Previous works have suggested that the impairment of platelet aggregation by halothane was partly related to a stimulation of cyclic adenosine monophosphate (cAMP) production, to an inhibitory effect on Ca2+ signaling, or both. Intracellular Ca2+ measurements therefore were undertaken, first to determine the critical steps in the platelet CaZ+ signaling cascade most likely to be affected by halothane or by an increase in cAMP production, and second to establish if the effect of halothane involves aggregation-related biochemical pathways triggered by an increase in internal Ca2+.
Human washed platelets were treated with halothane or forskolin for 5 min before application of either platelet-activating factor, thrombin, U46619, or thapsigargin. The cytosolic Ca2+ concentration ([Ca2+]i) was measured with the fluorescent Ca2+ indicator fura-2. Nephelometric measurements were also performed to assay the aggregation process.
Our results indicate that pretreating platelets with halothane leads to a partial impairment of the [Ca2+]i increase induced either by U46619, thrombin, or platelet-activating factor, but this had no significant effect on the [Ca2+]i response triggered by thapsigargin. In addition, our results show that halothane inhibits platelet aggregation triggered by U46619, but not by thapsigargin. Conversely, forskolin completely inhibited the [Ca2+]i response to U46619 and thapsigargin and prevented platelet aggregation induced by both agonists.
These results suggest that halothane and cAMP exert their effects on platelet aggregation and Ca2+ signaling through different mechanisms, and that halothane cannot impair platelet aggregation independently of phospholipase C stimulation.
AMONG volatile anesthetics, halothane is unique in its ability to alter platelet function at clinically relevant concentrations. In fact, halothane increases bleeding time in vivo and impairs platelet aggregation in vitro. [3,4]More recently, it was reported that halothane protects against thrombus formation in stenosed coronary arteries in the dog. However, the cellular mechanism underlying the action of halothane on platelet function still remains poorly circumscribed.
Platelet aggregation is a complex phenomenon that likely involves several intracellular biochemical pathways. There is increasing evidence that internal Ca2+stores play a central role in the response of platelets to activating agents. It is thus possible that part of the observed effect of halothane on platelet aggregation results from an impaired Ca2+response. In fact, halothane is already known to affect Ca (2+) signaling in many cell types, [8–10]including human platelets. There are, however, several possible ways by which halothane can interfere with Ca2+signaling and platelet aggregation. For instance, halothane could affect the production of second messengers linked to Ca2+mobilization, or inhibit directly the internal Ca2+release plus agonist-induced Ca2+influx initiated after platelet stimulation. In the latter case, the halothane effect would not require phospholipase C activation. In addition, halothane might affect the biochemical pathways that are triggered by internal Ca2+and that lead to platelet aggregation. Under these conditions, halothane would impair platelet aggregation independently of the mechanisms responsible for the increase in cytosolic Ca (2+).
Several studies have also provided evidence that cyclic AMP (cAMP) is involved in the action of halothane on many cell types, including platelets. [12,13]In these preparations, halothane was shown to promote cAMP formation by stimulating adenylate cyclase without affecting phosphodiesterases. It is known that cAMP is one of the most potent platelet function inhibitors, and investigators have proposed that the inhibitory action of halothane on platelet aggregation resulted from an increased level of cAMP. Because cAMP impairs Ca2+mobilization in platelets, an effect of halothane on aggregation may also arise from an enhanced cAMP production, which would in turn affect internal Ca2+mobilization.
Thus the purpose of this study was to determine more precisely the mechanism by which halothane affects Ca2+signaling and platelet aggregation. Our results indicate first that halothane and cAMP exert their inhibitory effect through distinct mechanisms, and second that the action of halothane in these cells requires phospholipase C stimulation.
Materials and Methods
Solutions and Drugs
The acid-citrate dextrose anticoagulant was composed of 85 mM sodium citrate, 78 mM citric acid, and 111 mM dextrose. The control solution was composed of 10 mM HEPES (pH 7.4), 145 mM NaCl, 5 mM KCl, 1 mM MgCl2, and 10 mM dextrose. Fura-2-AM, BAPTA (1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid), acetylsalicylic acid, and digitonin were purchased from Sigma Chemical Company (St. Louis, MO); U46619 was from Pharmacia and Upjohn (Kalamazoo, MI); thrombin (human plasma), forskolin, and platelet activating factor C-16 (1-O-palmityl-2-acetyl-sn-glycero-3-phosphocholine) were obtained from Calbiochem (San Diego, CA). Stock solutions of 24 mM halothane (Ayerst, Montreal, Quebec, Canada) were prepared by vigorous stirring of halothane dissolved in control solutions for 2 h.
Fura-2 - loaded platelets were prepared according to the method of Sage et al., with minor modifications. Blood from healthy nonmedicated volunteers was drawn in plastic tubes containing 15% vol/vol acid-citrate dextrose anticoagulant. Blood was centrifuged at 350g for 10 min, and the resulting platelet-rich plasma was treated with 100 [micro sign]M aspirin to ensure that the observed agonist-evoked effects did not result from the secondary formation of prostaglandins and thromboxanes. No phosphodiesterase inhibitors were used. Platelets in platelet-rich plasma were loaded with the fluorescent probe fura-2-AM (4 [micro sign]M) for 40 min at room temperature. They were sedimented by centrifugation at 437.5 g for 15 min and resuspended in the control solution to achieve an approximate concentration of 1.2 x 106platelets per milliliter.
Washed platelets (1.4 ml) were introduced into a 1.8-ml gas-tight silicone-coated glass bottle that serves as a “gas-tight spectrofluorimeter cuvet.” CaCl2was added to a final concentration of 1 mM 5 min before fura-2 measurements. After the addition of Ca2+, 100 [micro sign]l of the 24 mM halothane stock solution were injected into the cuvet using a gas-tight syringe, for a final concentration of 1.6 mM. This concentration of halothane corresponds to a gaseous partial pressure of 5% at 37 [degree sign]C, but to 3% in the current case because the experiments were done at 22 [degree sign]C. The effective halothane concentration was tested in experiments in which the halothane content of control solutions was measured by gas chromatography, as previously described. A similar procedure was used in experiments in which platelets were pretreated with forskolin (0.1 to 10 [micro sign]M). Platelets were then stimulated with either 1 [micro sign]M U46619, 10 nM platelet-activating factor, 1 U/ml thrombin, or 1 [micro sign]M thapsigargin. In some experiments, 10 mM of the Ca2+chelating agent BAPTA was added 50 s before the addition of the platelet stimulator to elicit a response in the absence of external Ca2+.
Fluorometric measurements were performed using a dual-excitation spectrofluorometer (Spex Fluorolog II, Spex Industries, Edison, NJ). The excitation wavelengths were set at 340 nm and 380 nm, and emission was monitored at 505 nm. The cytosolic Ca2+concentration ([Ca2+]i) was calculated from the ratio of the fluorescence measured at 340 nm and 380 nm. The ratios, Rminand Rmax, were obtained by measuring the fluorescence in 5 mM EGTA plus digitonin (50 [micro sign]M) at pH 9.0 in the absence and the presence of 10 mM CaCl2, respectively.
Qualitative Aggregation Measurements
For the aggregation measurements, washed platelets were prepared as described before and the same protocols were applied. Nephelometric measurements were used to assay the aggregation, with the increase in light transmission proportional to the extent of the aggregation. All experiments were done at room temperature.
Each set of experiments was repeated at least three times, and the results are presented as mean values +/- SD. Statistical significance was assessed by Student's t test. The significance level was set at P < 0.05.
Effect of Halothane on Ca2+Signaling
The effect of halothane on basal [Ca2+]iwas assayed by adding 1.6 mM halothane to resting platelet suspensions. In 75% of the experiments performed (32 of 42), a small transient [Ca2+]iincrease of 17 +/- 5 nM was detected (data not shown). This halothane-evoked Ca2+increase was observed in the presence and in the absence of external Ca2+, suggesting that the observed Ca2+increase was related to a mobilization of Ca2+from intracellular stores.
(Figure 1) shows examples of fura-2 recordings obtained from experiments aimed at investigating the effect of halothane on the Ca2+response induced by various platelet activators. Figure 2summarizes the results of these experiments. In conditions in which platelets were exposed for 5 min to 1.6 mM halothane, there was a significant reduction of the initial U46619-evoked [Ca2+]iincrease from 227 +/- 115 nM to 124 +/- 89 nM (n = 10;Figure 2A). A significant decrease of the initial [Ca (2+)]ipeak from 286 +/- 80 nM to 196 +/- 67 nM (n = 6) was also observed after halothane treatment, when platelets were stimulated with 10 nM platelet-activating factor (Figure 2B). Similar results were obtained with thrombin (1 U/ml), with a reduction of the initial Ca2+increase from 252 +/- 128 to 149 +/- 42 (n = 4), respectively (data not shown).
The agonist-evoked Ca2+response in platelets is a biphasic process characterized by a release of Ca2+from internal stores coupled to a Ca2+influx from the external medium. To determine if halothane could selectively inhibit the release of Ca2+from internal pools, platelets were stimulated in the absence of external Ca2+. In these experiments, BAPTA, a Ca2+-chelator, was added 50 s before exposure to U46619, as shown in Figure 1C. Under these conditions, halothane caused a significant decrease of the initial Ca2+peak induced by U46619 from 210 +/- 110 nM to 104 +/- 25 nM (n = 7;Figure 2C), indicating an action of halothane on the mechanisms underlying the release of Ca2+from internal pools. Similar results were obtained with platelet-activating factor and thrombin (data not shown).
To determine if the observed action of halothane on Ca2+signaling requires the production of second messengers related to the phosphoinositide pathway, halothane experiments were performed in which platelets were stimulated with the sarcoplasmic-endoplasmic reticulum Ca2+ATPase inhibitor, thapsigargin. Thapsigargin selectively block the internal Ca2+sequestration process, leading to an uncompensated Ca2+leak from internal stores. A mobilization of internal Ca2+is generated under these conditions without producing the second messenger myo inositol-1,4,5 trisphosphate (InsP3). In addition, this progressive and constant emptying of internal Ca2+pools has been shown to trigger a Ca (2+) entry from the external medium, called capacitative Ca2+influx. [7,19,20]The results in Figure 1D and Figure 2D show that halothane, at a supraclinical concentration of 1.6 mM, failed to significantly affect the [Ca2+]imobilization induced by thapsigargin, with a change from 227 +/- 135 nM to 195 +/- 124 nM (n = 7) of the Ca2+level measured 200 s after thapsigargin application. These results suggest that stimulation of phospholipase C is essential for halothane to inhibit the mobilization of Ca2+from internal Ca2+pools and the related capacitative Ca2+influx.
Effect of Forskolin on Ca2+Signaling
Because halothane increased cAMP production in platelets, experiments were done to compare the action of forskolin and halothane on the Ca2+response induced by U46619 or thapsigargin. Figure 1A and Figure 1C show that a 5-min treatment with 1 [micro sign]M forskolin completely abolished the Ca2+increase triggered by U46619 in the presence (n = 4) or in the absence (n = 4) of external Ca2+, respectively. However, in contrast to halothane, 1 [micro sign]M forskolin succeeded also in inhibiting the Ca2+response induced by thapsigargin (Figure 1D). The results in Figure 1A indicate furthermore that the addition of 0.1 [micro sign]M forskolin caused a 50% reduction of the mean U46619-evoked [Ca2+]iincrease with values of 250 +/- 35 nM (n = 3) in control conditions and 123 +/- 20 nM (n = 3) after forskolin treatment. An inhibitory effect of equivalent potency was observed after a 5-min exposure to 1.6 mM halothane (Figure 1A). However, forskolin at 0.1 [micro sign]M appeared more potent than halothane in inhibiting the Ca2+response initiated by thapsigargin, with a 42 +/- 7%(n = 3) reduction of the internal Ca2+level measured 200 s after thapsigargin application (Figure 1D). These results suggest that halothane and cAMP operate in different ways on Ca2+signaling in platelets.
Effect of Halothane and Forskolin on the Capacitative Ca2+Influx
The capacitative Ca2+entry in platelets is characterized by an external Ca2+influx that is intimately regulated by the emptying of internal Ca2+stores. [7,19]To investigate the effect of halothane and forskolin on the capacitative Ca2+entry in platelets, fura-2 experiments were performed in which thapsigargin was used to generate a release of Ca2+from internal pools in the absence of InsP3production. In a first series of experiments, platelets were first treated for 5 min with either 1.6 mM halothane or forskolin (0.1 to 1 [micro sign]M) before the addition of thapsigargin (1 [micro sign]M) in the absence of external Ca2+. The Ca2+influx was initiated by the subsequent addition (100 s after thapsigargin) of 1 mM CaCl2to the external medium (Figure 3A). Pretreatment with 0.1 [micro sign]M forskolin decreased by 50 +/- 7%(n = 4) the internal Ca2+level measured 100 s after the addition of external Ca2+, whereas total inhibition of the Ca2+response was observed in platelets exposed to 1 [micro sign]M forskolin (data not shown). In contrast, under the same experimental conditions, halothane had a nonsignificant effect on the thapsigargin-evoked capacitative Ca2+influx, with a reduction of the related Ca2+increase of 10 +/- 6%(n = 4).
In a second series of experiments, halothane and forskolin were added at the peak of the Ca2+response after thapsigargin stimulation. The importance of the Ca2+influx to the thapsigargin-induced Ca2+increase was estimated in this case by chelating the external Ca2+with BAPTA 50 s after thapsigargin exposure. Figure 3B shows that the addition of BAPTA resulted in a slow decay of the internal Ca2+level to a lower steady-state value, indicating an absence of Ca2+entry in external Ca (2+) free conditions. Similarly, a dose-dependent decrease of the fura-2 signal was observed after the addition of forskolin at concentrations ranging from 0.1 to 10 [micro sign]M (Figure 3B). Figure 4summarizes the results of these experiments. The presence of BAPTA caused a 75 +/- 5%(n = 4) inhibition of the Ca2+influx, compared with 49 +/- 7%(n = 4) and 27 +/- 4%(n = 4) with forskolin at 10 [micro sign]M and 1 [micro sign]M, respectively. As observed previously, halothane had no effect (5 +/- 5%, n = 4) on thapsigargin-induced Ca2+influx. These results provide additional evidence for a differential effect between halothane and forskolin on Ca2+signaling machinery in platelets.
Acute effect of Halothane on Ca2+Signaling
The inhibition of the initial agonist-induced Ca2+increase by halothane illustrated in Figure 1A to Figure 1C was measured after a 5-min pretreatment with halothane. Experiments were also performed in which halothane was added 30 s after the mobilization of Ca2+in response to several platelet activators. This protocol allows the internal stores to deplete rapidly and transitionally, with a maximal and sustained contribution of the Ca2+influx process. The results presented in Figure 5A to Figure 5C indicate that the acute addition of halothane to U46619-, platelet-activating factor-, and thrombin-stimulated cells initiated a small increase in the rate at which the fura-2 signal decayed to a lower steady state value. These observations suggest that halothane can also rapidly affect the sustained agonist-mediated Ca2+entry.
Effect of Halothane on Platelet Aggregation
(Figure 6A and Figure 6B) show the effect of halothane and forskolin on platelet aggregation caused by U46619 and thapsigargin, respectively. The results indicate that halothane and forskolin acted differently, depending on the platelet activator used. A high concentration of forskolin (1 [micro sign]M) completely inhibited platelet aggregation induced by either U46619 (n = 3) or thapsigargin (n = 4). In contrast, 1.6 mM halothane impaired the aggregation triggered by U46619 (n = 3) but failed to affect the thapsigargin-mediated aggregation process (n = 4).
This study investigated the effect of halothane on platelet Ca2+mobilization and aggregation. Our results essentially indicate that halothane inhibits the aggregation and initial Ca2+release in platelets stimulated with agonists linked to the phosphatidylinositol pathway, but affect minimally the Ca2+response and the aggregation process induced by thapsigargin.
Effect of Halothane on Agonist-evoked Ca2+Responses
The results presented in Figure 1A, Figure 1B, Figure 2A, and Figure 2B indicate that halothane inhibits the internal Ca2+increase induced by U46619 and platelet-activating factor. These results support the original observations of Kohro and Yamakage, who reported a dose-dependent inhibition of the Ca2+release induced by thrombin in human platelets. In their study, halothane was effective at clinically relevant concentrations (<1 mM), and complete inhibition of the Ca2+increase triggered by thrombin was observed at 2 mM halothane, with half inhibition measured at 1 mM. There was no effect of isoflurane. In the current work, a near 50% inhibition was obtained at 1.6 mM halothane, depending on the agonist used as a stimulating agent. A higher halothane concentration in our case may be explained in part by differences in experimental conditions, such as the temperature (room temperature in the current study compared with 37 [degree sign]C in the work of Kohro and Yamakage ), because the same concentration of 1.6 mM halothane corresponds to a gaseous partial pressure of 5% at 37 [degree sign]C and 3% at 22 [degree sign]C. Given the negative results obtained by Kohro and Yamakage, we did not investigate isoflurane further in the current study.
The action of halothane may involve various specific mechanisms, including inhibition of the internal Ca2+release from internal stores, [9,21]decrease of the Ca2+influx, or impairment of the Ca (2+) extrusion processes. In platelets, the Ca2+response to U46619 stimulation results from a fast release of Ca2+from internal stores and a sustained Ca2+influx from the external medium. [7,23]The results in Figure 1B and Figure 2B provide clear evidence that halothane interferes strongly with the initial Ca2+release process as already reported in some cell preparations. [9,21]This impaired Ca2+release may result from several mechanisms, including an inhibition of the InsP3production, a direct action on the InsP3-dependentCa2+release from internal pools, or both. In this regard, evidence has already been provided for a reduced InsP3production in thrombin-stimulated human platelets exposed to halothane (2 mM). In addition, the absence of a halothane-dependent inhibition of the Ca2+increase initiated by thapsigargin argues for an effect of halothane on the production of second messengers linked to phospholipase C stimulation.
Further, when halothane was added after U46619 or thrombin or platelet-activating factor, there was a clear increase in the rate at which the [Ca2+]idecayed to a lower steady state value (Figure 5A Figure 5B Figure 5C). Because Ca2+influx is responsible for the sustained [Ca2+]i7increase observed under these conditions, halothane thus appears to inhibit Ca2+influx, as reported in endothelial cells. However, halothane had no effect on the capacitative Ca2+influx induced by thapsigargin (Figure 3A and Figure 3B) suggesting first that halothane does not significantly modify the Ca2+extrusion process, and second that part of the Ca2+influx in U46619-stimulated cells is mediated by a second messenger-dependent Ca2+entry process distinct from the capacitative Ca2+influx and that would be halothane sensitive. Kinetic experiments have already shown that Ca (2+) influx precedes Ca2+release, suggesting that the capacitative Ca2+influx is not exclusively responsible for the overall Ca2+influx observed in U46619-stimulated platelets. Therefore, a direct correlation seems to exist between the production of second messengers, which participate in Ca2+mobilization processes in platelets, and the ability of halothane to interfere with the Ca2+signaling machinery.
The Effect of Forskolin on Ca2+Signaling
Previous studies have shown that halothane increases cAMP production in platelets. Because cAMP alters the platelet Ca2+signaling process, the effects of halothane we observed could have been indirectly caused by an increase in cAMP production. This mechanism was tested by comparing the Ca2+response of platelets to various Ca2+-mobilizing agonists, after treatment with either halothane or forskolin, an agent that directly stimulates the formation of cAMP. Our results confirmed that forskolin at concentrations of 0.1 to 1 [micro sign]M attenuates or abolishes (or both), in a dose-dependent manner, the Ca2+mobilization initiated by U46619 (Figure 1A). Furthermore, the action of cAMP appeared to be independent of phospholipase C stimulation, because, in contrast to halothane, a pretreatment with forskolin markedly reduced the Ca2+increase initiated by the Ca2+pump inhibitor thapsigargin (Figure 1D). These results suggest that the observed effect of halothane on Ca2+release is not compatible with a stimulation of cAMP formation by halothane. The mechanism underlying the action of forskolin on Ca2+signaling in platelets is not, however, fully elucidated. A report by Koike et al. has indicated that the Ca2+influx induced in human platelets by thapsigargin could be partly suppressed by pretreatment with phosphatase inhibitors, such as okadaic acid, calyculin, and tautomycin, suggesting an effect of phosphorylation on the Ca2+influx evoked by thapsigargin in these cells. These observations point to a different mechanism of action between forskolin and halothane on Ca2+response.
Effect of Halothane and Forskolin on Platelet Aggregation
Because cAMP is recognized as a potent platelet aggregation inhibitor, [7,14]experiments were done to test the hypothesis that the effect of halothane on platelet aggregation [2–4]was mediated, at least in part, by cAMP. The results in Figure 6A and Figure 6B do not, however, support this hypothesis. For instance, 1.6 mM halothane completely inhibited the aggregation induced by U46619, an effect that could be mimicked by 1 [micro sign]M forskolin (Figure 6A). The results in Figure 6B indicate, however, that, in contrast to forskolin at 1 [micro sign]M, halothane did not inhibit platelet aggregation initiated with thapsigargin, even when it was used at a supraclinical concentration of 1.6 mM. A model in which the action of halothane would be mediated exclusively by an increased cAMP production should have yielded identical results in both cases. Thus our observations correspond with the findings reported by Kohro and Yamakage and do not support a cAMP-based effect of halothane on platelet aggregation (Figure 6A).
The mechanism underlying the action of halothane remains, however, ill defined. It is clear from our observations that cytosolic Ca2+seems to play a key role in the effect of halothane on platelet aggregation. In fact, we observed a strong correlation between the ability of halothane to affect the Ca2+response in stimulated platelets and its potency to impair platelet aggregation. For instance, halothane had no effect on Ca2+mobilization and aggregation triggered by thapsigargin. In contrast, halothane significantly inhibited the platelet aggregation process in response to U46619 stimulation (Figure 6A), in agreement with the potent inhibitory action of halothane on the Ca2+response induced by this agonist (Figure 1A and Figure 1C). A similar relation between Ca2+signaling and platelet aggregation can also be inferred from the results obtained from forskolin-treated platelets. Forskolin impaired the increase in intracellular Ca2+triggered by both thapsigargin and U46619 (Figure 1A and Figure 1D), supporting the platelet aggregation measurements presented in Figure 6A and Figure 6B. It is also apparent from these observations that the mechanisms underlying the action of halothane and forskolin on Ca2+signaling and aggregation differ. In the case of halothane, our results point to a requirement for a phospholipase C-related second messenger formation for an effect on internal Ca2+and platelet aggregation, as reported by Kohro and Yamakage. Such a requirement was not observed in thapsigargin-stimulated platelets exposed to forskolin. In addition, our results showed that halothane does not affect the events leading to platelet aggregation downstream from the release of Ca2+from internal pools and the initiation of a Ca2+influx. Even when halothane was used at high concentrations (1.6 mM) and caused an inhibition of the Ca (2+) response of platelets to Ca2+mobilizing agonists, there was no effect of halothane on the thapsigargin-induced platelet aggregation, despite an increase of Ca2+in this case. Thus halothane is likely to affect events before the Ca2+release process. For instance, halothane may alter phospholipase C activation and decrease the production of both InsP3and diacylglycerol. This would in turn lead to a reduced InsP3-mediatedCa2+release while providing a mechanism whereby the effect of halothane on aggregation is mediated by a decreased protein kinase C (PKC) activity. In fact, PKC activation by phorbol esters has been shown to induce platelet aggregation through a Ca2+-independentprocess. [26,27]It is thus possible that part of the action of halothane on platelet aggregation may be the result of an inhibition of the PKC-activation pathway. In support of this model, evidence has been provided for an inhibitory action of halothane on phospholipase C activation triggered by adenosine diphosphate and isoproterenol in erythrocyte membranes. 
Our study reveals no clear evidence that the impaired Ca2+release and aggregation in the presence of halothane is mediated by an increase in cAMP production. The action of halothane on Ca2+signaling and platelet aggregation appeared to be closely linked to the production of phospholipase C-related second messengers. In contrast, forskolin can directly inhibit the capacitative Ca2+influx and platelet aggregation induced by thapsigargin. These results indicate that the increase in cytosolic Ca2+is directly responsible for the aggregation in aspirin-treated blood platelets activated by thapsigargin. Halothane cannot, however, impair the platelet aggregation process if the Ca2+increase results from a phospholipase C-independent mechanism.
The authors thank Line Garneau and Dr. Lucie Parent for their critical reading of the manuscript.