29108-29114
18. Coughlin, S. R. (1999) Proc Natl Acad Sci U S A 96, 11023-11027
19. Kim, S., Foster, C., Lecchi, A., Quinton, T. M., Prosser, D. M., Jin, J., Cattaneo, M., and
Kunapuli, S. P. (2002) Blood 99, 3629-3636.
20. Milligan, G., Mullaney, I., and Mitchell, F. M. (1992) FEBS Letters 297, 186-188
21. Offermanns, S., Toombs, C. F., Hu, Y.-H., and Simon, M. I. (1997) Nature 389, 183-
186
22. Klages, B., Brandt, U., Simon, M. I., Schultz, G., and Offermanns, S. (1999) J Cell Biol
144, 745-754
23. Paul, B. Z. S., Daniel, J. L., and Kunapuli, S. P. (1999) J. Biol. Chem. 274, 28293-28300
24. Bauer, M., Retzer, M., Wilde, J. I., Maschberger, P., Essler, M., Aepfelbacher, M.,
Watson, S. P., and Siess, W. (1999) Blood 94, 1665-1672
25. Hirsch, E., Bosco, O., Tropel, P., Laffargue, M., Calvez, R., Altruda, F., Wymann, M.,
and Montrucchio, G. (2001) Faseb J 15, 2019-2021.
by guest on July 12, 2020http://w
ww
.jbc.org/D
ownloaded from
22
26. Banfic, H., Tang, X., Batty, I. H., Downes, C. P., Chen, C., and Rittenhouse, S. E.
(1998) J Biol Chem 273, 13-16
27. Li, Z., Xi, X., and Du, X. (2001) J Biol Chem 276, 42226-42232.
28. Bauer, M., Maschberger, P., Quek, L., Briddon, S. J., Dash, D., Weiss, M., Watson, S.
P., and Siess, W. (2001) Thromb Haemost 85, 331-340.
29. Rasmussen, U. B., Gachet, C., Schlesinger, Y., Hanau, D., Ohlmann, P., Van
Obberghen-Schilling, E., Pouyssegur, J., Cazenave, J. P., and Pavirani, A. (1993) J Biol
Chem 268, 14322-14328
30. Ohkubo, S., Nakahata, N., and Ohizumi, Y. (1996) Br J Pharmacol 117, 1095-1104.
31. Simpson, A. W., Hallam, T. J., and Rink, T. J. (1986) FEBS Lett 201, 301-305.
32. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) J Biol Chem 260, 3440-3450.
33. Kunapuli, S. P., Fen Mao, G., Bastepe, M., Liu-Chen, L. Y., Li, S., Cheung, P. P.,
DeRiel, J. K., and Ashby, B. (1994) Biochem J 298, 263-267
34. Salomon, Y. (1979) Adv..Cyc. Nucl. Res. 10, 35-55
35. Hung, D. T., Wong, Y. H., Vu, T.-K. H., and Coughlin, S. R. (1992) J.Biol.Chem. 267,
20831-20834
36. Jin, J., Daniel, J. L., and Kunapuli, S. P. (1998) J. Biol. Chem. 273, 2030-2034
37. Shattil, S. J., Hoxie, J. A., Cunningham, M., and Brass, L. F. (1985) J Biol Chem 260,
11107-11114.
38. Shenker, A., Goldsmith, P., Unson, C. G., and Spiegel, A. M. (1991) Journal of
Biological Chemistry 266, 9309-9313
39. Nieswandt, B., Schulte, V., Zywietz, A., Gratacap, M. P., and Offermanns, S. (2002) J
Biol Chem
by guest on July 12, 2020http://w
ww
.jbc.org/D
ownloaded from
23
40. Clapham, D. E., and Neer, E. J. (1997) Annu Rev Pharmacol Toxicol 37, 167-203
41. McNicol, A., Philpott, C. L., Shibou, T. S., and Israels, S. J. (1998) Biochem Pharmacol
55, 1759-1767.
42. Wilkinson, S. E., Parker, P. J., and Nixon, J. S. (1993) Biochem J 294, 335-337
43. Quinton, T. M., Kim, S., Dangelmaier, C., Dorsam, R. T., Jin, J., Daniel, J. L., and
Kunapuli, S. P. (2002) Biochemical Journal (In press)
44. Rosado, J. A., and Sage, S. O. (2001) J Biol Chem 276, 15659-15665.
45. Pasquet, J. M., Noury, M., and Nurden, A. T. (2002) Thromb Haemost 88, 115-122.
46. Woulfe, D., Jiang, H., Mortensen, R., Yang, J., and Brass, L. F. (2002) J Biol Chem 277,
23382-23390
by guest on July 12, 2020http://w
ww
.jbc.org/D
ownloaded from
24
Figure Legends
Figure 1. Characterization of YFLLRNP-mediated human platelet responses (A) Platelet
shape change induced by YFLLRNP was measured in a washed human platelet system using
lumi aggregometer. The sample was incubated with 10 µM Y-27632 for 10 minutes at 37°C
before addition of agonist. The additions are indicated by arrows. Data are representative of
tracings obtained from three different donors. (B) (C). Cyclic AMP formation was measured
after stimulation with 20 µM forskolin, and either 10 µM ADP or 60 µM YFLLRNP. Data is
expressed as percent of total [3H] adenine nucleotides, and is the mean ± S.E. of three separate
experiments performed on different donors.
Figure 2. The effect of combined G12/13 and Gi signaling on human platelet aggregation.
Samples (0.5 mL) of aspirin-treated and washed human platelets were placed in a cuvette in the
presence of 1 mg/mL human fibrinogen. In cases of multiple agonists, either 60 µM YFLLRNP
+ 10 µM epinephrine or 60 µM YFLLRNP + 10 µM ADP were added simultaneously. The
P2Y1 antagonist 1 mM A3P5P was added to samples prior to stimulation with YFLLRNP +
ADP. Tracings are respresentative of three experiments.
Figure 3. The effect of combined G12/13 and Gi signaling on PAC-1 mAb binding Aspirin-
treated and washed human platelets were added to tubes containing 5 µL of PAC-1 mAb and the
agonists noted. Platelets were stimulated for 10 minutes each, diluted with Tyrode’s and
immediately analyzed on a FACSCAN flow cytometer for increases in fluorescence that
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correlate with GPIIb/IIIa activation. Data was calculated as median fluorescence by multiplying
the median point of the cell population with the percentage of the cell population in the marker.
Each bar is the average of three experiments ± S.E from three donors. * denotes p < 0.05. NS =
statistically not significant.
Figure 4. Selecive stimulation of the G12/13 pathway via TP receptor causes aggregation
when combined with Gi signaling. Platelet aggregation was measured as described in the
methods. The arrows indicate the addition of agonists. Addition of multiple agonists was done
simultaneously. The P2Y1 antagonist 1 mM A3P5P was added to samples prior to stimulation
with 10 nM U46619 + 10 µM ADP. Tracings are representative of three experiments from three
different donors.
Figure 5. The role of calcium in platelet aggregation caused by combined G12/13 and Gi
stimulation. A) Intracellular calcium mobilization: The tracings are representative of each
concentration of agonist-mediated calcium mobilization of three experiments. Data are
compared to a single concentration of ADP (3 µM). B) Platelet aggregation caused by selective
activation of Gq and Gi pathways with small increase in intracellular calcium. Platelets
stimulated with agonists as noted; and C) effect of dimethyl BAPTA: Aspirin-treated, washed
human platelets were pre-incubated with 1 µM dimethyl BAPTA for 3 min at 37°C. After pre-
incubation, samples were stimulated with G12/13 and Gi signaling via 60 µM YFLLRNP + 10
µM epinephrine. Tracings are representative of three experiments from three different donors.
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Figure 6. The effect of protein kinase inhibitors on platelet aggregation caused by
combined G protein stimulation. Aspirin-treated, washed human platelets were pre-incubated
with the inhibitors as follows: 3 minute pre-incubation with 10 µM Ro31-8220 or 25 µM LY
294002, at 37°C, 10 minute pre-incubation with 10 µM U0126, or 10 µM Y-27632 at 37°C.
After pre-incubation, samples were stimulated with A) G12/13 and Gi signaling via 60 µM
YFLLRNP + 10 µM epinephrine or B) Gq and Gi signaling via ADP (10 µM). Tracings are
representative of three experiments from three different donors. Addition of agonist(s) is
indicated by an arrow.
Figure 7. Model depicting GPIIb/IIIa activation caused by co-stimulation of the G12/13
and Gi pathways. The G12/13-coupled receptor (on left) represents either the TP receptor,
which is stimulated by thromboxane A2, or PAR-1 receptor, which is stimulated by thrombin
and YFLLRNP. The Gi, Gz-coupled receptor (center) represents either the a2A adrenergic
receptor, which is stimulated by epinephrine, or the P2Y12 receptor, which is stimulated by
ADP. The Gq coupled receptor (on the right) represents TP receptor, PAR-1 or P2Y1 receptor
which is stimulated by ADP. The double bars represent the inhibitory activity of Y-27632 on
p160ROCK activity.
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Fig. 1. Dorsam et al
60 µM YFLLRNP 60 µM YFLLRNP +10 µM Y-27632
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Fig. 1. Dorsam et al.
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Figure 2. Dorsam et al.
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0
50
100
150
200
Mea
n Fl
uore
scen
ce
Unst
imul
ated
60 m
M Y
FLLR
NP
10 m
M E
pine
phrin
e
10m
M A
DP
10 m
M A
DP +
1
mM
A3P
5P
60 m
M Y
FLLR
NP +
10 m
M E
pine
phrin
e
60 m
M Y
FLLR
NP +
1 m
M A
3P5P
+10
mM
ADP
*
*
*
NS
NS
NS
Fig. 3. Dorsam et al.
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Fig. 4. Dorsam et al.
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0.3 µM ADP
0.3 µM ADP
0.3 µM ADP +10 µM Epinephrine
1 µM AR-C66096
1 µM AR-C 69931
B
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60 µ
M Y
FLL
RN
P +
10 µ
M E
pine
phrin
e
60 µ
M Y
FLL
RN
P +
10 µ
M E
pine
phrin
e
1 µM dimethyl BAPTA
CControl
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Fig. 6. Dorsam et al.
60 µM YFLLRNP +10 µM Epinephrine (G12/13 + Gi)
10 mM Ro 31-822010 mM Y-2763225 mM LY 294002Control 10 mM U0126
10 mM Ro 31-822010 mM Y-2763225 mM LY 294002Control 10 mM U0126
10 µM ADP (Gq + Gi)
A
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Fig. 7. Dorsam et al.
GPIIb/IIIa Activation
EpinephrineADP
Gi, Gz
U46619 (low dose)YFLLRNP (low dose)Thrombin
G12/13
Rho A
p160 ROCK
Shape Change
Y-27632
PLC?
PI-3 Kinase
?
U46619 (high dose)ADPThrombinYFLLRNP (high dose)
Gq
?
PI-3 Kinaseg
Inhibition ofadenylyl cyclase
Ca++
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Robert T. Dorsam, Soochong Kim, Jianguo Jin and Satya P. KunapuliGPIIb/IIIa in human platelets
Coordinated signaling through both G12/13 and Gi pathways is sufficient to activate
published online September 23, 2002J. Biol. Chem.
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