α2A-Adrenergic Receptor Stimulation Potentiates Calcium Release in Platelets by Modulating cAMP Levels*

α2A-Adrenergic receptor-mediated Ca2+ signaling and integrin αIIbβ3 exposure were investigated in human platelets under conditions where indirect, thromboxane- or ADP-mediated effects were absent. The α2-adrenergic receptor agonists, UK14304 and epinephrine (EPI), were unable to raise cytosolic levels of inositol 1,4,5-trisphosphate (InsP3) or Ca2+but potentiated the [Ca2+] i rises evoked by other agonists that act through stimulation of phospholipase C (thrombin or platelet-activating factor) or stimulation of Ca2+-induced Ca2+ release (CICR) in the absence of InsP3generation (thimerosal or thapsigargin). In addition, α2-adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with Gsα-stimulating prostaglandin E1, EPI increased the Ca2+ signal evoked by either phospholipase C- or CICR-stimulating agonists mainly through modulation of the cAMP level. The stimulating effects of UK14304 and EPI on platelet Ca2+ responses, and also on integrin αIIbβ3 exposure and platelet aggregation, were abolished by pharmacological stimulation of cAMP-dependent protein kinase, and these effects were mimicked by inhibition of this activity. In permeabilized platelets, UK14304 and EPI potentiated InsP3-induced, CICR-mediated mobilization of Ca2+ from internal stores in a similar way as did inhibition of cAMP-dependent protein kinase. In summary, a Giα-mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of α2A-adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the Giα-coupled α2A-adrenergic receptors does not result in phospholipase C activation but rather in modulation of the Ca2+ response by relieving cAMP-mediated suppression of InsP3-dependent CICR.

␣ 2A -Adrenergic receptor-mediated Ca 2؉ signaling and integrin ␣ IIb ␤ 3 exposure were investigated in human platelets under conditions where indirect, thromboxane-or ADP-mediated effects were absent. The ␣ 2 -adrenergic receptor agonists, UK14304 and epinephrine (EPI), were unable to raise cytosolic levels of inositol 1,4,5-trisphosphate (InsP 3 ) or Ca 2؉ but potentiated the [Ca 2؉ ] i rises evoked by other agonists that act through stimulation of phospholipase C (thrombin or plateletactivating factor) or stimulation of Ca 2؉ -induced Ca 2؉ release (CICR) in the absence of InsP 3 generation (thimerosal or thapsigargin). In addition, ␣ 2 -adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with G s␣ -stimulating prostaglandin E 1 , EPI increased the Ca 2؉ signal evoked by either phospholipase C-or CICR-stimulating agonists mainly through modulation of the cAMP level. The stimulating effects of UK14304 and EPI on platelet Ca 2؉ responses, and also on integrin ␣ IIb ␤ 3 exposure and platelet aggregation, were abolished by pharmacological stimulation of cAMP-dependent protein kinase, and these effects were mimicked by inhibition of this activity. In permeabilized platelets, UK14304 and EPI potentiated InsP 3 -induced, CICR-mediated mobilization of Ca 2؉ from internal stores in a similar way as did inhibition of cAMP-dependent protein kinase. In summary, a G i␣ -mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of ␣ 2A -adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the G i␣ -coupled ␣ 2A -adrenergic receptors does not result in phospholipase C activation but rather in modulation of the Ca 2؉ response by relieving cAMP-mediated suppression of InsP 3 -dependent CICR.
In most cell types, the ␣ 2A -adrenergic receptor is linked to a G i protein, and thus receptor occupation inhibits adenylate cyclase activity in a pertussis toxin-sensitive manner. In human platelets, containing various isoforms of both ␣and ␤-adrenergic receptors, it appears to be mainly the ␣ 2A -receptor type that is responsible for the platelet-activating effect of epinephrine (EPI) 1 and other catecholamines (1)(2)(3)(4). Thus, in platelets, EPI causes activation of G i␣2 followed by adenylate cyclase inhibition (5,6). Consequently, EPI efficiently antagonizes the cAMP-elevating effect of G s␣ -stimulating agents like prostacyclin and prostaglandin E 1 (PGE 1 ) (7)(8)(9). In addition, EPI evokes a range of functional platelet responses, such as activation of encrypted integrin ␣ IIb ␤ 3 (fibrinogen) receptors followed by platelet aggregation and, in the presence of other platelet agonists, increased exocytosis (10 -13). However, which signaling events, putatively downstream of G i , underlie these platelet reactions has long remained unclear.
Earlier data from the literature suggest that a fall in cAMP level as such is insufficient to activate platelets (14 -16), implying that EPI may activate other, G i -independent pathways. For instance, EPI can induce a transient increase in cytosolic [Ca 2ϩ ] i in platelets charged with the Ca 2ϩ -sensitive photoprotein, aequorin, although this is not the case for platelets loaded with the Ca 2ϩ probes Quin-2 or Fura-2 (7,17). In addition, EPI potentiates phosphoinositide hydrolysis evoked by other receptor agonists, due to a stimulation of ADP release and/or thromboxane A 2 formation (18 -20), or by enhancing the coupling of the other receptors with phospholipase C (16,21). Another early proposal is that EPI may act through stimulation of the Na ϩ /H ϩ exchanger in the plasma membrane (22), although this effect could later be ascribed to an involvement of protein kinase C (23). A final suggestion is that EPI may act by inhibiting the GTPase-activating protein, Rap1B-GAP (24). Intriguingly, however, most or all of these EPI effects are also under control of the cAMP concentration (7,24), which may point to a possible G i -mediated effect.
In a variety of cell types other than platelets, ␣ 2A -adrenergic receptor activation leads to a potent increase in cytosolic [Ca 2ϩ ] i , which is mediated by G i activation. In erythroleukemia cells equipped with endogenous ␣ 2A -adrenergic receptors and also in cell lines expressing transfected ␣ 2A -receptors, this Ca 2ϩ signal is a consequence of activation of phospholipase C␤ via G␤␥ subunits that are released upon receptor-G i coupling (25,26). Since also in platelets G␤␥ subunits can activate phospholipase C␤2 and ␤3 isoforms (27,28), the question arises whether this G␤␥ signaling pathway contributes to the effects of ␣ 2A -receptor stimulation in platelets.
Here, we examined this subject using aspirin-treated human platelets. It appeared that, similarly to EPI, the specific ␣ 2adrenergic agonist UK14304 was unable to elicit detectable increases in cytosolic InsP 3 or Ca 2ϩ concentration, whereas both compounds potentiated the release of Ca 2ϩ induced by other agonists and caused aggregation of platelets, even in the absence of phospholipase C activation. Using a variety of pharmacological agents, we were able to show that ␣ 2A -receptor stimulation activates the platelets, at least in part, through modulation of cytosolic cAMP and cAMP-dependent protein kinase.
Free Ca 2ϩ concentrations were measured in saponin-permeabilized platelets, basically as described elsewhere (31). Platelets (6 ϫ 10 8 /2 ml) were freshly suspended in calcium-free Hepes/KCl buffer, pH 7.4, composed of 100 mM KCl, 100 mM sucrose, 20 mM Hepes, 1.4 mM MgCl 2 , 1.25 mM NaN 3 , 7.5 mM phosphocreatine, 1 mM ATP, 1 mM KH 2 PO 4 , 30 g/ml creatine kinase, 0.6 g/ml oligomycin, and 1 M Fluo-3. The platelets were treated with EPI or UK14304, if indicated, and then permeabilized by a 10-min incubation with 30 -40 g of saponin. The [Ca 2ϩ ] was then adjusted to 120 nM by stepwise additions from a concentrated CaCl 2 solution, after which InsP 3 was added. Fluorescence intensities (F) were continuously recorded at 488-nm excitation and 526-nm emission wavelengths (slits of 4 nm), using an SLM-Aminco DMX-1100 spectrofluorometer (Rochester, NY). Calibrations were performed by the addition of excess amounts of CaCl 2 or EGTA/Tris (1:1, mol/mol) to obtain F max and F min values, respectively. The level of [Ca 2ϩ ] in the medium was calculated from the binding equation Ultrapure, calcium-free water was used for preparation of buffers, supplements, and agonists.
Measurement of InsP 3 and cAMP Concentrations-Levels of InsP 3 were determined in samples of resting or activated platelets (180 l, 3.5 ϫ 10 8 cells). Cellular activity was stopped by adding 75 l of ice-cold 10% (w/v) HClO 4 . After standing on ice for 30 min and centrifuging at 2000 ϫ g for 10 min (strictly at 4°C), supernatants were collected and neutralized to pH 7 with a solution of 1.7 M KOH in 75 mM Hepes. After another 30-min incubation on ice, precipitated KClO 4 was removed by centrifugation at 2000 ϫ g for 10 min (4°C). In the supernatants, mass amounts of InsP 3 were measured using a Biotrak radioreceptor assay system (Amersham Pharmacia Biotech) with freshly dissolved InsP 3 as standard.
For determination of intracellular cAMP levels, samples of 200 l of platelets (0.4 ϫ 10 8 ) in suspension were withdrawn from incubations of [Ca 2ϩ ] i or aggregation measurements, stopped with ice-cold ethanol (70 volume % final concentration), and frozen in liquid nitrogen. After thawing, the samples were centrifuged, and supernatants were used to measure cAMP, using the Biotrak cAMP enzyme immunoassay system from Amersham Pharmacia Biotech.
Platelet Aggregation and Tyrosine Phosphorylation-Aggregation of aspirin-treated, washed platelets and platelets in plasma was measured in 500-l portions by recording changes in light transmission at 37°C. Affinity modulation of integrin ␣ IIb ␤ 3 was quantified in 10ϫ diluted platelet-rich plasma using fluorescein-labeled PAC1 antibody directed against activated integrin ␣ IIb ␤ 3 (11), with a Becton Dickinson FACStar flow cytometer (Mountain View, CA).
Tyrosine phosphorylation of platelet proteins was determined in 100-l samples taken from aggregation cuvettes. Reactions were stopped by adding 10 mM citrate, 5 mM EGTA, and 5 mM EDTA (final concentrations). The stopped incubation mixtures were centrifuged at 10,000 ϫ g for 20 s, and the pellets dissolved into 100 l of sample buffer, which was composed of 63 mM Tris, 4% (v/v) ␤-mercaptoethanol, 10% (v/v) glycerol, and 3% (w/v) SDS (pH 6.8). Samples were heated at 90°C for 5 min and subjected to electrophoresis on 8% (w/v) polyacrylamide gels. Prestained electrophoresis markers from Bio-Rad (Hertfordshire, United Kingdom) were run in the same gel. Protein tyrosine phosphorylation was detected on Western blots with immunostaining using the phosphotyrosine-specific monoclonal antibody 4G10, as described before (32).

Potentiation of Platelet Ca 2ϩ Responses by ␣ 2 -Adrenergic
Stimulation-To determine the involvement of ␣ 2A -adrenergic signaling in mobilization of Ca 2ϩ in the cytosol, platelets were loaded with Fura-2 and stimulated with EPI, as a general adrenergic agonist, or with UK14304, which is a specific ␣ 2adrenergic activator (25). Indirect effects due to endogenously released thromboxane A 2 or ADP were prevented by treating the platelets with aspirin and using ADP-degrading apyrase in the suspension medium. Under these conditions, neither EPI nor UK14304 (10 -10,000 nM) was capable of inducing a detectable rise in [Ca 2ϩ ] i (Fig. 1A). This clearly contrasts with the situation in native erythroleukemia cells or in CHO cells transfected with ␣ 2A -adrenergic receptors, where low concentrations of either agonist (10 nM) were already sufficient to evoke significant Ca 2ϩ responses (25,26). On the other hand, in platelets, both UK14304 and EPI had a marked, increasing effect on the [Ca 2ϩ ] i rises induced by low doses of G q -stimulating receptor agonists like thrombin (1 nM), platelet-activating factor (20 nM), and lysophosphatidate (1 M) ( Fig. 1A; see below). This is in agreement with earlier reports (16). The potentiating effect was seen upon application before or after thrombin (Fig. 1A). In case of preincubation, the potentiation was dose-dependent up to levels of 122 Ϯ 4 and 124 Ϯ 5% (mean Ϯ S.E., n ϭ 3), compared with the thrombin-evoked response, for 10 M UK14304 and 10 M EPI, respectively. Immediately after platelet isolation, the effect was already detectable at 2 nM EPI, and it had an EC 50 value of 75 nM. As a confirmation of the identity of the receptors involved, it appeared that the ␣ 2 -adrenergic antagonists (33) yohimbine (Fig. 1A) and RX-821002 (not shown) completely inhibited the EPI-induced potentiation of Ca 2ϩ mobilization.
Increased Ca 2ϩ signal generation was not only seen in combination with G q -coupled receptor agonists, but also with Ca 2ϩmobilizing agents acting independently of phospholipase C. Using aspirin-treated platelets bathed with apyrase, ␣ 2 -adrenergic agonists evoked a strong potentiation of the Ca 2ϩ signal induced by 100 nM thapsigargin (Fig. 1B), a compound inhibiting endomembrane Ca 2ϩ -ATPases (30). The EC 50 values of both UK14304 and EPI were now in the range of 75-100 nM. Potentiation was observed with EPI added either before or after thapsigargin, while yohimbine (Fig. 1B) and RX-821002  (not shown) were again completely inhibitory. Both adrenergic agents also caused a 2-fold increase in the Ca 2ϩ response evoked by 10 M thimerosal (Fig. 2). Thimerosal is a membrane-permeable sulfhydryl reagent that, independently of phospholipase C, sensitizes InsP 3 receptors and thereby stimulates the process of Ca 2ϩ -induced Ca 2ϩ release (CICR). As checked with platelet agonists of various types (i.e. thrombin, platelet-activating factor, thapsigargin, and thimerosal), the magnitude of the EPI-mediated rise in [Ca 2ϩ ] i was independent of the presence or absence of extracellular CaCl 2 (Fig. 2), demonstrating that the principal effect of EPI is increase of the Ca 2ϩ mobilization from intracellular stores instead of modulation of Ca 2ϩ influx. On the other hand, in combination with ionomycin (5 M) (i.e. a compound directly permeating the cellular membranes for Ca 2ϩ ), EPI did not change the increase in [Ca 2ϩ ] i (102 Ϯ 5% (mean Ϯ S.E., n ϭ 3)), which is in agreement with earlier findings (16). We observed some donorto-donor variability in the magnitude of the actions of EPI and also noted desensitization of the effects within 2 h of platelet isolation (see below). Taken together, the comparable effects of UK14304 and EPI and the efficient suppression of these effects by yohimbine and RX-821002 strongly indicate that a single class of ␣ 2 -adrenergic receptors is involved in the potentiating effect of EPI on Ca 2ϩ signal generation. The complete lack of Ca 2ϩ responses with UK14304 or EPI alone suggests that these agents are unable to activate phospholipase C isoforms in platelets. This was verified by measuring InsP 3 levels. While thrombin evoked the expected increase in InsP 3 concentration, UK14304 and EPI were completely ineffective, even when given in combination with thapsigargin (Table I). This agrees well with the earlier noted absence of phosphoinositide turnover in EPI-stimulated platelets (18 -20). Apparently, platelets differ from other cells expressing ␣ 2A -adrenergic receptors, where compounds like UK14304 cause potent increases in both InsP 3 and [Ca 2ϩ ] i (25,26). Involvement of cAMP in ␣ 2 -Adrenergic Stimulation of Ca 2ϩ Signaling-The above results prompted us to re-examine effects of ␣ 2 -adrenergic stimulation on the classical G i␣ /adenylate cyclase pathway. Using freshly isolated, aspirin-treated platelets, 10 M EPI caused a small, but nevertheless significant, reduction in cAMP concentration of about 20% (Table II). In comparison, a low dose of thrombin, i.e. another G i -stimulating agonist, induced a smaller decrease in cAMP, whereas thapsigargin was without influence on the cAMP level. Thus, EPI might act by a G i␣ -mediated reduction in cAMP level and subsequent decrease in cAMP-dependent protein kinase activity. This possibility was tested in a number of ways.
Since both G s␣ -and adenylate cyclase-stimulating agents (e.g. PGE 1 and forskolin), which raise the cAMP level, are known to down-regulate the Ca 2ϩ responses of platelets (31,34), this type of intervention is expected to oppose the Ca 2ϩ release-stimulating effect of ␣ 2A -adrenergic agonists. Indeed, both EPI and UK14304 (10 M each) efficiently antagonized the inhibitory effect of PGE 1 on the [Ca 2ϩ ] i rises evoked by thrombin or thapsigargin, i.e. both in the presence and in the absence of phospholipase C activation (Fig. 3). Plots constructed of the thrombin-induced increase in [Ca 2ϩ ] i versus the cAMP concentration at the time of Ca 2ϩ measurement reveal a nonlinear relationship, in which the Ca 2ϩ response steeply declines with the increase in cAMP level (deflection point around 3 pmol of cAMP/10 8 platelets). Typically, the relation between Ca 2ϩ response and cAMP level was similar in the presence and absence of EPI (Fig. 4). When thapsigargin was used as co-agonist instead of thrombin, essentially the same results were obtained (data not shown).
This type of experiment gave more information on the apparent time-dependent desensitization of the EPI effects. In all cases where tested, platelets that after more than 2 h of isolation did not respond to EPI by an increased Ca 2ϩ signal were well able to do so when pretreated with a low dose of PGE 1 (data not shown). This suggests that the observed desensitization is not a consequence of changed receptor binding or transduction properties but, instead, of a time-dependent change in basal cAMP level. Indeed, in two experiments, basal cAMP (in the absence of agonists) was found to decrease from 3.5 to 2.6 pmol/10 8 platelets (mean values) in a time period of 90 min. Second, experiments were conducted in which cAMPdependent protein kinase activity was stimulated by interfering in the signaling pathway downstream of adenylate cyclase. Platelets were therefore treated with the cAMP-dependent phosphodiesterase inhibitor, isobutyl 1-methylxanthine (400 M), to block cAMP degradation (14). This treatment resulted in a 2-fold increase in cAMP level and diminished the thrombin-and thapsigargin-evoked rises in [Ca 2ϩ ] i . Moreover, it abolished the stimulating effect of EPI on the Ca 2ϩ signal (Fig.  5). This suggests that resting platelets have a basal, non-zero activity of cAMP-dependent protein kinase, as indeed assessed by others (35). Platelets were also treated with the phosphodiesterase-resistant cAMP analog, (S p )-cAMPS, which specifically activates cAMP-dependent protein kinase (36). This treatment again reduced the Ca 2ϩ responses with thrombin and thapsigargin and totally inhibited the Ca 2ϩ -potentiating effect of EPI (Fig. 5).
Third, effects were determined of inhibition of platelet cAMPdependent protein kinase. For this purpose, initially the reported adenylate cyclase inhibitors, 2Ј,5Ј-dideoxyadenosine and SQ22536, were used (15,37). However, in our hands, these compounds (even high concentrations (500 M)) diminished the PGE 1 -evoked reduction of the thrombin-induced Ca 2ϩ responses with no more than 12 and 25%, respectively, which points to only moderate adenylate cyclase inhibition. Platelet treatment with SQ22536 was only of little influence on the EPI-mediated Ca 2ϩ release (Fig. 6). Clearer results were ob-tained with selective inhibitors of cAMP-dependent protein kinase, i.e. the structurally dissimilar hydrolysis-resistant cAMP analog, (R p )-8-CPT-cAMPS (200 M) (38), and the kinase active-site inhibitor, KT5720 (2.5 M) (39). Both compounds caused appreciable abrogation of the PGE 1 -induced reduction of the Ca 2ϩ response, with 55 and 70%, respectively. In addition, they had a marked effect on the Ca 2ϩ signal in the absence of PGE 1 . (R p )-8-CPT-cAMPS and KT5720 raised the basal [Ca 2ϩ ] i from 45 Ϯ 3 nM to 65 Ϯ 4 and 70 Ϯ 2 nM (mean Ϯ S.E., n ϭ 3), respectively. The compounds increased both the thrombin-and thapsigargin-evoked Ca 2ϩ responses and canceled the EPI-mediated potentiation of these responses (Fig. 6). Similarly, the time curves of the [Ca 2ϩ ] i transients indicated that (R p )-8-CPT-cAMPS (not shown) and KT5720 (Fig. 7A) mimicked the effects of EPI. As a comparison, we monitored effects of the protein kinase C inhibitor, Ro-318220 (3 M), and the protein-tyrosine kinase inhibitor, genistein (100 M) (40). However, neither of these substances altered the effect of EPI on the thrombin-evoked Ca 2ϩ response (117 Ϯ 2 and 120 Ϯ 1% of control values, respectively, with EPI alone stimulating to a level of 120 Ϯ 3%).
Fifth, since the InsP 3 receptor channel in platelets is known to be cAMP-sensitive, we determined the effect of ␣ 2 -adrenergic agents on InsP 3 -evoked Ca 2ϩ release from intracellular stores in saponin-permeabilized platelets. As shown in Fig. 7B, prestimulation with EPI before the addition of saponin led to a potent increase in the Ca 2ϩ -releasing effect of InsP 3 . Preincu-FIG. 6. Conditions inhibiting cAMPdependent protein kinase mimic ␣ 2adrenergic effect. Fura-2-loaded platelets were activated with 1 nM thrombin (A) or 100 nM thapsigargin (B) with or without 10 M EPI, as described for Fig. 1. The platelets were pretreated with vehicle (Control) or agents with a suppressing effect on cAMP-dependent protein kinase. Pretreatment was with 500 M SQ22536 (10 min), 200 M (R p )-8-CPT-cAMPS (10 min), or 2.5 M KT5720 (2 min). Where indicated, EPI was then given, followed by thrombin or thapsigargin. Maximal changes in [Ca 2ϩ ] i were measured (in nM). Data (mean values Ϯ S.E., n ϭ 3-5) are percentages of the Ca 2ϩ responses relative to the control condition. *, significantly different from the corresponding condition without EPI (p Ͻ 0.05, t test, one-sided).
bation of the platelets with KT5720 had a similar effect. When using freshly isolated platelets, UK14304 and EPI potentiated the InsP 3 -evoked Ca 2ϩ mobilization to 140 Ϯ 6 and 161 Ϯ 8% of the control value, respectively (mean Ϯ S.E., n ϭ 6 -8, p Ͻ 0.01). At 2 h after platelet isolation, the stimulating effect of EPI was decreased to 111 Ϯ 5% (n ϭ 10, p Ͻ 0.04). Taken together, these results suggest that, in freshly isolated platelets, InsP 3 -induced Ca 2ϩ mobilization is partially down-regulated by tonic activity of the cAMP-dependent protein kinase and that ␣ 2 -adrenergic stimulation can relieve this down-regulation.
Involvement of cAMP in ␣ 2 -Adrenergic Mediated Integrin ␣ IIb ␤ 3 Activation-As an extension of the Ca 2ϩ measurements, we monitored effects of ␣ 2 -adrenergic stimulation on the exposure of active integrin ␣ IIb ␤ 3 on the platelet surface by using fluoresceinisothiocyanate-labeled PAC1 antibody, which only binds to this activated integrin form. Confirming an earlier report (11), platelet stimulation with 10 M EPI or UK14304 resulted in a considerable increase in fluorescein-PAC1 binding (Fig. 8). The EPI-induced integrin ␣ IIb ␤ 3 activation was completely blocked by yohimbine, isobutyl 1-methylxanthine, or PGE 1 but not by Ro-318220. Since integrin exposure causes platelet aggregation when fibrinogen is present, and extensive protein tyrosine phosphorylation (12,13), these platelet responses were also measured. Yohimbine, isobutyl 1-methylxanthine, PGE 1 , or (S p )-cAMPS almost completely suppressed the EPI-induced aggregate formation (Fig. 9A) and tyrosine phosphorylation events (Fig. 9B).

DISCUSSION
The present results demonstrate that, in platelets, the signaling pathway upon ␣ 2A -adrenergic stimulation differs from that reported for other cells expressing this receptor, i.e. erythroleukemia cells and embryonic kidney 293 cells with native receptors (41,42), and Chinese hamster ovary and COS-7 cells transfected with ␣ 2A -receptors (26). In Fura-2-loaded platelets, which respond to minor increases in InsP 3 level by a clear Ca 2ϩ signal (30), neither UK14304 nor EPI appears to elevate InsP 3 or Ca 2ϩ concentrations, which agrees well with the reported lack of EPI to induce hydrolysis of radioactive labeled phosphoinositides (18 -20). This suggests that, in platelets, the G␤␥-mediated pathway of phospholipase C␤ stimulation, as identified for the other cell types (26,42), does not play an important role in the ␣ 2 -adrenergic signaling cascade. On the other hand, our data confirm those from others that ␣ 2 -adrenergic receptor (EPI) stimulation of platelets causes G i activation with consequent inhibition of adenylate cyclase (1-9), ultimately leading to the exposure of integrin ␣ IIb ␤ 3 (11)(12)(13).
Compatible with a G i␣ -mediated action, we detected a significant decrease in basal cAMP level in the EPI-treated platelets (Table II). Various sets of experiments suggest that this cAMP change is crucial in the effect of EPI to potentiate the Ca 2ϩ FIG. 7. Stimulation of Ca 2؉ mobilization by EPI and KT5720. A, aspirin-treated, Fura-2-loaded platelets were stimulated with thapsigargin (TG), EPI, and/or KT5720, as indicated for Fig. 6. B, platelets in KCl/ATP medium were pretreated with EPI (10 M) and/or KT5720 (2.5 M), as indicated. Subsequently, the cells were permeabilized with saponin, and mobilization of Ca 2ϩ from stores was measured with Fluo-3. After adjustment of the Ca 2ϩ level of the medium to 110 nM, InsP 3 (100 nM) was added as indicated. Data are from a representative experiment out of five or more performed. responses with phospholipase C-stimulating (e.g. thrombin) and phospholipase C-independent (e.g. thapsigargin) platelet agonists ( Figs. 1 and 2). For instance, both UK14304 and EPI efficiently antagonize the G s /cAMP-mediated suppression of the Ca 2ϩ signal (Fig. 3). In addition, when comparing intracellular levels of Ca 2ϩ and cAMP in platelets that were pretreated with various doses of PGE 1 , the Ca 2ϩ responses appear to decline steeply with an increase in cAMP concentrations independently of the presence or absence of EPI, with a reflection point that is close to the cAMP level of resting platelets (Fig. 4). Other evidence that cAMP and cAMP-dependent protein kinase are involved in this ␣ 2A -adrenergic response comes from the observation that agents that stimulate the kinase suppress the Ca 2ϩ responsiveness and inhibit the EPI effect (Fig. 5); and also from data that inactivation of the kinase results in increased Ca 2ϩ responses while blocking the EPI effect (Fig. 6). In addition, both UK14304 and EPI appear to have a highly stimulatory effect on InsP 3 -induced Ca 2ϩ mobilization in permeabilized platelets (Fig. 7), a reaction that is particularly sensitive to the cAMP and Ca 2ϩ concentrations (31,34). 2 The latter adrenergic effect can be mimicked by inhibition of cAMPdependent protein kinase, which suggests that ␣ 2 -adrenergic agents act by relieving the partial suppression of the Ca 2ϩ mobilization by the basal cAMP level. Taken together, these results suggest that ␣ 2A -adrenergic receptor stimulation in platelets influences the Ca 2ϩ responses primarily by modulation of the cAMP concentration.
The present data indicate that the channel activity of the platelet InsP 3 receptors is altered by EPI action. It seems that EPI reduces the basal, cAMP-dependent phosphorylation of InsP 3 receptors and, thereby, increases their responsiveness toward InsP 3 -generating and Ca 2ϩ -mobilizing agonists. This explains why the ␣ 2 -adrenergic agonists potentiate the Ca 2ϩ responses even in combination with thapsigargin and thimerosal, compounds that operate independently of phospholipase C stimulation. Indeed, in platelets, the process of CICR by which InsP 3 receptors mobilize Ca 2ϩ in a Ca 2ϩ -stimulated way appears to be influenced not only by increased Ca 2ϩ leakage fom stores (by thapsigargin) or sensitization of the InsP 3 receptor (by thimerosal), but also by changes in intracellular cAMP. 2 Although the platelet InsP 3 receptors have been recognized as major cAMP sensors in Ca 2ϩ signaling (31), it is not unlikely that EPI may also influence other cAMP-sensitive steps involved in the regulation of the Ca 2ϩ signal, e.g. by stimulating phosphoinositide turnover (44) or reducing plasma membrane Ca 2ϩ -ATPase activity (45,46).
The current proposal that small changes in (basal) cAMP concentration significantly modulate the Ca 2ϩ responses agrees well with published evidence that the estimated level of cAMP in unstimulated platelets (about 4.4 M) is somewhat higher than the apparent cAMP dissociation constant of cAMPdependent protein kinases (47) and only slightly lower than the intracellular concentration of cAMP-binding sites on the ki-2 R. van Gorp, unpublished results.  (35, 48). Thus, cAMP-mediated protein phosphorylation in platelets can be considered as a signal transduction pathway of both high sensitivity and high capacity (48). On the other hand, it is clear from our findings and the literature that a decrease in cAMP as such is insufficient to produce full platelet activation. For instance, we found that the kinase inhibitors (R p )-8-CPT-cAMPS and KT5720 potentiate platelet activation but do not evoke a major Ca 2ϩ signal or aggregation of the platelets. This is in agreement with published evidence that the use of 2Ј,5Ј-dideoxyadenosine or SQ22536 to inhibit adenylate cyclase is insufficient to induce aggregate formation (9,15,37). Although under our conditions these compounds were only weak adenylate cyclase inhibitors, it is likely that other signaling events than a decrease in cAMP level are needed for full platelet activation.
A typical effect of ␣ 2A -adrenergic receptor stimulation in other cell types than platelets is the G␤␥-mediated activation of a mitogen-activated protein kinase cascade (26,42). Such a signaling route, although not yet proven, might also occur in EPI-stimulated platelets. In particular, this might be involved in the EPI-induced exposure of integrin ␣ IIb ␤ 3 and, thus, in platelet aggregation. Nevertheless, also for this ␣ 2 -adrenergic response, modulation of the cAMP level seems to play an ultimate controlling role, as apparent from the integrin-and tyrosine phosphorylation-inhibitory effects of cAMP-elevating interventions ( Figs. 8 and 9).
In summary, we propose the following scheme of ␣ 2A -adrenergic receptor stimulation in platelets (Fig. 10). Receptor occupation results in G i␣ -mediated inhibition of adenylate cyclase. Consequent lowering in cAMP level and decreased activity of cAMP-dependent protein kinase relieves the tonic, suppressive effect of cAMP-dependent phosphorylation on the Ca 2ϩ channel function of the InsP 3 receptors. These channels allow Ca 2ϩ release into the cytosol in a InsP 3 -and Ca 2ϩ -dependent way according to the CICR mechanism. In addition to this G i␣mediated effect, the cAMP level is controlled by G ␣s -mediated activation of adenylate cyclase and cAMP hydrolysis by phosphodiesterases. Signaling through G␤␥ subunits may contribute to the platelet-activating effect of ␣ 2A -receptor agonists; however, not by activation of phospholipase C␤.