Ketamine Suppresses Platelet Aggregation Possibly by Suppressed Inositol Triphosphate Formation and Subsequent Suppression of Cytosolic Calcium Increase

The study was conducted in accordance with the human research standards of the Kyoto University Ethics Committee (Kyoto, Japan). Venous blood was obtained from nine healthy volunteers who had taken no drugs known to affect platelet aggregation for at least 2 weeks. After adding a 10% volume of 3.8% (weight/volume) trisodium citrate, the blood was centrifuged at 160 g  for 10 min to prepare platelet-rich plasma (PRP) or at 1,600 g  for 30 min to prepare platelet-poor plasma (PPP). After 1 h of storage, PRP was used for the aggregation studies and Ca²⁺studies. To prepare washed platelets, a 10% volume of 100 mm EDTA (pH 7.4) was added to PRP, and the mixture was centrifuged at 900 g  for 15 min. The platelet pellet was suspended in buffer A (8 mm Na²HPO⁴, 2 mm NaH²PO⁴, 10 mm EDTA, 5 mm KCl, and 135 mm NaCl [pH 7.2]) and was then recentrifuged at 900 g  for 15 min. Platelet pellets for the binding assay were resuspended in buffer B (25 mm Tris-HCl, 1 mm EGTA, 5 mm MgCl², and 138 mm NaCl [pH 7.5]). The platelet counts were adjusted to 1 × 10⁶/μl. Platelet pellets for IP³assay, Ca²⁺measurement, and aggregation study were resuspended in HEPES buffer (10 mm HEPES, 145 mm NaCl, 5 mm KCl, 0.5 mm Na²HPO⁴, 1 mm CaCl², and 6 mm glucose [pH 7.5]) and were adjusted to 1 × 10⁶/μl, 3 × 10⁵/μl, and 3 × 10⁵/μl, respectively. Washed platelets were stored in Ca²⁺-free buffer at 4°C for 1 h. A few minutes before use, the Ca²⁺concentration of platelet suspension was adjusted to 1 mm by adding CaCl², except for [³H]-labeled TXA²receptor antagonist, 5Z-7-(3-endo-([ring-4-³H] phenyl) sulfonylamino-[2.2.1.] bicyclohept-2-exo-yl) heptenoic acid ([³H]S145) binding assay, and (+)-9,11-epithia-11,12-methano-TXA²(STA²) binding assay.

Aliquots (200 μl) of PRP or platelet suspension in HEPES buffer, with or without ketamine (0.25–2.0 mm), were placed in siliconized glass tubes 2 min before each experiment and were kept at 37°C. PRP was used for STA²-, ADP-, epinephrine-, and A23187-induced aggregation studies. Platelet suspension in HEPES buffer was used for thrombin-induced aggregation study to avoid coagulation. Platelet aggregation induced by STA²(0.5 μm), ADP (0.5 μm), epinephrine (1.0 μm), A23187 (calcium ionophore; 0.5 μm), or thrombin (0.02 U/ml) was measured at 37°C by light transmission using an eight-channel aggregometer (MCM Hematracer 212; MC Medical Inc., Tokyo, Japan; n = 5 each). The light transmission of PPP or HEPES buffer was taken as 100%. The extent of aggregation in steady states (1 min after epinephrine stimulation and 3 min after stimulation with other stimulants) was compared between groups.

After loading of Fura-2/AM, washed platelets were prepared as described. For the study of intracellular free Ca²⁺concentration ([Ca²⁺]ⁱ) increase, Fura-2/AM–loaded PRP was washed twice and then resuspended in 1 ml HEPES buffer. After 1 h of incubation on ice, the platelet count was adjusted by adding PPP for STA²-induced or HEPES buffer for thrombin-induced [Ca²⁺]ⁱincrease study. After addition of CaCl²(final, 1 mm), an aliquot (800 μl) of washed platelets or PRP was placed in a siliconized glass cuvette and preincubated for 1 min in a fluorometer (CAF-110; Japan Spectroscopic, Tokyo, Japan). The samples were stimulated with thrombin (0.02 U/ml) or STA²(0.5 μm) for 5 min with or without various concentrations of ketamine that had been added 3 min before stimulation. The intensities of 500-nm fluorescence induced by 340- and 380-nm excitation were monitored. To obtain the minimum and maximum fluorescences, the cells were treated with 1 mm EDTA or 2 μm ionomycin, respectively. The luminescent signal was converted to [Ca²⁺]ⁱby using a Fura-2–Ca²⁺dissociation constant of 224 nm as described by Grynkiewicz et al.  11 

Aliquots of washed platelets (120 μl; counts, 1,000,000/mm³) were stimulated by STA²(1 μm) or thrombin (0.02 U/ml) after preincubation at 37°C with or without ketamine (1 mm) for 3 min. Ketamine had been added 3 min before stimulation. At various time points after stimulation (0, 5, 10, and 60 s), the samples were added with 50 μl ice-cold perchloric acid (10%) to terminate the reaction, were kept in an ice bath for 30 min, and were then centrifuged at 1,800 g , 4°C, for 10 min. After adjusting the pH to 7.4, the supernatants were placed in the ice bath for more than 30 min and were then centrifuged at 8,000 g , 4°C, for 3 min. The IP³concentrations of the supernatants were measured using a commercially available IP³assay kit according to the manufacturer's protocol.

Ketamine (0 or 2 mm) was added directly to test tubes containing 100 μl washed platelets (1,000,000/mm³) and 700 μl buffer B for 10 min before addition of [³H]S145. After incubation for 10 min at 37°C, [³H]S145 (1–5 nm) was added to the test tubes (n = 3 each) and was then incubated for 30 min. For analysis of STA²binding affinity, a 100-μl platelet suspension was added to the test tube containing 1 mm ketamine, 1 nm [³H]S145, various concentrations of STA²(1 nm–1 μm), and buffer B (total 200 μl) (n = 4 each) and was preincubated for 10 min at room temperature. After 30 min of incubation at 37°C, the samples were added to 5 ml ice-cold Tris-HCl buffer (5 mm; pH 7.4) to terminate the reaction, were filtered in vacuo  through a Whatman GF/C filter, and were washed three times with 5 ml precooled Tris-HCl buffer. The radioactivity of each filter was determined using a liquid scintillation analyzer (Tri-Carb 1900 CA; Packard Instrument Co., Meriden, CT). Scatchard analysis studies were conducted, and equilibrium dissociation constant (K^d) and maximal concentration of binding sites (B^max) of each experiment were calculated.

Platelet pellets were suspended in 5 mm Tris-HCl, pH 7.4 at a density of 10,000,000 platelets/mm³and were homogenized by two strokes in a Potter-Elvehjem homogenizer to prepare the membrane fraction. The homogenate was centrifuged at 100,000 g  for 30 min. Precipitates were suspended in 20 mm Tris-HCl, pH 7.4, at 5 mg protein/ml. Platelet high-affinity GTPase activity was assessed essentially by the method of Ushikubi et al.  12In brief, the reaction mixture contained 50 μg membrane protein, 3 mm MgCl², 1 mm EGTA, 0.2 mm ATP, 2 mm AMP-PNP, 1 mm phosphocreatine, 50 units creatine phosphokinase, 0.2 mm dithiothreitol, 100 mm NaCl, 0.1 μm [γ-³²P]GTP (222 TBq/mm), 20 mm Tris-HCl (pH 7.4), several concentrations of ketamine (0–1.0 mm), and an agonist (0.02 U/ml thrombin or 300 nm STA²) in a final volume of 50 μl. The reaction mixture had been preincubated for 3 min before the agonist was added. High-affinity GTPase activity was defined as that inhibited by the addition of 1 mm GTP in the assay. The assay mixture was incubated for 10 min at 37°C and added to 1 ml ice-cold Norit A (5%) in 20 mm phosphate buffer (pH 7.4). After vortex and centrifugation (20,000 g , 5 min), aliquots (500 μl) of the supernatant were counted for radioactivity by a liquid scintillation counter. A blank value was less than 5% of the total [γ-³²P]GTP added.

The drugs used in this study were as follows: ketamine, thrombin, EGTA, ATP, AMP-PNP, phosphocreatine, dithiothreitol (Sigma Chemical Co., St. Louis, MO); acetoxymethyl ester of Fura-2 (Fura-2/AM; Dojindo, Kumamoto, Japan); and creatine phosphokinase (Nacalai tesque, Kyoto, Japan). [³H]S145 and STA²were gifts from Shionogi Research Laboratories (Osaka, Japan) and Ono Pharmaceuticals (Osaka, Japan), respectively. GF/C filter was obtained from Whatman International (Maidstone, United Kingdom). The IP³assay kit was a D-myo-IP³[³H] assay system, code TRK 1000 (Amersham International Plc, Buckinghamshire, United Kingdom). [γ-³²P]guanosine triphosphate (GTP) (222 TBq/mm) was obtained from New England Nuclear (Boston, MA).

The data were analyzed using an unpaired t  test for aggregation study, measurements of [Ca²⁺]ⁱ, IP³assay, and measurement of GTP hydrolysis. Data are expressed as mean ± SD. For TXA²receptor binding assay, data of the Scatchard analysis was analyzed using an unpaired Student t  test, and data of the STA²binding affinity was analyzed using analysis of variance.

Figure 1shows typical recordings of aggregation, in the absence and presence of ketamine (500 μm), in response to ADP (0.5 μm), epinephrine (1.0 μm), STA²(0.5 μm), thrombin (0.02 U/ml), and A23187 (0.5 μm). Ketamine (up to 1 mm) did not affect primary aggregations induced by ADP and epinephrine. Ketamine (150–500 μm) suppressed aggregation of platelet after 3 min of stimulation with ADP (0.5 μm;fig. 2A), thrombin (0.02 U/ml;fig. 2B), and STA²(0.5 μm;fig. 2D). Ketamine (150–1,000 μm) suppressed the aggregation after 1 min of stimulation with epinephrine (1.0 μm;fig. 2C) in a dose-dependent manner, but only 1 mm ketamine affected aggregation after 3 min of epinephrine stimulation, suggesting that ketamine delayed the onset of epinephrine-induced secondary aggregation. Platelet aggregation induced by A23187 (0.5 μm) was not altered by ketamine (up to 1 mm;fig. 1).

Ketamine (250 μm, 500 μm, and 1 mm) inhibited [Ca²⁺]ⁱincrease induced by thrombin (0.02 U/ml) and STA²(0.5 μm) significantly and in a dose-dependent manner (fig. 3).

Scatchard analysis of [³H]S145 binding showed that ketamine (2 mm) did not affect B^maxor K^d(fig. 4A). Figure 4Bshows that the STA²(agonist) binding affinity was not changed by ketamine. These results suggested that the inhibitory effect of ketamine on STA²-induced platelet aggregation was not due to the inhibition of the receptor expression or binding affinity.

Figure 5shows the time course and the effects of 1 mm ketamine on intracellular IP³formation in human platelets stimulated by STA²(1 μm) or thrombin (0.02 U/ml). The intracellular IP³concentration ([IP³]ⁱ) at time 0 was 0.394 ± 0.369 pmol/10⁹platelets (n = 5) and was not significantly changed with ketamine (0.450 ± 0.522 pmol/10⁹platelets). STA²(1 μm) induced a rapid increase of [IP³]ⁱ. [IP³]ⁱpeaked at 5 s after stimulation, and the mean peak value was 4.39 ± 2.41 pmol/10⁹platelets. Ketamine (1 mm) inhibited the rapid increase of [IP³]ⁱ(1.90 ± 0.86 pmol/10⁹platelets, P < 0.05;fig. 5A). In the same manner, thrombin (0.02 U/ml) induced an [IP³]ⁱincrease (n = 4 each). The maximum [IP³]ⁱwas at 10 s after stimulation, and the mean value was 6.60 ± 1.819 pmol/10⁹platelets. Ketamine (1 mm) also reduced the [IP³]ⁱincrease induced by thrombin (2.41 ± 0.98 pmol/10⁹platelets, P < 0.05;fig. 5B).

Figure 6shows the effect of ketamine on GTP hydrolysis. Ketamine (≤ 125 μm) dose dependently suppressed platelet membrane–bound GTPase activity in STA²- or thrombin-stimulated conditions; GTPase activities in the presence of ketamine (1 mm) were 50.3 ± 3.2% and 67.5 ± 5.5%, respectively (n = 4 each), of those in the absence of ketamine.

Several anesthetic agents, including volatile and intravenous anesthetics, were reported to affect human platelet aggregation through different mechanisms. 2,5,7,13Kohro and Yamakage 5demonstrated that halothane strongly suppresses thrombin-induced human platelet aggregation by suppressing [Ca²⁺]ⁱincrease without affecting agonist receptor binding. They concluded that this suppressing effect of halothane on [Ca²⁺]ⁱwas likely to be mediated by decreased IP³formation, by increased cytosolic free cyclic adenosine monophosphate, or by both. We previously reported that propofol, although it enhances STA²-induced IP³formation, 7inhibits both ADP- and epinephrine-induced secondary aggregation by suppressing cyclooxygenase-1 activity. 7 

As for the effect of ketamine on platelet aggregation, Atkinson et al.  8demonstrated the inhibitory effects on thromboxane synthesis. In the current study, ketamine suppressed secondary aggregation induced by ADP and epinephrine without altering primary aggregation. Furthermore, ketamine suppressed thrombin- and STA²-induced platelet aggregation as well. Obviously, the suppression of the effects of thrombin and a TXA²analog shown in the current study cannot be ascribed to the suppression of TXA²formation. The results of our aggregation study thus strongly suggested that ketamine affects the binding of TXA²and thrombin to their receptors or mechanisms that follow the receptor activation.

However, the radioligand binding assay showed the absence of the effect of ketamine on the K^dand B^maxof S145. Furthermore, STA²binding assay showed that ketamine had no effect on the STA²-TXA²receptor binding affinity. These results indicated that, at least in the platelet TXA²signaling pathway, the inhibitory effects of ketamine are not due to suppression of TXA²receptor binding.

The current [Ca²⁺]ⁱstudy showed clearly that ketamine suppresses thrombin- and STA²-induced [Ca²⁺]ⁱincrease, concomitant with the suppression of aggregation. Moreover, the finding that ketamine did not affect calcium ionophore (A23187)–induced platelet aggregation indicates that ketamine does not affect the pathway that follows the cytosolic free calcium increase.

Thromboxane A²receptors on platelets, as well as thrombin receptors, are known to be members of the seven transmembrane domain family that interacts with G-protein. 14–17The membrane-bound G-proteins coupled with these receptors are known to have α subunits belonging to the Gq family. 18,19This Gq activates platelet phospholipase Cβ. 20Phospholipase C hydrolyzes phosphatidyl inositol 4,5-bisphosphate and produces IP³and diasylglycerol. 21IP³is known to release Ca²⁺from the dense tubular system of platelets, 22–24which is noted as the first peak of [Ca²⁺]. 25In an attempt to elucidate the mechanism by which ketamine suppresses [Ca²⁺]ⁱincrease, we tested and demonstrated the suppressive effect of ketamine on IP³formation. We further demonstrated suppression of GTP hydrolysis by ketamine in homogenized platelet membrane stimulated with thrombin and STA². These results may suggest that ketamine suppresses receptor-coupled mechanisms, including G-protein, to decrease the activity of phospholipase Cβ.

An intravenous dose of 2 mg/kg ketamine in clinical use was reported to cause a peak plasma concentration of approximately 60 μm. 26In the current study, 150 μm ketamine significantly suppressed epinephrine-induced platelet aggregation, and 250 μm suppressed thrombin-induced platelet aggregation and [Ca²⁺]ⁱincrease. Our experiments using higher concentrations of ketamine were performed mainly in an attempt to elucidate the underlying mechanism for pharmacologic interests. One may think that these high concentrations of ketamine could be cytotoxic, exerting irreversible effects on platelets. However, Akata et al.  27reported the reversibility of the inhibitory effect of ketamine (up to 3 mm) on vascular smooth muscle contraction. We also confirmed, in a preliminary study, the reversibility of the effects of ketamine on platelets. In brief, platelets were incubated with 2 mm ketamine for 15 min and were then washed by centrifugation. These platelets were aggregated normally by STA², clearly denying the irreversible or cytotoxic effect of ketamine on platelets in our experiments.

In contrast to our study, Atkinson et al.  8used clinically relevant concentrations of ketamine in their ex vivo  experiments with baboons and showed that ketamine, even at clinical concentrations, clearly suppresses platelet aggregation. One may argue that anesthesiologists do not notice bleeding tendency in a clinical situation using ketamine. It is possibly because in most situations, bleeding during surgery is more dependent on surgical technique than on the pharmacologic action of anesthetics. Furthermore, ketamine is known to affect plasma concentration of catecholamines by suppression of released catecholamine uptake, 28,29and therefore, it is also possible that this effect of ketamine ameliorates the antiaggregatory effect of ketamine observed in vivo . Therefore, we speculate that the antiaggregatory effect of ketamine may not have clinical importance in most cases. In spite of these facts, we still consider that the antiaggregatory effect of ketamine may have clinical importance in critically ill patients or patients with hematologic disorders.

In conclusion, ketamine has inhibitory effects on the thrombin- and TXA²-related Ca²⁺release system of platelets. It was strongly suggested that the antiaggregatory effects of ketamine are at least partly ascribable to the inhibition of thrombin and TXA²-induced GTPase activation.