Although halothane directly inhibits platelet aggregation, the mechanisms of this effect are still unknown. The current study aimed to clarify the inhibitory mechanisms of halothane on thrombin-induced human platelet aggregation by measuring (1) platelet-surface glycoprotein Ib expression, (2) the concentration of intracellular free Ca2+ ([Ca2+]i) measured simultaneously with aggregation, (3) the concentration of intracellular inositol 1,4,5-triphosphate, and (4) the concentration of intracellular cyclic 3',5'-adenosine monophosphate ([cAMP]i).


Washed platelet suspensions, obtained from healthy volunteers, were preincubated with halothane (0-2 mM) for 2 min and then exposed to 0.02 units/ml thrombin for 3 min. The glycoprotein Ib bound to fluorescein-labeled antibody was measured by fluorescence flow cytometry. [Ca2+]i was measured, simultaneously with aggregation, in Fura-2 (Ca2+ indicator)-loaded platelets by use of a fluorometer. Inositol 1,4,5-triphosphate and [cAMP]i were measured by radioimmunoassay.


Halothane had no effect on glycoprotein Ib expression with or without thrombin. Halothane decreased the thrombin stimulated [Ca2+]i transient and inhibited platelet aggregation in a dose-dependent manner, both in the presence and in the absence of external Ca2+. Isoflurane had no apparent effect on either platelet aggregation or [Ca2+]i in the absence of external Ca2+. Halothane inhibited the increase in inositol 1,4,5-triphosphate induced by thrombin. Halothane moderately but significantly increased [cAMP]i, but the adenylate cyclase activator forskolin (which has the same inhibitory ability on aggregation as halothane) increased [cAMP]i to a much greater extent than did halothane.


Halothane inhibits thrombin-induced human platelet aggregation by decreasing [Ca2+]i without inhibiting agonist-receptor binding; the inhibitory effect of halothane on [Ca2+]i might be mediated by a decrease in inositol 1,4,5 triphosphate and in part by an increase in [cAMP]i.

Key words: Anesthetics, volatile: halothane. Blood, platelet: human. Ions: calcium. Receptors: glycoprotein Ib. Second messenger: cyclic 3',5'-adenosine monophosphate; inositol 1,4,5-triphosphate.

HALOTHANE, in concentrations used clinically, directly inhibits platelet aggregation in vivo [1,2]and in vitro. [1,3,4]However, because the aggregation pathways are complex and the platelet function per se is easily activated by many factors, the mechanism of this effect of halothane is still far from clear. [5]Walter et al. [4]demonstrated that halothane increased platelet adenylate cyclase activity with the inhibition of aggregation and concluded that the direct inhibitory effect of halothane could be mediated in part by a resultant increase in the concentration of intracellular cyclic 3',5'-adenosine monophosphate ([cAMP]i). Cyclic 3',5'-adenosine monophosphate inhibits platelet aggregation and this inhibitory effect is thought to be mediated through two pathways: (1) a decrease in the concentration of intracellular free Calcium2+ ([Calcium2+]i), [6,7]and (2) an inhibition of myosin light chain kinase activity by the activation of cAMP-dependent protein kinase [8](Figure 1).

As in other tissues, platelet processes are regulated by Calcium2+ (Figure 1). [9]Increasing the free Calcium2+ concentration in its cytoplasm is a necessary and sufficient event in the cell's activation that involves a shape change and exocytotic processes leading to aggregation. [10,11]Conversely, many aggregating agonists (e.g., thrombin) increase the concentration of intracellular inositol 1,4,5-triphosphate ([IP3]i) liberated from phosphatidylinositol 4,5-bisphosphate [12,13](Figure 1) and intracellular IP3is a major second messenger to regulate agonist-induced cytosolic Calcium2+ responses in platelets through release of Calcium2+ from dense tubular systems (DTS). [14,15]There is some evidence in other tissues that halothane regulates the concentration of this second messenger at clinically used concentrations. [16,17]Furthermore, it has been reported that halothane reduces the ligand-binding affinity of the platelet-surface thromboxane A2receptor. [18]Thus, to study the inhibitory mechanisms of halothane on platelet aggregation, it is important to know the effect of halothane on the concentrations of essential intracellular second messengers and its effect on the ligand-binding affinity of aggregating agonists.

The current study was designed to clarify the inhibitory mechanisms of halothane on thrombin-induced human platelet aggregation by: (1) measuring the glycoprotein Ib (GpIb) expression on the platelet surface to investigate the possible interruption of the binding of thrombin to the receptor by halothane, (2) measuring [Calcium2+]isimultaneously with platelet aggregation during exposure to thrombin and halothane, (3) measuring [IP3]iduring exposure to thrombin and halothane, and (4) measuring [cAMP]iin the presence of halothane or forskolin.

Preparation of Platelet-rich Plasma and Washed Platelet Suspension

This study was approved by the Sapporo Medical University Ethical Committee on Human Research. After informed consent was obtained, blood (approximately 15 ml) was drawn by antecubital venipuncture from 80 healthy volunteers who had not taken any medications for at least 14 days before the donation. The mean age of the volunteers was 26.8 yr (range 22-30 yr). Blood was collected into plastic syringes containing acid-citrate-dextrose solution. The acid-citrate-dextrose solution contained 85 mM sodium citrate, 70 mM citric acid, and 110 mM glucose. The ratio of blood to acid-citrate-dextrose solution was 17:3, and the final concentration of citrate in the whole blood was approximately 13 mM. Processing of blood samples was begun within 20 min of venipuncture.

Platelet-rich plasma and washed platelet suspension (WPS) were prepared by a modification of previously published methods. [19,20]Briefly, the whole blood was centrifuged at 150g for 10 min and the upper two thirds of the platelet-rich plasma was collected. The WPS was prepared by centrifuging the platelet-rich plasma at 600g for 10 min and resuspending the pellet in a modified Tyrode's solution. The Tyrode's solution contained (in mM) 140 NaCl, 2.7 KCl, 0.23 MgCl2, 0.4 NaH2PO4, 12 NaHCO3, 5 glucose, and 10 N-(2-hydroxy-ethyl)piperazine-N'-2-ethanesulfonic acid; pH level was adjusted to 7.4 with NaOH. Platelets were counted by a Coulter Counter STKS (Coulter, Hialeah, FL) for adjustment of the concentrations of platelet-rich plasma and WPS. All procedures were performed at room temperature (22-24 degrees C). The experiments were started after preincubation with or without 1 mM Calcium2+ for 1 min at 37 degrees Celsius and were completed within 3 h after venipuncture. Siliconized glassware and plastic tools were used throughout.

Measurement of the Platelet-surface Glycoprotein Ib Expression

To investigate the interruption of thrombin-receptor binding by halothane, we measured platelet-surface nonbinding GpIb, a part of the thrombin receptor, [21]by fluorescence flow cytometry. [22]After the preincubation with 1 mM Calcium2+ for 1 min, samples of WPS were incubated with halothane-containing solution (final concentration: 0, 0.5, 1.0, 1.5, or 2.0 mM) for 2 min and were then stimulated with or without 0.02 units/ml thrombin for 3 min at 37 degrees C. Platelet preparations were stirred at 1,000 rpm during this protocol. Each incubation was stopped by the addition of an equal volume of 1% (vol/vol) paraformaldehyde. After the fixation, the paraformaldehyde-treated platelets were washed twice by centrifugation (600g for 5 min) and resuspension in phosphate-buffered saline (composition in mM: 120 NaCl, 2.7 KCl, 8 Na2HPO4, and 2 KH2PO4; pH 7.4). The fixed platelets were then incubated with an isothiocyanate fluorescein-labeled mouse monoclonal immune globulin G antibody (SZ-2-FITC, 2 micro gram/5 x 106platelets) for 30 min at room temperature, washed twice, and resuspended in phosphate-buffered saline at a final concentration of 5 x 107platelets/milliliter. The GpIb antibody used in this study (SZ-2) binds specifically to the human platelet GpIb alpha-subunit with mean K sub d = 6.6 x 10 sup -10 M. [23]The labeled platelets were analyzed with an argon ion laser fluorescence flow cytometer (FACScan 440; Becton Dickinson, Mountain View, CA) using a 488-nm wavelength at 300 mW. Fluorescence was detected through a 530+/-15 nm band-pass filter. The fluorescence histograms were analyzed on an attached computer (Consort 30; Becton Dickinson). The determinations were made in duplicate of the channel-weighted mean fluorescence intensity for 104platelets, and the results are expressed as the fluorescein intensity ratio to the control without thrombin or halothane.

Measurements of Intracellular Free Calcium sup 2+ Concentration and Aggregation

The method of Pollock and Rink [24]was followed. Platelet-rich plasma was incubated with Fura-2/AM (5 micro Meter) for 30 min at room temperature. After the Fura-2 loading, WPS was obtained as described earlier. Approximately 1 ml of the WPS (approximately 1.5 x 107platelets per cuvette) was added to the stirred (1,000 rpm) cuvette attachment of a fluorometer (CAF-110; JASCO, Tokyo, Japan). After the WPS was preincubated with 1 mM Calcium2+ or 50 micro Meter EGTA for 1 min, the samples were incubated with various concentrations of halothane-containing solution (range of final concentration: 0-2.0 mM) for 2 min and then stimulated with 0.02 units/ml thrombin for 3 min. To further investigate the effect of other anesthetics on the increase of [Calcium2+]idue to Calcium2+ release from intracellular stores (DTS), we performed additional experiments using isoflurane (range 0-2.0 mM) in the absence of external Calcium2+ as well.

The changes of [Calcium2+]i, indicated by Fura-2 fluorescence, and of platelet aggregation were simultaneously measured with the CAF110 fluorometer. The platelet samples were illuminated alternately (128 Hz) at the excitation wavelengths of 340 and 380 nm. The intensities of 500-nm fluorescence induced by 340-nm excitation and by 380-nm excitation were monitored. At the end of each experiment, the cells were treated with 0.1% (vol/vol) Triton X-100 followed by the addition of 10 mM ethylene glycol-bis-(beta-amino ethylether)-N, N, N'-tetraacetic acid (EGTA) to obtain the maximum and minimum fluorescence, respectively. Absolute values of [Calcium2+]iwere determined by the formula of Grynkiewicz et al. [25]using a Fura-2-Calcium2+ dissociation constant of 224 nM. The background fluorescence, measured by adding 1 mM MnCl2, was negligible. Platelet aggregation was assessed using a spectrophotometer adjusted to 0% transmittance for control WPS and to 100% transmittance for distilled water.

Measurement of Intracellular Inositol 1,4,5-triphosphate Concentration

The technique of Uemura et al. [26]was used to measure the intracellular IP3concentration ([IP3]i). After preincubation with 1 mM Calcium2+ for 1 min, WPS (0.5 ml, 1 x 109platelets/ml) was first incubated with halothane-containing solution (final concentration: 0, 1, or 2 mM) for 2 min and then stimulated with thrombin (0.02 units/ml). The reactions were terminated after 0, 5, 10, 15, 30, 60, or 120 s of thrombin stimulation by the addition of 0.2 volume of ice-cold 20% (vol/vol) perchloric acid solution. The samples were kept in an ice bath for 20 min and were then centrifuged at 2,000g for 15 min to remove insoluble materials. The pH level of the supernatant was adjusted precisely to 7.5 with 10 N KOH and insoluble precipitates (primarily KClO4) were removed by centrifugation at 2,000g for 10 min. The resultant supernatant was lyophilized and stored at -20 degrees Celsius. The lyophilized samples were dissolved in 100 micro liter distilled water, and the amount of IP3was measured using the Amersham IP3assay system (code TRK 1000; Amersham Japan, Tokyo, Japan). This assay is based on competition between unlabeled IP3in the sample and a fixed quantity of tritium-labeled IP3for a limited number of high affinity binding sites on a specific IP3binding protein. [27]The determinations were made in duplicate and the results are expressed as pmol/5 x 108platelets.

Measurement of Intracellular Cyclic 3',5'-adenosine Monophosphate Concentration

Washed platelet suspension (1 ml, 5 x 108platelets/milliliter) was incubated with 1 mM Calcium2+ for 1 min. Intracellular cAMP concentrations ([cAMP]i) were measured under four conditions 6 min after addition of Calcium2+: without exposure to halothane or thrombin; or with 5 min exposure to halothane (0, 1, and 2 mM, respectively) with additional exposure to 0.02 units/ml thrombin for the final 3 min. In experiments involving addition of forskolin, a potent adenylate cyclase activator, instead of halothane, concentrations of this drug were used that produced the same effect as halothane (1 and 2 mM) on the inhibition of the platelet aggregation. Measurement of the [cAMP]iof platelets was done by a modification of previously published methods. [28]At the end of the incubations, the reactions were stopped by the addition of an equal volume of ice-cold 0.1 N HCl solution and the suspensions were immediately homogenized for 60 s at 4 degrees C with a VC-50 ultrasonic homogenizer (Sonic and Material, Danbury, CT). The homogenized samples were then centrifuged at 3,000g for 15 min at 4 degrees C and 100 micro liter of the supernatant was used for the measurement of the [cAMP]i. We analyzed [cAMP]iby a sensitive radioimmunoassay method (Yamasa cyclic adenosine monophosphate assay system; Yamasa, Chiba, Japan). The determinations were made in duplicate and the results are expressed as pmol/108platelets.

Determination of Anesthetic Concentration

Because of the possibility that halothane or isoflurane bubbling per se affects platelet aggregation and its measurement, we introduced the anesthetic into the cuvette using a 50-micro liter microsyringe (MS-50P4AF; Ver Werk, Ilmenau, Germany) to deliver preconcentrated anesthetic in the Tyrode's solution. To prevent anesthetic volatilization from WPS in the cuvette, we sealed the top of the cuvette with mineral oil and circulated the expected concentration of the anesthetic over the cuvette (approximately 1 l/min). Anesthetic concentrations in the cuvette were analyzed in each experiment using a gas chromatograph (GC-12A; Shimadzu, Kyoto, Japan) equipped with a flame ionization detector (FTD-8; Shimadzu) and an integrator (Chromatopac C-R 3A; Shimadzu). Both halothane and isoflurane concentrations in the cuvette were constant during the experiment for more than 5 min (data not shown). In addition, we measured the halothane and isoflurane concentrations in WPS, which were bubbled extensively with known gas concentrations of the anesthetic, determined with a calibrated infrared anesthetic gas monitor (5250 RGM; Ohmeda, Madison, WI). After equilibration, the mean halothane concentrations in the solutions (1.0, 2.0, 3.0, and 4.0% halothane in the gas phase) were 0.8, 1.8, 2.5, and 3.5 mM, respectively; whereas the mean isoflurane concentrations in the solutions (2.0 and 4.0% isoflurane in the gas phase) were 1.2 and 2.2 mM, respectively. The concentration of the anesthetics in the solution had close linear correlation with the concentration of the agent in the gas phase.


The following drugs and chemicals were used in this study: halothane (ICI, Dighton, MA); isoflurane (Abbott Laboratories, North Chicago, IL); acetoxymethyl ester of Fura-2 (Fura-2/AM; Dojindo, Kumamoto, Japan); Triton X-100 and sodium citrate (Katayama Chemical, Tokyo, Japan); citric acid (Kanto Chemical, Tokyo, Japan); EGTA, thrombin, paraformaldehyde, Triton X-100 and mineral oil (Sigma Chemical, St. Louis, MO); isothiocyanate fluorescein-labeled mouse monoclonal IgG antibody (SZ-2-FITC; Immunotech, Marseilles, France) and phosphate-buffered saline (Life Technologies, Grand Island, NY).

Statistical Analysis

Data are expressed in scatter diagrams or as means+/-SEM. The effects of halothane on the aggregation or on the [Calcium2+]iof platelets were assessed by analysis of the variance of regression coefficients. Comparisons for all of the other data were analyzed with unpaired, two-tailed t test or one-factor analysis of variance with Fisher's a posteriori test. In all comparisons, P < 0.05 was considered significant.

Effect of Halothane on Platelet-surface Glycoprotein Ib Expression

We measured the level of thrombin-receptor binding indirectly by estimating the presence of platelet-surface nonbinding GpIb with a fluorescence technique. The control fluorescence intensity of isothiocyanate-labeled platelets without thrombin or halothane was 51.7 +/-4.8/104platelets (n = 7). Table 1shows the effects of thrombin and halothane on the GpIb expression. Halothane (0-2 mM) did not alter the binding between GpIb and the fluorescein-labeled antibody SZ-2-FITC in the absence of thrombin (P = 0.6). Thrombin (0.02 units/ml) significantly decreased labeled nonbinding GpIb by 25+/-4% (P < 0.01). Halothane did not interfere with the binding between GpIb and thrombin in the range 0-2 mM (P = 0.7), but at a high concentration (5.5 mM), the anesthetic exhibited a small (21+/-5%) but significant inhibitory effect on the GpIb-thrombin binding (P < 0.05, data not shown in Table 1).

Effects of Halothane and Isoflurane on Intracellular Free Calcium sup 2+ Concentration and Aggregation

(Figure 2) shows the effects of halothane on [Calcium2+]iand on the aggregation of thrombin-stimulated human platelets in the presence of 1 mM extracellular Calcium2+. The resting [Calcium2+]iwas 106+/-6 nM (n = 5), and halothane did not change [Calcium2+]iwhen tested at concentrations up to 2 mM. [Calcium sup 2+]iwas rapidly increased by 0.02 units/ml thrombin with a biphasic response: a first peak was seen at approximately 15 s and a second at approximately 2 min after exposure to thrombin. The [Calcium2+]iat the first and second peaks were 520+/-18 and 453 +/-22 nM, respectively (n = 5). The thrombin-stimulated platelets were rapidly aggregated, reaching a peak (approximately 60% aggregating ratio) at approximately 3 min. We show representative effects of halothane at 0.9 and 1.8 mM on the platelet aggregation and [Calcium2+]iin Figure 2. In this case, both peaks of [Calcium2+]iinduced by thrombin were substantially decreased by halothane, but the time courses of changes in [Calcium2+]iand of aggregation did not appear to change.

The relationships between halothane concentration (mM) and changes in (A) percentage of aggregation at 3 min after exposure to thrombin and (B) [Calcium2+]iof the first peak in the presence of 1 mM external Calcium2+ are shown in Figure 3. Halothane (0-2 mM) significantly inhibited aggregation and caused a dose-dependent decrease in [Calcium2+]i. Linear relationships were observed both between halothane concentrations and percent aggregation (r = -0.94, n = 50, P < 0.01) and between halothane concentrations and [Calcium2+]i(r = -0.92, n = 50, P < 0.01).

We also investigated the effects of halothane and isoflurane on [Calcium2+]iand the aggregation of thrombin-stimulated human platelets in the absence of external Calcium2+ (solution containing 50 micro Meter EGTA instead). As shown in Figure 4(A), the resting [Calcium2+]iwas 73+/-5 nM (n = 5) and was significantly less than that obtained in the presence of 1 mM external Calcium2+ (P < 0.05). Halothane did not change the resting [Calcium sup 2+]iin the concentration range of 0-2 mM. As observed in the presence of external Calcium2+, [Calcium2+]iwas rapidly increased by exposure to 0.02 units/ml thrombin. However, in contrast to the observation with external Calcium2+, this initial increase was followed by a substantial reduction and there was no second peak. The time course of the initial change in [Calcium2+]iwas similar to that seen in the presence of 1 mM external Calcium2+ with a peak occurring approximately 15 s after exposure to thrombin. However, the peak [Calcium sup 2+]i(293+/-14 nM) was significantly less than that of the first peak obtained with extracellular Calcium2+ (P < 0.01, n = 5). Platelets smoothly aggregated, reaching their respective peaks (approximately 30% aggregating ratio) at approximately 3 min. Halothane (1.0 and 2.0 mM) decreased the induced changes in [Calcium2+]iand inhibited aggregation in the absence of external Calcium2+. To determine whether these effects were induced by other anesthetics as well, we performed additional experiments using isoflurane in the absence of external Calcium2+ (Figure 4(B)). Isoflurane at concentrations up to approximately 2 mM (= approximately 3.6% in gas phase) had no apparent effect on either platelet aggregation (inhibited by approximately 5 +/-5% at 1 mM and approximately 8+/-6% at 2 mM, respectively) or the increase in [Calcium2+]i(inhibited by approximately 3+/-5% at 1 mM and approximately 6+/-8% at 2 mM, respectively) induced by thrombin (n = 5 at each point).

(Figure 5) shows the relationships obtained in the absence of external Calcium2+ between halothane concentration and changes in (A) percentage of aggregation at 3 min and (B) peak [Calcium2+]i. Halothane (0-2 mM) significantly inhibited aggregation and caused a dose-dependent decrease of [Calcium2+]i. Linear relationships were observed both between halothane concentrations and percent aggregation (r = -0.95, n = 40, P < 0.01) and between halothane concentrations and [Calcium2+]i(r = -0.93, n = 40, P < 0.01). Halothane, at approximately 2 mM, almost completely inhibited thrombin-induced platelet aggregation and decreased the [Calcium2+] sub i to the resting value.

Effect of Halothane on Intracellular IP sub 3 Concentration

The time course and effects of 2 mM halothane on intracellular IP3concentrations ([IP3]i) in thrombin-stimulated human platelets are shown in Figure 6. The [IP3]iat time 0 was 1.4+/- 0.2 pmol/5 x 108platelets (n = 6) and did not change with the addition of halothane (1.3+/-0.3 pmol/5 x 108platelets at 1 mM and 1.2+/-0.2 pmol/5 x 108platelets at 2 mM halothane). Thrombin (0.02 units/ml) produced a rapid increase in the [IP sub 3]i, reaching its maximum (5.9+/-0.9 pmol/5 x 108platelets) at 10 s after the stimulation. The rapid increase in [IP3] sub i induced by thrombin was followed by a rapid and substantial decrease to a concentration of approximately 2 pmol/5 x 108platelets. Halothane (2 mM) significantly inhibited the increase of [IP3]iinduced by thrombin at 5-15 s after thrombin stimulation without an apparent change in the time course of [IP3]i. The inset of Figure 6summarizes the effects of various concentrations of halothane (0, 1, and 2 mM) on the peak [IP3]iat 10 s after thrombin stimulation. Halothane significantly inhibited in a dose-dependent manner the increase in [IP3]iinduced by thrombin.

Effect of Halothane on Intracellular Cyclic 3',5'-adenosine Monophosphate Concentration

The effect of various concentrations of forskolin on thrombin (0.02 units/ml)-induced platelet aggregation and on intracellular cAMP concentrations ([cAMP]i) were compared with those of halothane. As shown in Figure 3(A), halothane at 1 and 2 mM decreased thrombin-induced platelet aggregation to approximately 36% and approximately 4% of control, respectively. Forskolin at 10 and 21 micro Meter concentrations decreased thrombin (0.02 units/ml)-induced platelet aggregation to 35+/-4% and to 6+/-3%, respectively (n = 5). There was no significant difference between halothane and forskolin with respect to the extent of inhibition of platelet aggregation.

The effects of halothane and forskolin at these concentrations on [cAMP]iof thrombin-stimulated human platelets are shown in Figure 7. The [cAMP]iin the resting state without thrombin was 1.6 +/-0.2 pmol/108platelets (n = 6). After stimulation with 0.02 units/ml thrombin, [cAMP]itended to decrease to 1.4+/- 0.1 pmol/108platelets, but this change was not significant. Halothane at 1 and 2 mM moderately but significantly increased [cAMP]iby approximately 20% and approximately 40%, respectively. In contrast, forskolin at 10 and 21 micro Meter caused significantly greater increases in [cAMP]i(P < 0.05, n = 6) than did halothane at concentrations equieffective for inhibition of aggregation.

Ligand-receptor Interaction

Physiologic platelet activation or inhibition involves interaction of an extracellular signaling molecule with the platelet surface via ligand-receptor coupling. Based on the interactions of thrombin with the glycocalicin component of GpIb, [29]GpIb is proposed to be a functionally significant thrombin receptor (Figure 1). [21]As reported previously, [30]we found using flow cytometry with fluorescent labeling of the platelet surface proteins [22]that thrombin (0.02 units/ml) significantly reduced the amount of antibody binding to GpIb. Halothane did not affect the thrombin-induced decrease in antibody labeling of GpIb, suggesting that halothane within the range of 0-2 mM (0-2.4% in gas phase) does not interfere with the interaction of thrombin with its receptor. This is in agreement with the general observation that volatile anesthetics, at clinically used concentrations, have little or no effect on agonist binding. [31]Although Hirakata et al. [18]demonstrated that halothane reduced the receptor-binding affinity of a potent aggregating agonist thromboxane A2in human platelets, this effect likely required the extremely high concentration of halothane (14 mM [nearly equal] to 16% in gas phase) that they used.

Effects of Halothane on Activating Second Messengers: Calcium sup 2+ and Inositol 1,4,5-triphosphate

As is established in other tissues, Calcium2+ is thought to be an important regulator of platelet activation (Figure 1). [9,32]Results from the current study also support a central role for Calcium2+ in platelet aggregation (Figure 2and Figure 4). Our results are similar to those observed by others with platelet stimulation by thrombin, [19,33]collagen, [33]or adenosine diphosphate. [34]An increase in [Calcium2+]iactivates myosin light chain kinase, which initiates platelet aggregation through increased phosphorylation of myosin light chain. [10,34]In platelets, the increase in [Calcium2+]iinvolves the release of Calcium2+ from intracellular stores, especially DTS, and later Calcium2+ influx from the extracellular space. [35]As shown in Figure 2, [Calcium2+]iinduced by aggregating agonists (e.g., thrombin) usually induces a biphasic response. The first peak of [Calcium2+]iis thought to result mainly from Calcium2+ release from DTS and the second peak of [Calcium2+] sub i is thought to reflect Calcium2+ influx from the extracellular space. [24,36]Our measurement of [Calcium2+]iwith or without external Calcium2+ support this model of Calcium2+ regulation and its role as a regulator of platelet activation.

Halothane decreased [Calcium2+]iin parallel with its inhibition of platelet aggregation in the presence of external Calcium2+ (Figure 2and Figure 3). Our data support the hypothesis that halothane inhibits platelet aggregation mainly by suppressing the increase of [Calcium2+]iinduced by aggregating agonists. Halothane also decreased the peak [Calcium2+]iin the absence of external Calcium2+. There is some evidence that Calcium2+ release from intracellular stores may be more essential for platelet activation than is Calcium2+ influx from the extracellular space. [20,24,36]Therefore, it is possible that the inhibition of the increase in [Calcium sup 2+]idue to intracellular stores is the main mechanism by which halothane inhibits platelet aggregation. Interestingly, isoflurane had little effect on the [Calcium2+]iin the absence of external Calcium2+. This observation parallels the clinical observation that halothane is more effective than other anesthetics at inhibiting platelet aggregation at clinically used concentrations. [5].

Hossain and Evers [37]reported that in clonal (GH3) pituitary cells halothane increased the resting [Calcium2+]ithrough Calcium2+ release from IP3-gated intracellular stores. Although we did not observe any increase in [Calcium2+]iby halothane in the absence of external Calcium2+, our data do not exclude the possibility that the decrease in thrombin-induced increase of [Calcium2+]iby halothane results partly from the depletion of the Calcium2+ stores by preincubation with the anesthetic. [38,39]Our data do indicate that halothane inhibits Calcium2+ influx through the platelet membrane, but the role of Calcium2+ influx from the extracellular space in the aggregation response of platelets to activation remains uncertain. [40]Further studies are required to clarify the significance of this effect.

Stimulation of the thrombin receptor activates the G protein-linked phospholipase C, resulting in hydrolysis of phosphatidylinositol 4,5-bisphosphate to the two potent stimulatory second messengers IP3and 1,2-diacylglycerol (Figure 1). [12,13,41]While 1,2-diacylglycerol activates Calcium2+ /phospholipid-dependent protein kinase at the membrane, [41]IP3mobilizes Calcium2+ from intracellular organella DTS. [14,15]Because, in platelets, IP3is the primary regulator for Calcium2+ release from intracellular stores and because the time course of the increase in [IP3]iinduced by thrombin was very similar to that of the change in [Calcium2+]i(Figure 2and Figure 4), we suggest that IP3is an important determinant of [Calcium2+]iduring agonist stimulation. Furthermore, there is evidence that IP3can directly enhance Calcium2+ influx from the extracellular space. [26]Therefore, inhibition of increases in [IP3]iby halothane might in itself account for the observed effects on both [Calcium2+]iand aggregation.

Our results are in general agreement with studies in a variety of cell types, in which halothane treatment has been associated with inhibition of the increase in [Calcium2+]imediated by IP3. [16,42,43]These studies have demonstrated that halothane alters Calcium2+ homeostasis, an action that underlies the in vivo effect of the anesthetic. However, Smart et al. [17]and Rooney et al. [44]showed that halothane induced IP3formation in SH-SY5Y human neuroblastoma cells and turkey erythrocytes, respectively. Rooney and colleagues also showed activation of phospholipase C activity by halothane. [44]These apparent discrepancies may result from the differences of cell types and species and/or the selective effects of halothane on certain receptors, G-proteins, or phospholipase C isozymes.

Effects of Halothane on Inhibitory Second Messengers: Cyclic 3',5'-adenosine Monophosphate

Cyclic 3',5'-adenosine monophosphate is another mechanism known to regulate [Calcium2+]i. In this study, halothane moderately but significantly increased the [cAMP]iof human platelets. This result is consistent with the previous suggestion by Walter et al. [4]that the impairment of platelet aggregation observed with halothane incubation might result from halothane-induced activation of platelet adenylate cyclase, resulting in a higher cAMP concentration in the cytosol. Several effects of cAMP on platelet Calcium sup + metabolism have been established, [45,46]including stimulation of Calcium2+ efflux [6]and stimulation of Calcium2+ uptake into DTS, [7]both of which result in a decrease in [Calcium2+]i. In addition, cAMP-dependent protein phosphorylation has been reported to attenuate IP sub 3 -mediated Calcium2+ release from DTS. [47]However, more recent data obtained with a pure preparation of the catalytic subunit of protein kinase seem to contradict this evidence. [48]Thus, the molecular mechanism by which cAMP regulates IP3-mediated Calcium2+ responses in intact platelets is far from fully established. Primarily as a consequence of its effect on [Calcium2+]i, cAMP influences certain other platelet responses, including phosphorylation of myosin light chain kinase [10]: cAMP-dependent protein kinase phosphorylates myosin light chain kinase and inhibits its activity in vitro [49]via a mechanism independent of Calcium2+ (Figure 1).

In this study, forskolin was used to investigate the role of cAMP in the inhibition by halothane of platelet aggregation. When forskolin was used at concentrations that inhibited platelet aggregation to the same extent as halothane (1 and 2 mM), the [cAMP]iincreased to a much greater extent than with halothane (Figure 7). Thus, the inhibitory effect of halothane on platelet aggregation appears to involve additional mechanisms other than an increase of [cAMP]i.

In summary, halothane in clinical concentrations inhibits thrombin-induced human platelet aggregation mainly by decreasing [Calcium sup 2+]iwithout inhibiting agonist-receptor binding. The inhibitory effect of halothane on [Calcium2+]imay be mediated both by a decrease in [IP3]iand by an increase in [cAMP]i. We suggest that, while cAMP both suppresses [Calcium2+]iand acts via a Calcium2+ -independent pathway to inhibit platelet aggregation, IP3is a crucial regulator of aggregation in platelets and that the inhibitory action of halothane on aggregation is explicable by its effects on IP3.

The authors thank A. Namiki, M.D., Ph.D., Professor and Chairman of Anesthesiology, Sapporo Medical University School of Medicine, for directing the research and reviewing the manuscript; H. Kanoh, M.D., Ph.D., Sapporo Medical University, K. Matsuno, M.D., Ph.D., Hokkaido University, T. L. Croxton, Ph.D., M.D., T. S. Kickler, M.D., and P. A. Stephens, Ph.D., The Johns Hopkins Medical Institutions, for comments and suggestions concerning this manuscript; and T. Mitani, Ph.D., Hokkaido Red Cross Blood Center, for technical assistance.

Dalsgaard-Nielsen J, Risbo A, Simmelkjaer P, Gormsen J: Impaired platelet aggregation and increased bleeding time during general anaesthesia with halothane. Br J Anaesth 1981; 53:1039-42.
Fyman PN, Triner L, Schranz H, Hartung J, Casthely PA, Abrams LM, Keaney AE, Cottrell JE: Effect of volatile anaesthetics and nitrous oxide-fentanyl anaesthesia on bleeding time. Br J Anaesth 1984; 56:1197-200.
Ueda I: The effects of volatile general anesthetics on adenosine diphosphate-induced platelet aggregation. ANESTHESIOLOGY 1971; 34:405-8.
Walter F, Vulliemoz Y, Verosky M, Triner L: Effects of halothane on the cyclic 3',5'-adenosine monophosphate enzyme system in human platelets. Anesth Analg 1980; 59:856-61.
Gibbs NM: The effect of anaesthetic agents on platelet function. Anaesth Intensive Care 1991; 19:495-520.
Johansson JS, Nied LE, Haynes DH: Cyclic AMP stimulates Calcium sup 2+ -ATPase-mediated Calcium sup 2+ extrusion from human platelets. Biochim Biophys Acta 1992; 1105:19-28.
Kaser-Glanzmann R, Jakabova M, George JN, Luscher EF: Stimulation of calcium uptake in platelet membrane vesicles by adenosine 3',5'-cyclic monophosphate and protein kinase. Biochim Biophys Acta 1977; 466:429-40.
Feinstein MB, Egan JJ, Opas EE: Reversal of thrombin-induced myosin phosphorylation and the assembly of cytoskeletal structures in platelets by the adenylate cyclase stimulants prostaglandin D sub 2 and forskolin. J Biol Chem 1983; 258:1260-7.
Salzman EW, Ware JA: Ionized calcium in blood platelets, Platelet Membrane Receptors: Molecular Biology, Immumology, Biochemistry, and Pathology. Edited by Jamieson GA. New York, Alan R. Liss, 1988, pp 419-30.
Nishikawa M, Tanaka T, Hidaka H: Calcium sup 2+ -calmodulin-dependent phosphorylation and platelet secretion. Nature (Lond) 1980; 287:863-5.
Feinstein MB: Release of intracellular membrane-bound calcium precedes the onset of stimulus-induced exocytosis in platelets. Biochem Biophys Res Commun 1980; 93:593-600.
Billah MM, Lapetina EG: Rapid decrease of phosphatidylinositol 4,5-bisphosphate in thrombin-stimulated platelets. J Biol Chem 1982; 257:12705-8.
Agranoff BW, Murthy P, Seguin EB: Thrombin-induced phosphodiesteratic cleavage of phosphatidylinositol bisphosphate in human platelets. J Biol Chem 1983; 258:2076-8.
O'Rourke FA, Halenda SP, Zavoico GB, Feinstein MB: Inositol 1,4,5-triphosphate releases Calcium sup 2+ from a Calcium sup 2+ -transporting membrane vesicle fraction derived from human platelets. J Biol Chem 1985; 260:956-62.
Brass LF, Joseph SK: A role for inositol triphosphate in intracellular Calcium sup 2+ mobilization and granule secretion in platelets. J Biol Chem 1985; 260:15172-9.
Sill JC, Uhl C, Eskuri S, Dyke RV, Tarara J: Halothane inhibits agonist-induced inositol phosphate and Calcium sup 2+ signaling in A7r5 cultured vascular smooth muscle cells. Mol Pharmacol 1991; 40:1006-13.
Smart D, Smith G, Lambert DG: Halothane and isoflurane enhance basal and carbachol-stimulated inositol(1,4,5)triphosphate formation in SH-SY5Y human neuroblastoma cells. Biochem Pharmacol 1994; 47:939-45.
Hirakata H, Ushikubi F, Narumiya S, Hatano Y, Nakamura K, Mori K: The effect of inhaled anesthetics on the platelet aggregation and the ligand-binding affinity of the platelet thromboxane A sub 2 receptor. Anesth Analg 1995; 81:114-8.
Jy W, Haynes DH: Thrombin-induced calcium movements in platelet activation. Biochim Biophys Acta 1987; 929:88-102.
Matsushima Y, Imai M, Ishimura Y, Ikeda Y. Inhibition by the antilipogenic antibiotic cerulenin of thrombin-induced activation of human platelets. Thromb Res 1988; 49:79-90.
Jandrot-Perrus M, Clemetson KJ, Huisse M-G, Guillin M-C: Thrombin interaction with platelet glycoprotein Ib: Effect of glycocalicin on thrombin specificity. Blood 1992; 80:2781-6.
Adelman B, Michelson AD, Handin RI, Ault KA: Evaluation of platelet glycoprotein Ib by fluorescence flow cytometry. Blood 1985; 66:423-7.
Ruan C, Du X, Xi X, Castaldi PA, Berndt MC: A murine anti-glycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen. Blood 1987; 69:570-7.
Pollock WK, Rink TJ: Thrombin and ionomycin can raise platelet cytosolic Calcium sup 2+ to micromolar levels by discharge of internal Calcium sup 2+ stores: Studies using Fura-2. Biochem Biophys Res Commun 1986; 139:308-14.
Grynkiewicz G, Poenie M, Tsien RY: A new generation of Calcium sup 2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985; 260:3440-50.
Uemura Y, Sakon M, Kambayashi J, Tsujinaka T, Mori T: Involvement of inositol 1,4,5-triphosphate in Calcium sup 2+ influx in thrombin stimulated human platelets. Biochem Int 1989; 18:335-41.
Palmer S, Hughes KT, Lee DY, Wakelam MJ: Development of a novel, Ins(1,4,5)P sub 3 -specific binding assay. Its use to determine the intracellular concentration of Ins(1,4,5)P sub 3 in unstimulated and vasopressin-stimulated rat hepatocytes. Cell Signal 1989; 1:147-56.
Homma M, Satoh T, Takezawa J, Ui M: An ultrasensitive method for the simultaneous determination of cyclic AMP and cyclic GMP in small-volume samples from blood and tissue. Biochem Med 1977; 18:257-73.
Okumura T, Hasitz M, Jamieson GA: Platelet glycocalicin. Interaction with thrombin and role as thrombin receptor of the platelet surface. J Biol Chem 1978; 253:3435-43.
Hourdille P, Heilmann E, Combrie R, Winckler J, Clemetson KJ, Nurden AT: Thrombin induces a rapid redistribution of glycoprotein Ib-IX complexes within the membrane systems of activated human platelets. Blood 1990; 76:1503-13.
Franks NP, Lieb WR: Selective actions of volatile general anaesthetics at molecular and cellular levels. Br J Anaesth 1993; 71:65-76.
Massini P, Kaser-Glanzmann R, Luscher EF: Movement of calcium ions and their role in the activation of platelets. Thromb Haemost 1978; 40:212-8.
Ryo R, Yoshida A, Sugano W, Yasunaga M, Nakayama K, Saigo K, Adachi M, Yamaguchi N, Okuma M: Deficiency of P62, a putative collagen receptor, in platelets from a patient with defective collagen-induced platelet aggregation. Am J Hematol 1992; 39:25-31.
Haslam RJ, Lynham JA: Relationship between phosphorylation of blood platelet proteins and secretion of platelet granule constituents. I. Effects of different aggregating agents. Biochem Biophys Res Commun 1977; 77:714-22.
Hallam TJ, Rink TJ: Insights into platelet function gained with fluorescent Calcium sup 2+ indicators, Platelets in Biology and Pathology III. Edited by MacIntyre DE, Gordon JL. New York, Elsevier, 1987, pp 353-72.
Tsunoda Y, Matsuno K, Tashiro Y: Spatial distribution and temporal change of cytoplasmic free calcium in human platelets. Biochem Biophys Res Commun 1988; 156:1152-9.
Hossain MD, Evers AS: Volatile anesthetic-induced efflux of calcium from IP sub 3 -gated stores in clonal (GH sub 3) pituitary cells. ANESTHESIOLOGY 1994; 80:1379-89.
Blanck TJJ, Peterson CV, Baroody B, Tegazzin V, Lou J: Halothane, enflurane, and isoflurane stimulate calcium leakage from rabbit sarcoplasmic reticulum. ANESTHESIOLOGY 1992; 76:813-21.
Connelly TJ, Coronado R: Activation of the Calcium sup 2+ release channel of cardiac sarcoplasmic reticulum by volatile anesthetics. ANESTHESIOLOGY 1994; 81:459-69.
Kroll MH, Schafer AI: Biochemical mechanisms of platelet activation. Blood 1989; 74:1181-95.
Kawahara Y, Takai Y, Minakuchi R, Sano K, Nishizuka Y: Phospholipid turnover as a possible transmembrane signal for protein phosphorylation during human platelet activation by thrombin. Biochem Biophys Res Commun 1980; 97:309-17.
Puil E, El-Beheiry H, Baimbridge KG: Calcium involvement in anesthetic blockage of synaptic transmission. Ann N Y Acad Sci 1991; 625:82-90.
Loeb AL, Longnecker DE, Williamson JR: Alteration of calcium mobilization in endothelial cells by volatile anesthetics. Biochem Pharmacol 1993; 45:1137-42.
Rooney TA, Hager R, Stubbs CD, Thomas AP: Halothane regulates G-protein-dependent phospholipase C activity in turkey erythrocyte membrane. J Biol Chem 1993; 268:15550-6.
Feinstein MB, Egan JJ, Sha'afi RI, White J: The cytoplasmic concentration of free calcium in platelets is controlled by stimulators of cyclic AMP production (PGD sub 2, PGE sub 1, forskolin). Biochem Biophys Res Commun 1983; 113:598-604.
Zavoico GB, Feinstein MB: Cytoplasmic Calcium sup 2+ in platelets is controlled by cyclic AMP: Antagonism between stimulators and inhibitors of adenylate cyclase. Biochem Biophys Res Commun 1984; 120:579-85.
Enouf J, Giraud F, Bredoux R, Bourdeau N, Levy-Toledano S: Possible role of a cAMP-dependent phosphorylation in the calcium release mediated by inositol 1,4,5-triphosphate in human platelet membrane vesicles. Biochim Biophys Acta 1987; 928:76-82.
O'Rourke F, Zavoico GB, Feinstein MB: Release of Calcium sup 2+ by inositol 1,4,5-triphosphate in platelet membrane vesicles is not dependent on cyclic AMP-dependent protein kinase. Biochem J 1989; 257:715-21.
Hathaway DR, Eaton CR, Adelstein RS: Regulation of human platelet myosin light chain kinase by the catalytic subunit of cyclic AMP-dependent protein kinase. Nature (Lond) 1981; 291:252-4.