Signaling mechanisms mediated by G-protein coupled receptors in human platelets¹

Sheikh Arshad SAEED², Huma RASHEED, Faisal A Wahed FECTO, Mohammad Ilyas ACHAKZAI, Rahmat ALI, John Dennis CONNOR, Anwar-ul-Hassan GILANI

Department of Biological and Biomedical Sciences, The Aga Khan University, Karachi-74800, Pakistan

¹ Project supported by the Research fund of the Aga Khan University, Karachi.

² Correspondence to Prof Saeed SA. Dr. Panjwani Institute of Molecular Medicine and Drug Research, University of Karachi, Karachi, Pakistan.

Received 2003-10-31 Accepted 2004-04-08

KEY WORDS platelet aggregation; epinephrine; arachidonic acid; cyclooxygenase; phospholipase C; calcium channel blockers; nitric oxide; MAP kinase

ABSTRACT

AIM: The present study deals with the investigation of mechanisms involved in the synergistic interaction between epinephrine and arachidonic acid (AA). METHODS: Venous blood was taken from healthy human volunteers reported to be free of medications for one week. Platelet aggregation was monitored at 37 ºC using Dual-channel Lumi-aggregometer. The resulting aggregation was recorded for 5 min by the measurement of light transmission as a function of time. RESULTS: The data show that a synergism in platelet aggregation mediated by subthreshold concentrations of epinephrine (1 µmol/L) and AA (0.2 µmol/L) was inhibited by the a₂-receptor antagonist (yohimbine, IC₅₀=0.6 µmol/L) and an inhibitor of AA-cyclooxygenase (COX), indomethacin (IC₅₀=0.25 µmol/L). In examining receptor influence on intraplatelet signalling pathways, it was found that the synergistic effect was inhibited by calcium channel blockers, verapamil (IC₅₀=0.4 mmol/L) and diltiazem (IC₅₀=2.5 µmol/L), as well as by low concentrations of inhibitors of phospholipase C (PLC) (U73122; IC₅₀=0.2 µmol/L) and mitogens activated protein kinase (MAPK) (PD 98059; IC₅₀=3.8 µmol/L). Herbimycin A, a specific inhibitor of tyrosine light chain kinase (TLCK), showed inhibition at IC₅₀ value of 15 µmol/L, whereas chelerythrine, a protein kinase C (PKC) inhibitor, had no effect up to 20 µmol/L. CONCLUSION: These data suggest that synergism between epinephrine and AA in platelet aggregation is triggered through receptors coupled to G-protein, which in turn, activate PLC, COX, and MAP kinase-signaling pathways.

INTRODUCTION

Platelet agonists, such as epinephrine, thrombo-xane, adenosine diphosphate (ADP), platelet activating factor (PAF), and thrombin interact with transmem brane receptors coupled to GTP binding proteins (G-proteins) on platelets. G-proteins mediate a variety of intracellular processes by affecting other effector molecules, including adenylyl cyclase, inositol phospholipid-specific phospholipase C (PLC) or ion channels^[1]. In human platelets, activation of Gi/adenylyl cyclase (eg, by epinephrine) decreases cAMP levels and increases platelet aggregation^[1]. However, stimulation of platelet receptors coupled with phosphoinositide C-linked G-proteins, Gq (eg, by 5-HT, PAF or thrombin) and Gi (eg, epinephrine and histamine) activates PLC and generates second messengers, diacylglycerol (DAG) and inositol-1,4,5-triphosphate (IP₃). These second messengers activate protein kinase C (PKC) and mobilize intracellular Ca²⁺, respectively^[2]. Both Ca²⁺ and PKC act in synergy to enhance platelet aggregation^[3,4], whereas deficiency of Gq protein impairs it^[5].

A number of agonists have been reported to act synergistically in platelet aggregation^[6]. However, few study has done in human platelets on the cooperative effects of epinephrine and arachidonic acid (AA)^[7]. In platelet membrane AA is the precursor for the synthesis of thromboxane A₂. Interactions of different stimuli could be physiologically important in vivo and in isolated platelet defects. It is now well documented that most platelet agonists act by stimulating G-protein coupled receptors (GPCRs). TXA₂, produced by the action of cyclo-oxygenase (COX) on AA, is a potent vasoconstrictor and mediator of platelet aggregation^[8]. It induces aggregation by binding to specific receptors on the platelet membrane^[9]. TXA₂ receptor stimulation activates phospholipase C and increases [Ca²⁺] via G-protein of the Gq/11 family; this leads to aggregation by Ca²⁺ influx^[10]. It has been postulated that the main proag-gregatory effects of TXA₂ are mediated by inhibition of an adenylate cyclase/cAMP complex^[11].

Epinephrine interacts with a₂-adrenergic receptors on platelets; activation of these receptors inhibits the adenylyl cyclase pathway^[12]. Epinephrine-mediated increase in platelet aggregation correlates with an increase in cytosolic free Ca²⁺, a process sensitive to Na⁺ concentration^[12]. Inward cytosolic Ca²⁺-influx is an important determinant in the signaling cascade mediating platelet aggregation^[13].

This study was conducted to examine the interaction between epinephrine and AA to elucidate the possible intracellular signaling mechanism(s) involved during synergism. We further evaluated the involvement of multiple signaling pathways including PLC/Ca²⁺, cyclooxygenase and mitogen activated protein kinase (MAPK) pathways in epinephrine and AA mediated platelet aggregation.

MATERIALS AND METHODS

Chemicals and drugs Arachidonic acid, epine-phrine, yohimbine, indomethacin, diltiazem, verapamil, genistein and chelerythrine were obtained from the Sigma Chemical Co (St Louis, Mo, USA). U73122 was obtained from Alexis LC Labs (UK). Herbimycin A was purchased from RBI (Natick, MA. USA). All other chemicals used were of the highest purity grade available.

Preparation of human platelets Venous blood was taken from healthy human subjects of either sex, aged (25±4) year and a body weight of 60±7 kg, reported to be free of medication for one week. Blood samples were mixed with 3.8 % (w/v) sodium citrate solution (9:1) and centrifuged at 260×g for 15 min at 20 ºC to obtain platelet rich plasma (PRP) and at 1200×g for 10 min to obtain platelet poor plasma (PPP). Aggregation studies were carried out at 37 ºC with PRP having platelet counts between 2.5 and 3.0×10⁹/L of plasma^[14].

Measurement of platelet aggregation Aggregation was monitored in 0.45 mL aliquots of PRP using a Dualchannel Lumiaggregometer (Model 400 Chronolog Corporation, Chicago, USA)^[14]. The final volume was made up to 0.5 mL with test drug dissolved either in normal saline or appropriate vehicle known to be devoid of any effect on aggregation. A sub-threshold concentration for aggregation by epinephrine was determined. To examine the synergistic effect of epinephrine and AA, we added low concentrations of these agonists (1 mmol/L and 0.2 mmol/L respec-tively). Anti-aggregatory effects of various inhibitors were studied by pretreating PRP with an inhibitor for 1 min, followed by addition of sub-threshold concentrations of epinephrine and AA. The resulting aggregation was recorded for 5 min after challenge by change in light transmission as a function of time. Once the anti-platelet activity of various inhibitors against agonists was established, dose-response curves were constructed to calculate 50 % inhibitory concentration of the inhibitors.

Thromboxane formation in platelets TXA₂ formation was studied with the co-addition of epinephrine (1 µmol/L) and AA (0.2 µmol/L) using radiochemical methods described previously^[15]. For these studies, human blood platelets having counts between 2.5 and 3.0×10⁹/L of plasma were routinely obtained in plastic bags from The Aga Khan University Hospital Clinical Laboratory, Karachi. The PRP was centrifuged at 260×g for 20 min and the sedimented platelets were washed twice with an ice-cold phosphate buffer (50 mmol/L, pH 7.4) containing sodium chloride (0.15 mol/L) and edetci acid (0.2 mmol/L). After centrifugation, washed platelets were resuspended in the same buffer without edetic acid and homogenized at 4 ºC using a polytron homogenizer for 15 s. The homogenate was centrifuged at 1200×g for 20 min and 300 mL of the supernatant (containing 0.4 mg of protein) was incubated with 10 mg unlabelled arachidonic acid (AA) and 3.7 MBq [1-¹⁴C]-arachidonic acid in the presence and absence of the agonists. After 15 min of gentle shaking in air at 37 ºC, the reaction was stopped by adding 0.4 mL of citric acid (0.4 mol/L) and ethyl acetate (7.0 mL). After mixing and centrifuging at 600×g for 5 min at 4 ºC, the organic layer was separated and evaporated to dryness under nitrogen. Residues were dissolved in 40 mL ethanol and 20 mL were applied to silica gel G thin layer chromatography (TLC) plates (Analtech, Delaware, USA). The solvent system used for the separation of TXB₂ in dried organic extracts of platelet incubates as above was ethyl acetate: isooctane: water and acetic acid (11:5:10:2, v/v, upper phase). Radioactive zones were located and quantified by Berthold TLC. linear analyzer and chromatography data system (Model LKB 511, Berthold W Germany). Protein concentration was determined using human serum allumin as standard^[16].

Data analysis IC₅₀=Concentration (µmol/L) producing 50 % inhibition of platelet aggregation (control response taken as 100 %). The 50 % inhibitory concentrations (IC₅₀) values were calculated as means±SD of 5-6 independent experiments.

Differences between control and test measurements were assessed by t-test.

RESULTS

Treatment of PRP with subthreshold concentrations of epinephrine (1 µmol/L) and AA (0.2 mmol/L) was synergistic with regard to aggregation (Fig 1). Synergism between epinephrine and AA was inhibited by pretreating PRP with a₂-adrenergic receptor blocker, yohimbine (IC₅₀=0.6 µmol/L) (Fig 2A), or AA-cyclooxygenase inhibitor, indomethacin (IC₅₀=0.25 µmol/L) (Fig 2B). We also determined the effect of these two agonists on COX activity by measuring TXA₂ formation in platelets exposed to epinephrine plus AA. Epinephrine (1 µmol/L) markedly potentiated the TXA₂ formation by low concentration of AA (0.2 mmol/L) (Fig 3).

Fig 1. Tracings from a representative experiment showing synergism with epinephrine (1 µmol/L) and arachidonic acid (AA= 0.2 mmol/L) on platelet aggregation, n=7.

Fig 2. (A) Synergism between epinephrine and arachidonic acid is blocked by a₂-receptor antagonist, yohimbine; (B) Synergism blocked by AA-cyclooxygenase inhibitor, indomethacin. Control response is platelet aggregation induced by epinephrine (1 µmol/L) plus arachidonic acid (0.2 mmol/L) without the antagonist. n=5.

Fig 3. Effects of subthreshold concentrations of epinephrine and arachidonic acid on thromboxane A₂ (TXA₂) formation by human platelets. n=7.

We used a PLC inhibitor (U73122) to find out if the effect of epinephrine and AA involves activation of PLC. Results show that pretreatment of PRP with U73122 completely inhibited the synergistic effect of epinephrine and AA with an IC₅₀ of 0.2 µmol/L (Fig 4). Therefore, we examined the effect of Ca²⁺ channel blockers (verapamil and diltiazem) on platelet aggregation. The synergistic effect of epinephrine and AA was inhibited by both diltiazem (Fig 5A) and verapamil (Fig 5B) and (IC₅₀=2.5 and 0.4 µmol/L respectively).

Fig 4. The effect of phospholipase C inhibitor, U73122 on the synergistic interaction of epinephrine (1 µmol/L) and AA (0.2 mmol/L). n=6.

Fig 5. The effect of calcium channel blockers, (A) diltiazem and (B) verapamil on the synergistic effect of epinephrine (1 µmol/L) and AA (0.2 mmol/L). n=7.

We used the selective MAP kinase inhibitor PD98059 in the epinephrine plus AA synergism model. Results show that pretreatment of PRP with PD98059 inhibited (IC₅₀=3.8 µmol/L) platelet aggregation usually produced by co-addition of subthreshold concentrations of epinephrine and AA (Tab 1 & Fig 6A).

Tab 1. Effect of various inhibitors on subthreshold concentration of epinephrine (1 µmol/L) and arachidonic acid (0.2 mmol/L)-induced platelet aggregation. n=5-7. Mean±SD.

Inhibitors	IC50/µmol·L-1	95 % Confidence limit
Yohimbine	0.60±0.09	0.49-0.71
Indomethacin	0.25±0.07	0.16-0.34
U73122	0.20±0.06	0.14-0.26
Diltiazem	2.5±0.7	1.85-3.15
Verapamil	0.40±0.09	0.32-0.48
SNAP	1.00±0.08	0.91-1.09
PD98059	3.8±0.5	3.18-4.42
Herbimycin A	15.0±2.5	11.90-18.10
Chelerythrine	NE	-

Data were indicated as half-maximal inhibitory effect (IC₅₀) of various agents. IC₅₀= concentration (µmol/L) producing 50 % inhibition of aggregation. IC₅₀ was calculated using tests done by 5-7 determinations of the inhibitors. NE=no effect.

Fig 6. (A) The effect of MAP kinase inhibitor, PD98059 and (B) tyrosine kinase inhibitor, herbimycin A on the synergistic interaction of epinephrine (1 µmol/L) and AA (0.2 mmol/L), n=5.

The NO donor, SNAP, completely blocked platelet aggregation mediated by the synergistic interaction of epinephrine and AA (Tab 1). These data suggest the idea that NO negatively modulates human platelet aggregation. Herbimycin A, a specific inhibitor of tyrosine kinase, also inhibited epinephrine and AA induced aggregation with IC₅₀ of 15 µmol/L, suggesting an involvement of tyrosine kinase in this cascade (Fig 6B).

However, an inhibitor of PKC (chelerythrine; 20 µmol/L) had no effect.

DISCUSSION

Our study shows that epinephrine and AA, when added to PRP in subthreshold concentration, acted synergistically to produce platelet aggregation. This effect was dependent on transmembrane a₂-receptors. Synergism between epinephrine and AA was inhibited by calcium channel blockers, an a₂-receptor antagonist and inhibitors of PLC, MAP kinase, and COX pathways. Platelet a₂-receptors are linked to Gi proteins, which, in turn, activate PLC. Recent studies show that bg-subunits of activated Gi protein can activate PLC^[17]. This sequence may explain why U73122, a selective inhibitor of PLC, inhibited platelet aggregation induced by co-activation of these agonists. Further, PLC activation leads to generation of IP₃, release of Ca²⁺ from internal stores and eventually store-depleted Ca²⁺ influx ^[13]. Ca²⁺ entry from the outside is inhibited by Ca²⁺ channel blockers, verapamil, and diltiazem. Moreover, an increase in cytosolic Ca²⁺ activates PLA₂ and COX-1 activity, thus stimulating TXA₂ synthesis^[13]. Since the synergism was inhibited by indomethacin, a COX-1 inhibitor, it seems that agonist-mediated synergism follows activation of COX-1 distal to PLC/Ca²⁺ activation. Recent studies suggest an important role for nitric oxide (NO) in modulating platelet aggregation^[18]. We tested the idea that increasing intracellular nitric oxide levels by using a NO donor, thus activating cGMP kinase, would inhibit platelet aggregation. Our results show that NO donor, SNAP, inhibited platelet aggregation (IC₅₀=1 mmol/L), suggesting that epinephrine/ AA synergism is sensitive to NO generation in platelets. However, a role for PKC seems unlikely, as PKC inhibition by chelerythrine chloride had no effect on the synergism.

Cyclic nucleotides, cAMP and cGMP, through activation of cGMP-dependent protein kinases, down-regulate Ca²⁺ responses and thus inhibit platelet aggregation^[13]. In fact, platelets contain cAMP and cGMP dependent protein kinases that can inhibit PLC induced IP₃ production through inactivation of IP₃ and TXA₂ receptors^[19]. Since stimulation of the G-protein/Ca²⁺ cascade leads to mitogen activated protein (MAP) kinase signaling^[13], inhibition of epinephrine/AA synergism by MAP kinase inhibitor, PD98059 suggests the involvement of MAP kinase downstream from Gq/PLC^[19]. Cytosolic PLA₂ is also a potential target for activation by an increase in cytosolic Ca²⁺. Taken together, it appears that both Ca²⁺ signaling and MAP kinase play an important role in the synergy.

The selective MAPK inhibitor, PD98059 inhibits COX-1 and COX-2 activities^[20]. Under our experimental conditions, PD98059 inhibited platelet aggregation with IC₅₀ of 3.8 µmol/L. Therefore it is possible that inhibition of agonist-induced platelet aggregation by PD98059 may be due to blockade of COX activity.

Activation of platelets by some agonists increases the level of tyrosine phosphorylation resulting in the appearance of a new set of tyrosine-phosphorylated proteins^[21]. To investigate the involvement of tyrosine kinase, we used herbimycin A, a known inhibitor of tyrosine kinase^[22]. Herbimycin A blocked epinephrine/AA-induced aggregation in a concentration-dependent manner (IC₅₀=15 µmol/L), showing that synergism may also involve tyrosine light chain kinase activation.

A common mechanism in synergism between various platelet agonists is thought to be due to activation of Ca²⁺ signaling cascade. A rise in intracellular Ca²⁺ concentration induced by the first agonist primes platelets for an enhanced functional response to the second agonist^[23]. Ca²⁺ plays a pivotal role in platelet aggregation. Synergism among various platelet agonists in the blood is of great clinical significance, as it could markedly potentiate platelet activation, producing cardiovascular pathology.

In conclusion, our studies show that subthreshold concentrations of epinephrine potentiate platelet aggregation mediated by AA. The synergism seems to derive from the PLC/Ca²⁺, COX, and MAP kinase pathways and is negatively modulated by a nitric oxide donor. The results serve to reinforce the concept that aggregation synergisms are multifactorial interactions between products of G-proteins stimulation.

ACKNOWLEDGEMENTS We thank Mr Ali MOOSA for expert editorial assistance.

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