Original Paper Journalof
Biomedical Science
Received:December3, 2003 Accepted:May 28, 2004
J Biomed Sci 2004;11:764-772 DOI: 10.1159/000081823
Mechanisms Involved in the Antiplatelet Activity of Ketamine in Human Platelets Yi C h a n g a,d T a - L i a n g C h e n b G o n g - J h e W u a G e o r g e H s i a o c M i n g - Y i S h e n d K u a n - H u n g Lin d D u e n - S u e y C h o u
c C h i e n - H u a n g Lin d
J o e n - R o n g S h e u c,d aDepartment of Anesthesiology, Shin Kong Wu Ho-Su Memorial Hospital, and Departments of bAnesthesiology and CPharmacology, and dGraduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan, ROC
Key Words Ketamine • Phospholipase C. Platelet aggregation • Protein kinase C. Thromboxane A2
Abstract The aim of this study was to systematically examine the inhibitory mechanisms of ketamine in platelet aggregation. In this study, ketamine concentration-dependently (100-350 gM) inhibited platelet aggregation both in washed human platelet suspensions and platelet-rich plasma stimulated by agonists. Ketamine inhibited phosphoinositide breakdown and intracellular Ca2+ mobilization in human platelets stimulated by collagen. Ketamine (200 and 350 gM) significantly inhibited thromboxane (Tx) A2 formation stimulated by collagen. Moreover, ketamine (200 and 350 gM) increased the fluorescence of platelet membranes tagged with diphenylhexatriene. Rapid phosphorylation of a platelet protein of Mr 47,000 (P47), a marker of protein kinase C activation, was triggered by phorbol-12,13-dibutyrate (100 nM). This phosphorylation was markedly inhibited by ketamine (350 gM). These results indicate that the antiplatelet activity of ketamine may be involved in the following
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pathways. Ketamine may change platelet membrane fluidity, with a resultant influence on activation of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and phosphorylation of P47, thereby leading to inhibition of intracellular Ca2+ mobilization and TxA 2 formation, ultimately resulting in inhibition of platelet aggregation. Copyright@2004NationalScienceCouncil,ROCand S. KargerAG, Basel
Introduction Platelet function may directly affect hemostasis during perioperative periods, and information regarding the effects of sedative-hypnotic drugs or anesthetics on platelet functions may be important for their clinical use. For example, triazolam and alprazolam inhibit platelet-activating factor (PAF)-induced aggregation [16], and the inhibition of PAF binding to platelets has been proposed to explain this anti-aggregatory effect [5]. Furthermore, the inhibitory effects of halothane and sevoflurane on human platelet aggregation have been shown in in vitro and in vivo studies [8, 15]. On the other hand, studies of ketamine in platelets have relatively rarely been c o m -
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pared with other sedative-hypnotic drugs or anesthetics. Ketamine, an anesthetic induction agent, is generally reserved for use in patients with severe hypotension or respiratory depression [21]. Atkinson et al. [1] reported that the anti-aggregatory effect of ketamine occurs through inhibition of thromboxane (Tx) A2 formation in human platelets. Recently, Nakagawa et al. [20] further demonstrated that ketamine (500 gM) possibly inhibits platelet aggregation through inhibition of inositol trisphosphate formation and [Ca2+]i mobilization. However, the detailed mechanisms underlying the signaling pathways of ketamine still remain obscure. We therefore systematically examined the influence of ketamine on washed human platelets in this study, and utilized the findings to characterize the mechanisms involved in this influence.
absence of ketamine). The degree of aggregation was expressed in light-transmission units. When measuring ATP release, 20 p.1 of a luciferin/luciferase mixture was added 1 min before the addition of agonists, and the ATP release was compared with that of the control.
Analysis of the Platelet Surface GIycoprotein lib~Ilia Complex by Flow Cytometry Triflavin, a specific fibrinogen receptor (glycoprotein IIb/IIIa complex) antagonist, was prepared as previously described [28]. Fluorescence-conjugated triflavin was also prepared as previously described [28]. The final concentration of FITC-conjugated triflavin was adjusted to 1 mg/ml. Human platelet suspensions were prepared as described above. Aliquots of platelet suspensions (4.5 x 108/ml) were preincubated with ketamine (200 and 350 gM) for 3 min, followed by the addition of 2 gl of FITC-triflavin. The suspensions were then incubated for another 5 min, and the volume was adjusted to 1 ml/tube with Tyrode's solution. Suspensions were then assayed for fluorescein-labeled platelets using a flow cytometer (Becton Dickinson, FACScan system, San Jose, Calif., USA). Data were collected from 50,000 platelets per experimental group. All experiments were repeated at least 4 times to ensure reproducibility.
Materials and M e t h o d s Materials Collagen (type I, bovine Achilles tendon), ADP, ketamine, epinephrine, EDTA, luciferin-luciferase, phorbol-12,13-dibutyrate (PDBu), Dowex- 1 (100-200 mesh; Xs, chloride form), prostaglandin Eb trichloroacetic acid, EGTA, bovine serum albumin, acrylamide, sodium pyruvate, ~-NADH, diphenylhexatriene (DPH), apyrase, heparin, and myoinositol were purchased from Sigma (St. Louis, Mo., USA). Fura 2-AM and fluorescein isothiocyanate (FITC) were purchased from Molecular Probe (Eugene, Oreg., USA). Trimeresurus flavoviridis venom was purchased from Latoxan (Rosans, France). Myo-2-[3H]-inositol was purchased from Amersham (Amersham, UK). TxB2, cyclic AMP, and cyclic GMP enzyme immunoassay (EIA) kits were purchased from Cayman (Ann Arbor, Mich., USA).
Preparation of Human Platelet Suspensions Human platetet suspensions were prepared as previously described [ 10]. In this study, human volunteers provided informed consent. In brief, blood was collected from healthy human volunteers who had taken no medicine during the preceding 2 weeks, and was mixed with acid/citrate/glucose (9:1, v/v). After centrifugation at 120 g for 10 min at room temperature, the supernatant (platelet-rich plasma) was supplemented with prostaglandin Ea (0.5 gM) and hepatin (6.4 IU/ml), then incubated for I0 min at 37 ° C and centrifuged at 500 g for 10 rain. Washed platelets were finally suspended in Tyrode's solution containing bovine serum albumin (3.5 mg/ml) and adjusted to about 4.5 x 108 platelets/ml. The final concentration of Ca 2+ in Tyrode's solution was 1 mM.
Platelet Aggregation The turbidimetric method used a Lumi-Aggregometer (Payton, Canada) as described previously [3]. In brief, platelet suspensions (0.4 ml) were prewarmed to 37 °C for 2 rain, and then ketamine was added for 3 min before the addition of platelet-aggregation inducers. The reaction was allowed to proceed for at least 6 min, and the extent of aggregation was expressed as a percentage of the control (in the
Antiplatelet Activity of Ketamine
Labeling of Membrane Phospholipids and Measurement of the Production of FH]-Inositol Phosphates The method was carried out as previously described [11]. Briefly, platelets were preincubated with [3H]-inositol (75 gCi/ml; Amersham) for 2 h followed by centrifugation, and finally by resuspension in Ca2+-fi'ee Tyrode's solution (5 x 10s platelets/ml). Ketamine was preincubated with 1 ml of loaded platelets for 3 min, and collagen (1 gg/ml) was then added to trigger aggregation. Finally, the inositol phosphates of the supernatants were separated using a Dowex-1 anion exchange column. Only [3H]-inositol monophosphate (IP) was measured as an index of total inositol phosphate formation.
Measurement of PIatelet [Ca2+]iMobilization by Fura 2-AM Fluorescence Citrated whole blood was centrifuged at 120 g for 10 rain. The supernatant was protected from light and incubated with Fura 2-AM (5 gM, Molecular Probe) at 37°C for 1 h. Human platelet suspensions were then prepared as described above. Finally, the external Ca 2+concentration of the platelet suspensions was adjusted to 1 mM. The [Ca2+]i rise was measured using a fluorescence spectrophotometer (CAF 110, Jasco, Tokyo, Japan) with excitation wavelengths of 340 and 380 nm, and an emission wavelength of 500 nm. [Ca2+]iwas calculated from the fluorescence measured using 224 nMas the Ca 2+Fura 2 dissociation constant [7].
Measurement of Membrane Fluidity with a Fluorescent Probe The intensity of fluorescence in human platelets was measured as described previously [13]. Briefly, platelets (4.5 x 108/ml) were preincubated with various concentrations of ketamine (200 and 350 gM) for 3 rain followed by the addition of a 0.5-gM DPH solution for another 6 rain. The relative fluorescence intensity ofplatelets was measured in a fluorescence spectrophotometer (Hitachi F4500, Tokyo, Japan) at 37°C.
Measurement of TxB2 Formation Washed human platelet suspensions (4.5 x 108/ml) were preincubated for 3 min in the presence (200 and 350 gM) or absence of
J Biomed Sci 2004;11:764-772
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o_
Fig.
1. Tracing curves of ketamine on collagen (1 gg/ml)-induced platelet aggregation in washed human platelet suspensions. Platelets were preincubated with ketamine (200 and 350 gM); collagen was then added to trigger aggregation (lower tracings) and ATP release (upper tracings). Profiles are representative examples of five similar experiments.
Ketamine
200,L l
T Collagen
ketamine before the addition of collagen (1 ~tg/ml). Six minutes after the addition of the agonist, 2 mM EDTA and 50 gM indomethacin were added to the reaction suspensions. Vials were then centrifuged for 3 min at 15,000 g. TxB2 levels of the supernatants were measured using an EIA kit (Cayman) according to the instructions of the manufacturer.
Determination of Lactate Dehydrogenase Lactate dehydrogenase (LDH) was measured according to a previously described method [31]. Platelets (4.5 x 108/ml) were preincubated with various concentrations of ketamine for 30 min, followed by centrifugation at 15,000 g for 5 min. An aliquot of supernatant was incubated with phosphate buffer containing 0.2 mg [~NADH for 20 min. Thereafter, 100 lal ofpyruvate solution were added, and the absorbance wavelength was read at 334 nm using a UVvisible recording spectrophotometer (UV-160; Shimazu, Kyoto, Japan). A maximal value of LDH was constructed from sonicated platelets.
Estimation of PlateIet CyclicAMP and Cyclic GMP Formation The method of Karniguian et al. [12] was followed. In brief, platelet suspensions were warmed to 37 °C for 1 min, then either prostaglandin E1 (10 gM), nitroglycerin (10 gM), or ketamine (200 and 350 gM) was added and incubated for 6 rain. The incubation was stopped, and the solution was immediately boiled for 5 min. After cooling to 4°C, the precipitated protein was collected as sediment after centrifugation. Fifty microliters of supernatant were used to determine the cyclic AMP and cyclic GMP contents by EIA kits (Cayman) following acetylation of the samples as described by the manufacturer.
Measurement of Protein Kinase C Activity Washed human platelets (2 x 109/ml) were incubated for 60 min at 37 °C with phosphorus-32 (0.5 mCi/ml, Amersham). Platelet sus-
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pensions were next washed twice with Tris-saline buffer. The 32p-labeled platelets were preincubated with ketamine (200 and 350 gM), then PDBu (100 riM) was added to trigger protein kinase C activation. Activation was terminated by the addition of Laemmli sample buffer, and analyzed by electrophoresis (12.5°/0; w/v) as described previously [6]. The relative intensities of the radioactive bands were analyzed using a Bio-imaging analyzer system (FAL2000, Fuji, Tokyo, Japan), and are expressed as photostimulated luminescence (PSL)/mm 2.
Statistical Analysis Experimental results are expressed as the mean -+ SEM and are accompanied by the number of observations. Data were assessed using analysis of variance (ANOVA). If this analysis indicated significant differences among the group means, then each group was compared using the Newman-Keuls method. A p value of
