Co-activation of Gi and Gq proteins exerts synergistic effect on human platelet aggregation through activation of phospholipase C and Ca2+ signalling pathways

Our previous studies have shown that subthreshold concentrations of two platelet agonists exert synergistic effects on platelet aggregation. Here we studied the mechanism of synergistic interaction of 5-h y d r o x y -tryptamine (5-HT) and epinephrine mediated platelet aggregation. We show that 5-HT had no or little effect on aggregation but it did potentiate the aggregation response of epinephrine. The synergistic interaction of 5-HT (1-5 M) and epinephrine (0.5-2 M) was inhibited by 2 - adrenoceptor blocker (yohimbine; IC 5 0 = 0.4 M ) , calcium channel blockers (verapamil and diltiazem with IC 5 0 of 10 and 48 mM, respectively), P L C inhibitor (U73122; IC 5 0 =6 M) and nitric oxide (NO) d o n o r, SNAP (IC 5 0 =1.6 M)). The data suggest t h a t synergistic effects of platelet agonists are recep-tor-mediated and occur through multiple signalling pathways including the activation PLC/Ca 2 Co-activation of Gi and Gq proteins exerts synergistic effect on human platelet aggregation through activation of phospholipase C and Ca 2+ signalling pathways


Introduction
Platelets react rapidly with a wide range of agonists to maintain vascular integrity and haemostasis. Upon vascular damage, platelets undergo rapid transformation; become more spherical, extrude pseudopodia and activate their fibrinogen receptors leading to aggregation. During this activation process, platelets release granule contents and substances which act in autocrine fashion to further enhance aggregation and haemostasis (Siess, 1989;Brass et al., 1993). The platelet agonists act in synergy to potentiate each other's effect (Ware et al. , 1987).
Most of the platelet agonists, like thrombin, ADP, PA F, epinephrine and 5-HT, interact with their transmembrane receptors on platelets coupled with GTP binding proteins (G proteins). The G-proteins mediate a variety of cellular processes by activating diff e r e n t e ffector molecules, in-cluding adenylyl cyclase, inositol phospholipid-specific phospholipase C (PLC) or ion channels (Siess et al., 1989;Exton, 1996). In human platelets, activation of Gs/adenylyl cyclase decreases platelet aggregation. In contrast, acti-vation of Gi/adenylyl cyclase (e.g. by epinephrine) leads decreases intracellular cAMP levels and increases platelet aggregation (Siess et al., 1989). In platelets, stimulation of receptors coupled with phosphoinositidase C-linked G-proteins (Gq) (e.g., by 5-HT, PAF or thrombin) leads to activation of PLC and thus generation of second messengers, diacylglycerol (DAG) and inositol-1,4,5triphosphate (IP 3 ). This results in the activation of protein kinase C (PKC) and the mobilization of intracellular Ca 2 + , respectively [Obberghen-Schilling and Pouyssegur, 1993). Both Ca 2+ and PKC act in synergy to enhance (Crabos et al ., 1992) whereas deficiency of Gq protein leads to impairment of platelet aggregation (Offermans et al. , 1997).
We and others have shown that subthreshold concentrations of platelet agonists potentiate aggregation response and thus exhibit synergistic effects (Ware et al., 1987;Saeed et al., 1997). This synergism is considered to occur mainly through priming of Ca 2+ responses, but the role of other effectors and second messengers is not well known. Here we show that synergistic inetraction of 5-HT and epinephrine can be inhibited by Ca 2 + -c h a n n e l blockers and PLC inhibitor suggesting the involvement of calcium and Gq/PLC pathways in this cascade. from Professor Graeme Milligan (Glasgow, UK). All other chemicals were of the highest purity grade available.

Preparation of human platelets
Blood was taken by veinpuncture from normal human volunteers 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˚C to obtain platelet rich plasma (PRP) with platelet counts between 2.5 and 3 ✕ 1 0 8 /ml of plasma. All experiments were performed within 2 h of PRP preparation.

Measurement of platelet aggregation
Aggregation was monitored using a Dual-channel Lumiaggregometer (Model 400 Chronolog Corporation, Chicago, U.S.A.) using 0.45 ml aliquots of PRP (Shah and Saeed, 1995). 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. Aggregation was induced with epinephrine and sub-threshold concentration determined. To obtain the syner-gistic effect of 5-HT and epinephrine, we added low concentrations of these agonists together. The anti-aggregatory effects were studied by pretreatment of PRP with various inhibitors for one minute followed by addition of the subthreshold concentrations of epinephrine and 5-HT. The resulting aggregation was recorded for 5 min after challenge by the change in light transmission as a function of time. Once the anti-platelet activity of various inhibitors against agonists was established, dose-res-ponse curves were constructed to calculate the IC 50 values of the test substances. Statistical analysis was done using Student's t-test.

Western blots
The generation and specifities of the various antisera used in this study are already described (Mitchell et al., 1991). Membrane samples were resolved by SDS/PA G E in 10% (w/v) acrylamide gels overnight at 60 V. For resolving Gqa and G11a, urea linear gradient gels were prepared and run overnight at 100 V as described in detail previously (Shah and Milligan, 1994). Proteins were transferred to nitrocellulose (Schleicher and Schuell) and blocked for 3 h in 5% (w/v) gelatin in phosphate buffered saline (PBS), pH 7.5. Primary antisera were added in 1% gelatin in PBS containing 0.2% Nonidet P-40 (NP-40) and incubated overnight. The primary antiserum was removed and blots washed extensively with PBS containing 0.2% NP-40. Secondary antiserum (donkey anti-rabbit IgG coupled to horseradish peroxidase; HRP) in 1% g e l a t i n / P B S / 0 . 2 % NP-40 was added and left for 3h. After removal of the second antiserum, blots were washed extensively as above and developed with o-dianisidine hydrochloride as substrate for HRP.

Results and Discussion
Platelet membranes were subjected to immunoblot analysis using antisera specific against the α-subunit of Gs, Gi and Gq/11 proteins. Western blots results in Figure 1A show that human platelets express predominantly 45 kD adenylyl cyclase-stimulatory G protein (GSα) as well as inhibitory (Gi2) G proteins. Using urea gradient g e l s under the conditions where Gq α and G11α proteins can be separated and detected by CQ2 antisera (Shah and Milligan, 1994), we find that platelets contain only G qα protein (42 kD) but not G11a ( Figure 1B). Normally Gqα and G11α are co-expressed in most of the body tissues (Mitchell et al., 1991;Shah and Milligan, 1994).
Treatment of PRP with 5-HT up to 10 μM had no appreciable effect on platelet aggregation, however,    and epinephrine (Epi). The Gqα and Giβ γ subunits converge at phospholipase C (PLC), which leads to generation of inositol triphosphate (IP3) and diacylglycerol (DAG) and thus release of Ca 2 + from internal stores and activation of protein kinase C (PKC). Both Ca 2 + and PKC are considered to be involved in the release of granule contents and activation of phospholipase A2, thus generation of potent agonist, thromboxane A2 ( T X A2) which in an autocrine fashion acts on platelets through Gq protein. Also Epimediated decrease in cAMP levels stimulate aggregation. NO from exogenously added SNAP (NO donor) inhibits platelet aggregation through production of cGMP from guanylate cyclase. The cGMP activates PKG which can phosphorylate PLC, Ca 2 +channels or IP3-receptor to inhibit Ca 2 + -mediated pro-aggregatory effects in platelets. epinephrine (0.2-20 μM) had concentration-dependent increase in platelet aggregation. The simultaneous addition of subthreshold concentrations of 5-HT (1-2 μM) and epinephrine (0.5-1 μM) exhibited synergistic e ffect (Figure 2A). 5-HT is considered a weak platelet agonist as it causes only shape change in platelets (Steen et al., 1993), however, subthreshold concentrations of 5-HT and epinephrine, when used together, can elicit a strong synergistic effect on platelet aggregation. Such a mechanism of synergism is known among other agonists too and is considered to occur due to activation of Ca 2+ signalling cascade. It has been reported that a rise in Ca 2 + induced by first agonist primes platelets for an enhanced functional response to the second agonist (Ware et al., 1987). Results show that the synergism between 5-HT and epinephrine is inhibited by pretreatment of PRP with yohimbine indicating that the effect is mediated through α 2 -adrenoceptors ( Figure 2B). The half-maximal inhibitory concentration (IC 5 0 ) of yohimbine against 5-HT plus epinpehrine was 0.4 μM.
Receptors for 5-HT in platelets are coupled with Gq/ PLC (De Chaffoy de Courcelles et al., 1987;Martin, 1994) whereas epinephrine interacts with α 2 -a d r e n o c e p t o r s coupled with Gi/adenylyl cyclase pathway (Steen et al ., 1993). Recent studies show that β γ-subunits of activated Gi protein can also activate PLC (Clapham and Neer, 1997;Banno et al., 1998). We used PLC inhibitor (U73122) to examine if 5-HT and epinephrine mediated eff e c t s involved PLC. Results show that pretreatment of PRP with U73122 inhibits the platelet aggregation mediated by synergistic effect of 5-HT and epinephrine ( Figure  2D). The IC 5 0 of U73122 was 6 μM (Figure 3). This raises the possibility of convergence of Gi and Gq pathways at PLC during platelet aggregation. A similar phenomenon has been observed in HEK 293 cells where it is shown that G β γ subunit-mediated α 2adrenoceptor and Gq/11 mediated α 1 B -a d r e n o c e p t o rcoupled MAP kinase path-ways converge at the level of PLC (Della Rocca et al., 1997). In fact the βγ-subunits of Gi2 and Gq proteins have similar efficacy in regulation of effector (PLC) in human platelets (Banno et al. , 1998).
Since activation of Gq/PLC pathway leads to an increase in cytosolic Ca 2 + due to its release from internal stores by IP 3 or through store-depleted calcium influx (Berridge, 1993;Heemskerk and Sage, 1994), we used Ca 2+ channel blockers (verapamil and diltiazem) to see if the Ca 2+ signalling is involved in aggregation following co-activation by 5-HT and epinephrine. We found that the synergistic effect of 5-HT and epinephrine is inhibited by both verapamil (IC 5 0 = 10 μM, Figure 2C) and diltiazem (IC 50 = 48 μM, Table 1). Verapamil has been shown to inhibit platelet aggregation, Ca 2 + fluxes and thromboxane production induced by ADP, collagen and thrombin (Brocchieri et al., 1995;Saeed et al., 1997). It is well known that Ca 2 + plays pivotal role in platelet aggregation (Heemskerk and Sage, 1994;Shah et al., 1996Shah et al., , 1998. A rise in cytosolic Ca 2 + levels accompanies platelet activation through stimulation of the enzymes which are not fully functional at low Ca 2+ concentration present in the resting platelets (Heemskerk and Sage, 1994). Interruption in the process of Ca 2+ activation either through C a 2 + -channels (Brocchieri et al., 1995;Shah et al., 1997) or Gi/Gq proteins (Offermans et al., 1997) can interfere in the activation of platelets.
Previously it was shown that cyclic nucleotides, cAMP and cGMP through activation of cAMP-and cGMPd e p e n -dent protein kinase, down-regulate the Ca 2 + responses and thus inhibit platelet aggregation. We tested if increasing intracellular nitric oxide (NO) levels by NO donor (SNAP) and thus activating cGMP kinase has any inhibitory effect on platelet aggregation induced by epinephrine and 5-HT. Results show that SNAP inhibits platelet aggregation at very low concentrations (IC 5 0 ; 1.6 μM) suggesting that epinephrine and 5-HT synergism is sensitive to NO generation ( Figure 3). In fact platelets contain an abun-dance of cAMP and cGMP-dependent protein kinases (El-Daher, et al. , 1996) which can inhibit PLC-induced inositol phosphate production and inactivate IP 3 and thromboxane receptors (see review by Heemskerk and Sage, 1994;Wa n g et. al ., 1998), thus inhibiting the platelet aggregation.
Multiple studies have shown that agents (like epinephrine) which decrease intracellular cAMP levels stimulate platelet aggregation (Brass et al., 1993;Siess et al., 1993). In fact epinephrine effects in platelets may involve multiple signalling pathways; stimulation of IP 3 production through stimulation of PLC by G protein β γ-subunits (Clapham and Neer, 1997;Banno et al., 1998), increase in Ca 2+influx, thus activation of phospholipase A2 (Heemskerk and Sage, 1994) and activation of some other proteins like Syk (Wang et al ., 1997). In conclusion, our results point to a new facet of 5-HT and epinephrine synergistic interaction and further demonstrate that such an effect is receptor mediated and occurs due to activation of Gq and Gi proteins which converge at PLC and Ca 2 + s i gnalling pathways as depicted in Figure 4. Table 1. Effect of various inhibitors on platelet aggregation mediated by synergistic interaction of 5-HT (1-5 μM) and epinephrine (0.5-2 μM). Data is mean ± SEM (n=6-7) and is indicated as half-maximal effect (IC50) of inhibitors.