coordinated signaling through both g12/13 i pathways is ... › content › early › 2002 › 09...

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

http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

2

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

by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

3

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

4

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

5

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-


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

6

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

7

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

8

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,


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

9

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,


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

10

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

11

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

12

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

13

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

14

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

15

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

16

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

17

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).


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

18

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

19

Acknowledgements: We thank Drs. James L. Daniel, Barrie Ashby, and Todd M. Quinton,

Temple University Medical School, for critically reading the manuscript.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

20

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.,


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

25

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

26

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.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

27

Fig. 1. Dorsam et al

60 µM YFLLRNP 60 µM YFLLRNP +10 µM Y-27632


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

28

Fig. 1. Dorsam et al.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

29

Figure 2. Dorsam et al.


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

30

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



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

31



ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

A


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

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

by guest on July 12, 2020 http://www.jbc.org/ Downloaded from

http://www.jbc.org/

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


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

34


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

B by guest on July 12, 2020http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

35


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++


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

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.

10.1074/jbc.M208778200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M208778200

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M208778200v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2002/09/23/jbc.M208778200.citation

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2002/09/23/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2002/09/23/jbc.M208778200.citation

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/

coordinated signaling through both g12/13 i pathways is ... › content › early › 2002 › 09...

Documents