Abstract

Objective

Levobupivacaine, an amide local anesthetic widely used in regional anesthesia, is reported in recent studies that it is a potent inhibitor of platelet functions. However, the concentrations of levobupivacaine were limitedly estimated in these reports. Additionally, the mechanisms by which it affects platelet function and blood coagulation is still not entirely known. The purpose of this study was to further investigate its effects on platelet function and the possible signaling mechanisms under various concentrations of levobupivacaine.

Methods

Blood samples collected from healthy volunteers were separated into whole blood, platelet-rich-plasma and washed platelets. The effect of levobupivacaine on platelet aggregation was studied using platelet function analyzer (PFA-100) and platelet aggregometer. Agonist-induced platelet adenosine triphosphate (ATP) release, cytosolic calcium mobilization, thromboxane B2 (TxB2) secretion and platelet P-selectin translocation under various concentrations of levobupivacaine were investigated.

Results

Our results indicated that levobupivacaine possessed negative effect on platelet aggregation. The closure times of (PFA-100) were lengthened and the agonist-induced platelet aggregation was significantly attenuated by levobupivacaine even at a low dose (50 μgml⁻¹). Pretreatment with levobupivacaine produced significant changes in agonist-induced platelet P-selectin translocation, ATP release, thromboxane A2 (TxA2) production, and calcium mobilization in a dose-dependent manner. The p38 mitogen-activated protein kinases (MAPK), protein kinase C (PKC) δ subtype, cytosolic phospholipase A2 (cPLA2), and protein kinase B (PKB or Akt) were involved in collagen-induced platelet signaling, which would be responsible for antiplatelet effects of levobupivacaine.

Conclusion

We explored possible targets of levobupivacaine on platelets aggregation signaling mechanisms. Our data revealed that p38 MAPK, PKC δ subtype, cPLA2, and Akt were pathways involved in collagen-induced platelet signaling, which might be responsible for antiplatelet effects of levobupivacaine. Our study did provide direct evidence bolstering the critical mechanisms of levobupivacaine within different contexts. Additionally, levobupivacaine imposed a negative effect on platelet aggregation through multiple signaling pathways.

Keywords

anesthetics, local: levobupivacaine; calcium; platelet aggregation; signal transduction;

1. Introduction

Previous publications reported that amide local anesthetics, such as lidocaine and ropivacaine, could impair blood clotting in a concentration-dependent manner by inhibiting platelet function and enhancing fibrinolysis.1, 2, 3 Levobupivacaine consisting of S (–)-enantiomer of bupivacaine, is an amino amide local anesthetic, which has recently been introduced to clinical practice and approved of use in local or regional anesthesia for surgery and obstetrics. It has also been approved of use in indicative pain management, which has not been granted to other local anesthetics.⁴ In recent pharmacodynamic reports, levobupivacaine has also been demonstrated to have lower cardiovascular and central nervous system toxicity and a greater margin of safety.5, 6

A recent study has reported that levobupivacaine is a potent inhibitor of platelet functions as reflected by thromboelastography in a dose dependent decrease in maximum amplitude.⁷ However, just only two concentrations of levobupivacaine (2.5 mg/ml and 2.5 mg/ml) were estimated in this report and it is not convincing enough to serve as solid evidence. Despite considerable information concerning the action of levobupivacaine in clinical setting, it is surprisingly that little is known about the actual anti-platelet mechanism effects.7, 8, 9 The purpose of this study was to assess the significant concentrations of levobupivacaine relative to anti-platelet effect and further investigate the possible signaling regulation pathways through widely functional studies and intracellular signaling molecules.

2. Materials and methods

2.1. Blood sampling

All the experimental protocols and procedures were approved by the Institutional Review Board and Ethics Committee of Chang Gung Memorial Hospital (Linkou, Taiwan, Republic of China). All volunteers signed an informed consent previous to drawing 36 ml whole blood (WB). They were healthy and passed a normal routine clinical clotting study with normal platelet counts and had a negative history of hematological diseases such as bleeding tendency, platelet, or coagulation disorders, and/or were not taking medication that might influence their hematologic function.

2.2. Chemicals

Collagen, thrombin, arachidonic acid (AA), adenosine triphosphate (ATP) and Chrono-Lume were obtained from Chrono-Log Co (Havertown, PA, USA).¹⁰ Adenosine diphosphate (ADP), stable thromboxane analogue, 9, 11-dideoxy-11a, 9a-epoxymethano-prostaglandin F2a (U46619), epinephrine, apyrase, prostaglandin E1 (PGE1), 3-isobutyl-1-methylxanthine (IBMX), ethylenediamine tetra acetic acid (EDTA), ingenol 3-angelate (I3A), phorbol 12-myristate 13-acetate (PMA), bovine serum albumin (BSA), and fura-2 were purchased from the Sigma Chemical Co. (St. Louis, MO, USA). Cyclic adenosine monophosphate (cAMP), cytotoxicity detection lactate dehydrogenase (LDH) kit, and thromboxane B2 (TxB2) EIA kits were purchased from Cayman Chemical (Ann Arbor, MI, USA). Collagen/epinephrine (CEPI) and collagen/ADP (CADP) cartridges were purchased from Dade Behring, Inc. (Deerfield, IL, USA). Levobupivacaine hydrochloride (5 mg/ml) in 0.9% normal saline buffer was obtained from Abbott Laboratories (Nycomed Pharma AS, Elverum, Norway) for levobupivacaine solution preparation. Cell-permeable signaling inhibitors, including Wortmannin, SB202190 and Rottlerin, were purchased from Calbiochem (Darmstadt, Germany). Phospho-specific and non-phospho-specific antibodies for phospholipase A₂ (cPLA₂), p38 mitogen-activated protein kinases (MAPK), protein kinase B (PKB or Akt), and PKCδ came from Cell Signaling Technology Inc. (Beverly, MA, USA). Gαq antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Fluorescein isothiocyanate (FITC)-labled anti-CD62P (P-selectin) antibody was obtained from BD Biosciences (San. Jose, CA, USA). All of the chemicals used in these experiments were of the highest purity available at each individual supplier.

2.3. PFA-100 analyzer studies

To evaluate the clot formation under dynamic conditions we utilized the platelet function analyzer, PFA-100 Analyzer (Dade, Miami, FL USA). WB samples were made anticoagulative with 3.2% buffered citrate and settled for 10 minutes at room temperature, and then incubated 5 minutes with levobupivacaine (50, 100 μg/ml) for blood clot analysis. All blood samples were analyzed for closure times according to the manufacturer's instructions using CEPI and CADP cartridges as described in our previous report.¹¹ The time required to reach a total occlusion of the aperture was defined as the collagen/ADP closure time (CADP-CT) or collagen/epinephrine closure time (CEPI-CT). Upper limit closure time was set at 300 seconds and all results greater than 300 seconds were recorded as 300 seconds.

2.4. Preparation of human platelets

Blood samples were separated into WB, platelet-rich-plasma (PRP), and washed platelets following a standard procedure described previously.¹² Blood was collected into 50 ml sample tubes containing 3.2% trisodium citrate (9:1) or acid citrate dextrose, at pH 4.6 (9:1) and mixed gently by inversion. PRP was prepared by centrifugating the blood sample at 300 g for 10 minutes and collecting the upper layer of the supernatant. PRP was adjusted with autologous PPP to 2 × 10⁸/ml. Washed platelets were prepared from the PRP. The platelet pellet was washed twice in Tyrode's buffer (137 mmol/L NaCl, 2 mmol/L KCl, 12 mmol/L NaHCO₃, 0.3 mmol/L NaH₂PO₄, 2 mmol/L CaCl₂, 1 mmol/L MgCl₂, 5.5 mmol/L glucose, 5 mmol/L Hepes, pH 7.3) containing 0.35% human serum albumin and then centrifuged at 1100 g for another 8 minutes. Finally, platelets were gently resuspended in the same buffer with 0.02 U/mL of the ADP scavenger apyrase. Platelets were kept at 37 °C throughout all experiments.

2.5. Flow cytometry analysis for P-selectin expression

Platelet surface CD62P (P-selectin) expression was assessed by flow cytometry. The washed platelet suspension (2 × 10⁸/ml) was preincubated with or without levobupivacaine (25, 50, 100, 200 μg/ml) at 37 °C for 5 minutes. Samples were then stimulated by U46619 (2 μM) at 37 °C for 5 minutes. Samples were fixed with 1% paraformaldehyde at 4 °C for 10 minutes, washed, and FITC-labeled CD62P antibody (Becton Dickinson, Mississauga, Canada) was added for 10 minutes at room temperature. Finally, analysis was performed using a fluorescence-activated cell sorting (FACS) Calibur instrument (Becton Dickinson, San Jose, CA, USA).

2.6. Platelet aggregation and ATP release reaction

The platelets aggregation and release of ATP responses were simultaneously measured by a Lumi-aggregometer (Model 560; Chrono-Log). For the platelet aggregation assay WB, PRP, or washed platelets were preincubated in the aggregometer for 1 minute at 37 °C and stirred at 1000 rpm before testing. Different concentrations of levobupivacaine (25 to 200 μg/ml) or vehicle were added 3 minutes prior to the addition of agonists. Aggregation was initiated by adding collagen (2 μg/ml). The change of light transmission was recorded for 10 minutes and analyzed using an aggrolink data processing system. The rate of inhibition of platelet aggregation was expressed as percentage over control. The release of ATP from platelets was detected by the Chrono-Lume luciferase reagent following manufacturer's instructions.

2.7. LDH release assay

Quantification of LDH release in cell culture medium with drug exposure can be used to detect drug cytotoxicity.¹³ The cytotoxic effect of levobupivacaine was assessed by detecting LDH release following exposure of the washed platelets at 37 °C for 10 minutes to 12.5, 25, 50, 100, 150, and 200 μg/ml levobupivacaine using aCytotoxicity Detection LDH kit (Cayman Chemical, Ann Arbor, MI, USA), according to the manufacturers' instructions. LDH release was compared with the total LDH activity of washed platelets treated with 0.1% Triton X-100 (Life Technologies, New York, NY, USA).

2.8. Measurement of thromboxane A2 (TxA2) secretion in platelets

The washed platelets (5 × 10⁸ platelets/ml) were prepared as described above, and different concentrations of levobupivacaine (50 μg/ml to 100 μg/ml) or vehicle were added 3 minutes before the addition of agonists. Aggregation was initiated by thrombin (0.1 U/ml), U46619 (2 μM), and collagen (2 μg/ml), AA (0.5 mM) or ADP (10 μM) at 37 °C for 10 minutes. The reaction was stopped by adding the equivalent-volume of cold EDTA solution with indomethacin. Samples were separated by centrifugation at 12,000 g for 10 minutes at 4 °C and supernatants were stored at –80 °C. Thromboxane B2, the stable downstream metabolite of TxA2, was measured using a TxB2 enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA), according to the instructions of the manufacturer.

2.9. Measurement of Ca2+ influx

The prepared PRP samples were centrifuged at 1100 g for 10 minutes at 37 °C and the platelet pellet was resuspended in Tyrode's buffer containing no calcium, at a density of 3 × 10⁸ platelets/ml. Platelet samples were loaded with 2 μM fura-2/AM for 30 minutes at 37 °C in dark conditions. After 30 minutes the samples were washed using Tyrode's buffer containing 0.35% bovine serum albumin and resuspended to 3 × 10⁸ platelets/ml at 22 °C with Tyrode's buffer containing 0.1% bovine serum albumin. Aliquots of fura-2/AM-loaded platelets were transferred to pre-warmed (37 °C) quartz cuvettes (10 × 10 mm). Cytosolic Ca²⁺ concentration was measured using a Hitachi F4500 fluorescence spectrophotometer with a dual wavelength program under continuous stirring. (Excitation was measured at 340 and emission at 510 nm).

2.10. SDS-PAGE and immunoblot

The washed platelets were stimulated by various agonists in the presence or absence of levobupivacaine (100 μg/ml). Reactions were terminated by adding equivalent volumes of ice-cold lysis buffer (50 mM Tris-HCl, 200 mM sodium chloride (NaCl), 2 mM ethylene glycol tetraacetic acid (EGTA), 2 mM Na₂ ethylenediaminetetraacetic acid (EDTA), 2 mM phenylmethyl sulfonyl fluoride, 2 μg/ml leupeptin, 1 μg/ml antipain, 2 mM dithiothreitol (DTT) and 2% Triton X-100), to the tested samples that were then placed in ice for 30 minutes. Proteins were subjected to 10%–12% sodium dodecyl sulfate polyacrylamide gels for electrophoresis and then transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with nonfat milk for one hour at room temperature, and incubated with the appropriate primary antibody at 4 °C overnight. Membranes were washed and incubated with horseradish peroxidase-conjugated secondary antibody. Proteins were detected by using enhanced chemiluminescence (Amersham Life Science) Arlington Heights, IL, USA.

2.11. Statistical analysis

The values were expressed as mean + standard error of mean for three to six experiments. The degree of significance of variations between control and experimental values for each experiment was assessed by analysis of variance test followed by the Dunnett or Fisher's Least Significant Difference (LSD) test using the Prism statistical software version 4.0 (GraphPad Software Inc., San Diego, CA, USA). Differences between the treated group and control group were analyzed by Student's t-test. Results were considered significant with p < 0.05.

3. Results

3.1. Levobupivacaine effect on clot formation through CEPI-CT and CADP-CT

We examined the specific effect of levobupivacaine-mediated anticoagulation by PFA-100 closure time measurement, which mimicked the in vivo microcirculation with applied shear stress. The WB samples incubated with levobupivacaine (50, 100 μg/ml) were exposed to epinephrine- or ADP-coated cartridges and the closure times were recorded as shown in Table 1. Levobupivacaine (100 μg/ml) significantly lengthened the closure times when samples passed through both epinephrine-coated columns (CEPI-CT > 300 seconds, control = 144.2 ± 3.38, p < 0.001) and ADP-coated columns (CADP-CT = 113.8 ± 4.51, control = 84.8 ± 2.27, p < 0.01). These data were accordant with previous reports, ascertaining the direct suppression effect of levobupivacaine on blood coagulation and platelet function.

Table 1. Effects of levobupivacaine on human whole blood by platelet function analyzer (PFA-100) parameters.

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3.2. Levobupivacaine effectively suppresses platelet activation-related surface marker CD62P (P-selectin) expression

CD62P (P-selectin) is a component of the platelet α granule membrane. Upon activation, P-selectin is expressed on the surface of activated platelets through α granule secretion.¹⁴ We explored whether levobupivacaine would affect the U46619 mediated platelet P-selectin expression by flow cytometry. P-selectin expression triggered by U46619 (2 μM) was suppressed with levobupivacaine exposure in a dose-dependent manner as shown in Fig. 1. These data indicate that levobupivacaine negatively regulates platelet function by inhibiting P-selectin mediated procoagulant activity.

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Fig. 1. Levobupivacaine decreased platelet P-selectin marker expression under U46619-induced activation. Wash platelets (2 × 108/ml) were preincubated or not with levobupivacaine (25, 50, 100, and 200 μg/ml) at 37 °C for 10 minutes prior to stimulation by U46619 (2 μM). Platelet surface CD62P (P-selectin) expression was assessed by flow cytometry. Dot plots shown are presented as the log of CD62P fluorescence intensity over forward scattering and representative of mean data presented in the bottom (n = 4). *p < 0.05 versus agonists control. U46619 = 9a-epoxymethano-prostaglandin F2a.

3.3. Levobupivacaine inhibits platelet aggregation triggered by collagen

To characterize the relative role of levobupivacaine in agonist-induced platelet aggregation, we examined platelet aggregation by using Lumi-aggregometer (Model 560, Chrono-Log). As shown in Fig. 2A, levobupivacaine that suppressed the washed platelet aggregation induced by collagen (2 μg/ml), significantly inhibited various agonists-induced aggregations at high doses (100–200 μg/ml) (data not shown). Interestingly, the collagen-induced aggregation was very sensitive to the levobupivacaine-mediated inhibitory effect and was significantly suppressed by levobupivacaine (25–200 μg/ml) in whole blood, PRP and washed platelet preparations (Fig. 2C, D, and E). By measuring LDH release, we could demonstrate that the antiplatelet effect of levobupivacaine is not due to drug cytotoxicity (Fig. 2B). These results strongly suggest that the collagen initiated platelet aggregation is directly inhibited by levobupivacaine.

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Fig. 2. Concentration dependent response for levobupivacaine-mediated inhibition in platelet aggregation and cytotoxicity. (A) Isolated platelets were incubated with control (normal saline, 0.9%) or different concentrations of levobupivacaine (25–200 μg/ml) at 37 °C for 3 minutes and then induced by collagen (2 μg/ml); (B) platelets were incubated with various concentrations of levobupivacaine (12.5–200 μg/ml) 37 °C for 10 minutes and the LDH activity released into solution was assessed. Cytotoxicity is expressed as the percentage of the LDH activity compared to total LDH activity released from platelets lysed by 0.1% Triton X-100. After levobupivacaine (25–200 μg/ml) pretreatment, separated blood samples included whole blood (panel C), platelet-riched plasma (panel D), and wash platelets (panel E) were triggered by collagen. Data are presented as means ± standard error of mean. *p < 0.05 and ** p < 0.01 as compared with respective control. LDH = lactate dehydrogenase.

3.4. Levobupivacaine inhibits ATP release in washed platelet aggregation induced by collagen

We further studied whether levobupivacaine had any influence on the platelet secretion upon aggregation. ATP release from washed platelet aggregations, induced by collagen (2 μg/ml) and pretreated with levobupivacaine at different doses (25, 50, 100, 150, 200 μg/ml) is shown in Fig. 3. Levobupivacaine inhibited ATP release from washed platelets upon activation in a dose dependent manner. ATP release was significantly reduced by levobupivacaine at doses of 25–200 μg/ml. These results demonstrate that the collagen induced ATP release in washed platelet aggregations were suppressed by levobupivacaine, suggesting that the inhibition of platelet aggregation by levobupivacaine was through the mechanism of suppressing platelet secretion.

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Fig. 3. Effects of various concentrations of levobupivacaine in (A) platelet aggregation; and (B) ATP release triggered by collagen. Wash platelets were preincubated with Chrono-Lume reagent for 2 minutes to achieve stable baseline and gain control set at 0.005×. After agonists (collagen: 2 μg/ml) induced aggregation, platelet ATP release was measured by Lumi-aggregometer Model 560 (Chrono-Log, Havertown, PA, USA). Values are presented as means ± standard error of mean (n = 5). ATP = adenosine triphosphate. * Statistic significant at p < 0.05 compared with collagen control group. ** Statistic significant at p < 0.01 compared with collagen control group.

3.5. Levobupivacaine suppresses TxB2 production triggered by U46619 and collagen and thrombin but not by ADP or AA

In order to further elucidate the levobupivacaine mediated platelet suppressive effect, we studied the TxB2 production triggered by ADP (10 μM), thrombin (0.1 U/ml), AA (0.5 mM), U46619 (2 μM), and collagen (2 μg/ml). Surprisingly, AA induced TxB2 production was not influenced by levobupivacaine. Only a partial attenuation of ADP induced TxB2 production was observed besides a high exposure to levobupivacaine (100 μg/ml; data not shown). Contrarily, U46619-, collagen- or thrombin-induced TxB2 production was significantly suppressed by levobupivacaine (100 μg/ml; Table 2). These results indicate that suppression of TxB2 production was through the inhibitory effect of levobupivacaine on collagen, thrombin and U46619 but was not through ADP and AA.

Table 2. Effects of levobupivacaine on wash platelet thromboxane B2 formation.

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3.6. Levobupivacaine significantly suppresses platelet intracellular calcium release triggered by collagen in a dose-dependent manner

We further examined whether levobupivacaine would attenuate the collagen-mediated platelet intracellular calcium release. Our data showed that levobupivacaine could suppress collagen (2 μg/ml) triggered platelet intracellular calcium release in a dose dependent manner (Fig. 4). These data indicate that the levobupivacaine-mediated platelet suppression is relevant to restrain calcium mobilization.

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Fig. 4. Effects of levobupivacaine on collagen-induced intracellular calcium mobilization. (A) Wash platelets (2 × 108/ml) were loaded with Fura-2-AM and the intracellular calcium mobilization was measured by spectrophotometer after addition of collagen (2 μg/ml) under levobupivacaine (100 μg/ml) pretreatment; (B–F) dose-related inhibition of levobupivacaine (12.5–100 μg/ml) in collagen (2 μg/ml) triggered calcium release was shown. The results are representative of three independent experiments. Data are presented as means ± standard error of mean. * p < 0.05 as compared with respective control.

3.7. Levobupivacaine suppressed cytosolic phospholipase A2 phosphorylation induced by collagen and thrombin but there were no changes observed in Gαq protein levels under various agonists

Molecules signaling mechanism of agonist-induced activation subject to levobupivacaine administration was also studied in washed platelets to probe into the presence of Gαq and cytosolic phospholipase A₂ (cPLA₂) phosphorylation. There were no changes observed in Gαq protein levels under the influence of various agonists (thrombin: 0.1 U/ml, PMA: 50 nM, AA: 0.5 mM, I3A: 100 nM, U46619: 2 μM, and collagen: 2 μg/ml; Fig. 5A). Platelet cPLA₂ phosphorylation displayed a significant decrease as triggered by thrombin and collagen under levobupivacaine pretreatment (Fig. 5B and C). The results suggest that levobupivacaine inhibits platelet aggregation possibly through cPLA₂ activity suppression.

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Fig. 5. Wash platelets were stimulated with various agonists (thrombin: 0.1 U/ml, collagen: 2 μg/ml, U46619: 2 μM, AA: 0.5 mM, PMA: 50 nM, I3A: 100 nM) in the presence or absence of levobupivacaine (100 μg/ml) pretreatment (5 minutes, stirring at 37 °C) in a platelet aggregometer. (A) Platelet lysate proteins were analyzed by Western Blot with anti-Gαq antibody; (B) platelet lysate proteins were analyzed by Western Blot with antiphospho-cPLA2 antibody or total kinase level anti-cPLA2 antibody. AA = arachidonic acid (AA); cPLA2 = cytosolic phospholipase A2; I3A = ingenol 3-angelate; PMA = phorbol 12-myristate 13-acetate; U46619 = 9a-epoxymethano-prostaglandin F2a. * p < 0.05 as compared with respective control.

3.8. Levobupivacaine significantly decreases platelet activation via p38 MAPK, PKC δ, and Akt phosphorylation

In platelets, MAPK signaling pathways triggered by collagen was shown to be regulated within the PKC family.14, 17 We assessed the phosphorylation of p38, PKC δ, and Akt pathways upon platelet activation under levobupivacaine (100 μg/ml) administration. As illustrated in Fig. 6A collagen (2 μg/ml) induced washed platelets activation was shown to have lower phosphorylation of p38, PKC δ, and Akt. By contrast, pretreatment with inhibitors such as Rottlerin (PKC inhibitor, 10 μM), SB202190 (p38 MAPK inhibitor, 10 μM), and Wortmannin (PI3 K/Akt inhibitor, 10 μM) showed the same inhibitory effect in washed platelet aggregation (Fig. 6B). These results indicate that collagen-induced signaling pathway plays a vital role in levobupivacaine induced antiplatelet effect.

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Fig. 6. Wash platelets were stimulated with collagen (2 μg/ml) in the presence or absence of levobupivacaine (100 μg/ml) pretreatment (5 minutes, stirring at 37 °C) in a platelet aggregometer.(A) The lysate was probed with antiphospho-p38 antibody, total kinase level anti-p38 antibody, antiphospho-Akt antibody, total kinase level anti-Akt antibody, antiphospho-PKCδ antibody or total kinase level anti-PKCδ antibody. The results are representative of three independent experiments. Data are presented as means ± standard error of mean; (B) wash platelets were incubated with the indicated pathway inhibitors (Rottlerin: 10 μM, SB202190: 10 μM, and Wortmannin: 10 μM) prior to stimulation with collagen.The Western analyses were shown in at least three independent experiments. * p < 0.05 as compared with respective control. Akt = Protein Kinase B; PKC = Protein Kinase C.

4. Discussion

In the present study, we observed that closure times of (PFA-100) were prolonged and the platelet aggregation was significantly attenuated by levobupivacaine at lower dose (50 μg/ml) and near totally suppressed at higher dose (200 μg/ml). Previous study reported that levobupivacaine could impair blood clotting in a concentration-dependent manner through inhibition of platelet function and enhancement of fibrinolysis.⁷ However, just only two concentrations of levobupivacaine (2.5 μg/ml and 2.5 mg/ml) were investigated using thromboelastography (TEG) in this report. Most important strength of our study was the use of the PFA-100 to quantify platelet function, which was a more specific method to provide a sensitive and reproducible measure of platelet function, and quantify functional defects than was TEG in which each parameter was influenced by several haemostatic factors.15, 16, 17 This finding was further confirmed by the results of pretreatment with levobupivacaine in which significant changes in platelet P-selectin translocation, ATP release, TxA2 production, and calcium mobilization were caused in a dose-dependent manner. These observations led us to speculate that levobupivacaine imposed a negative effect on platelet aggregation in a dose-dependent manner and the critical concentration, which could impair blood clot formation was extremely lower than was revealed by previous finding. To verify the reliability of this experiment, we identified the pharmacologic dosage range from 12.5 ug/ml to 200 ug/ml by LDH release experiments (Fig. 2B).

There is now widespread agreement that at least three signal-transduction pathways are present in platelets and the process involves intracellular pathways mediating platelet activation through phospholipase C (PLC) and the participation of TxA2 cascade.¹⁸ It is also known that platelet aggregation can be inhibited by an increase in the cytosolic concentration of cAMP, an effect mediated by a G-protein-dependent activation of adenylyl cyclase.19, 20, 21 We revealed that the presence of low concentrations of levobupivacaine significantly hampered platelet release reaction, as demonstrated by the reduction in the expression of platelet P-selectin (CD62) as well as significant inhibited the ATP-release reaction induced by collagen. Furthermore, pretreatment with levobupivacaine produced significant changes in agonist-induced calcium mobilization and TxA2 production indicated that both of PLC and PLA2 related pathways would be considered to be responsible for the observed anticoagulation effects of levobupivacaine. Therefore, we proposed that a differential suppression model in each aggregation pathway towards calcium homeostasis in which levobupivacaine did not trigger positive effects on platelet aggregation and signaling events.

Interaction of local anesthetics (LAs) with G-proteins coupled receptors has been suggested to be essential to mediate these pharmacological functions that cannot be explained by the blockade of sodium channels. Preliminary observations indicate that the lipophilic domain of tetracaine activated G-proteins and the C-terminal of Gα subunit can interact in the human promyelocytic leukemia (HL-60) cell.²² The inhibition of the lysophosphatidate signal but not the inositol triphosphate or angiotensin signal by lidocaine or bupivacaine was observed in the Xenopus oocyte model.23, 24 Moreover, lidocaine and bupivacaine inhibits calcium channels and inward rectifying potassium channels via G-protein-coupled receptors.25, 26, 27 Other studies also reported that lidocaine or bupivacaine could interfere with G-protein coupled receptor signaling by inhibiting Gαq proteins.28, 29, 30, 31 Interestingly, multiple target sites of bupivacaine suppressed the intracellular signaling pathway of the prostaglandin E2 (PGE2-EP1) activity which in turn mediated the PLC signal pathway, suggesting a molecular mechanisms of the bupivacaine mediated anti-inflammation and the authors have concluded that Gαq-protein coupled receptor signaling is the major target site for the observed LAs actions towards anti-thrombosis and anti-inflammation.29, 32 Based on our investigations and previous reports we postulated that Gαq-protein coupled receptor signaling would have a partial influence on ADP, thrombin, arachidonic acid and U46619 induced platelet aggregation. Thus, levobupivacaine should have different degree of suppression on platelet aggregation induced by ADP, thrombin, AA, and U46619, but not by collagen. We examined the effects of levobupivacaine on platelet aggregation initiated by different receptor-signaling pathways as discoursed in this report. Interestingly, levobupivacaine showed its strongest suppressive effect on platelet aggregation initiated by collagen than by others. This could indicate that some other extra-candidate molecules besides Gαq are involved as part of the pharmacological target sites for levobupivacaine mediated antithrombosis effect. Moreover, western data obtained from studies of the human platelets showed that levobupivacaine had more suppression effect on p38 MAPK and PKCδ rather than on Gαq.

Further support for the mechanism of levobupivacaine was derived from the observations of ADP-induced platelet aggregation and TxB2 production. That levobupivacaine had no influence on the AA triggered TxB2 production but did have a partial influence on those triggered by ADP was actually observed. Contrarily, collagen triggered TxB2 production was significantly inhibited by levobupivacaine. These results suggest that one of the pharmacological target sites of levobupivacaine is located above the AA in the PLC signaling pathway. It also appears to be a selective interaction of levobupivacaine with collagen-glycoprotein VI (GpVI)-PLC and subsequent phospholipase A2 signaling. In experiments of ADP induced platelet aggregation, a possible involvement of G protein coupled signaling, including Gαq, in the levobupivacaine-mediated platelet inhibition was also observed. ADP plays a significant role in platelet function and interacts with purinergic receptors such as P2Y₁ and P2Y₁₂ in platelets. It binds with P2Y₁ receptors and promotes Gαq-regulated PLC activation, which enhances the intracellular Ca²⁺ releases, and the suppression of adenylyl cyclase regulated by Gi-coupled P2Y₁₂ receptors. Actually, the P2Y₁ receptor is responsible for the stimulation of platelet aggregation via Ca²⁺ mobilizations, whereas P2Y₁₂ inhibits adenylyl cyclase. The initiation of ADP-P2Y₁₂ signaling blocks the formation of cAMP, which is a major intracellular inhibitor of platelet function, and leads to reinforce the P2Y₁-initiated platelet activation.33, 34, 35 ATP induces the P2X₁ receptors to change the shape of platelets and assisting in platelet activities mediated by collagen.¹⁰ Activation of each of these nucleotide receptors by ADP results in unique signal transduction pathways that are important in the regulation of thrombosis and homeostasis. Our data demonstrate that levobupivacaine inhibited ADP-induced platelet aggregation in a dose-dependent manner; however, ADP-induced TxB2 generation was only mildly affected by levobupivacaine. We hypothesize that levobupivacaine has a differential suppressive effect on the ADP induced platelet activation. The P2Y₁/Gαq/PLCβ signal pathways were possibly major target sites suppressed by levobupivacaine in a dose-dependent manner and P2Y₁₂/Gi/adenylate cyclase pathways were possibly minor target sites that were partially suppressed by levobupivacaine.

In clinical relevance, a convincing body of evidence indicates that epidural administration of local anaesthetics attenuates the postoperative hypercoaguable state and may decrease the incidence of thrombotic events in the postoperative period.36, 37, 38 Thromboelastograpic studies in major vascular surgeries showed a decrease in platelet-fibrinogen interactions with epidural analgesia.³⁶ Epidural analgesia might decrease platelet activation by the effects it exerts on leg blood flow, reduction in catecholamine secretion, and even a direct systemic hypocoaguable effect of LA itself.³⁹ We estimated that, 20 mL of 0.5% levobupivacaine would be fully equilibrated in a 70-kg human, that would amount to a dose concentration of approximately 20 μg/l; thus, bolus dose of levobupivacaine is used epidurally, so the plasma concentration would be much lesser than this. Our findings suggest that, at suitable plasma concentrations of levobupivacaine when a patient receives a clinical doses of epidural levobupivacaine, the platelet function is unaffected. Therefore, the mechanism proposed for the beneficial effects of epidural analgesia on postoperative fibrinolysis was due to side factors rather than due to a direct systemic pharmacological effect of LA agents.

In this study, we presented a more significant concentration of levobupivacaine, which attenuated platelet aggregation that was largely lower than previous finding. In clinical relevance, we speculate that in a patient with significant bleeding during epidural puncture attempts or catheterization, subsequently injection of loading dose levobupivacaine would attenuate local platelet aggregation, impaired hemostasis and induce hypo coagulopathy in the epidural space and even cause epidural hematoma formation. In our institution, 10–20 ml of levobupivacaine 0.5% is commonly used as an initial loading dose of epidural analgesia for general surgery. Levobupivacaine 0.1% is used as an initial loading dose for labor epidural analgesia; 0.1%–0.15% solution is used for continuous epidural infusion. The final concentrations of levobupivacaine in the epidural space after drug administration are difficult to determine as they are affected by initial concentration, relative systemic absorption, and variable compartment structure and volume of epidural space. Because the incidence of epidural hematomas is so low, it is difficult to investigate the specific risk associated with each individual local anaesthetic. Whatever the local anaesthetics, the balance between the benefits and risks is of central consideration. This balance not only depends on the pharmacology of each agent, but also on the dosage, timing of administration and patient characteristics.

In summary, our results demonstrated that levobupivacaine imposed a negative effect on platelet aggregation in a dose-dependent manner through multiple signaling pathways. Our data revealed that p38 MAPK, PKC δ subtype, cPLA₂, and Akt were pathways involved in collagen-induced platelet signaling, which might be responsible for anti-platelet effects of levobupivacaine (Fig. 7). The present study provides direct evidence bolstering the critical mechanisms of levobupivacaine within different contexts. Additionally, levobupivacaine imposed a negative effect on platelet aggregation through multiple signaling pathways. This novel knowledge could spawn a love of pharmacologic discoveries of the molecular mechanism of levobupivacaine and other local anesthetics by which a plausible explanation could be provided for other physiologic effects of local anesthetics besides the nerve blockade mechanism that we apply in the day-to-day practice.

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Fig. 7. Proposed antiplatelet model of levobupivacaine in collagen/thrombin-induced signaling pathways. Platelets stimulated with collagen- or thrombin-induced platelet phospholipase C subtypes phosphorylation and subsequently into a common pathway to activate PKC δ, p38 MAPK, TxA2, and calcium mobilization. Another pathway from PI3 K to Akt cascade was also, in part, involved with collagen-induced platelet activation. However, these platelet-activated signaling transductions may be negatively regulated under levobupivacaine administration through multiple targets inhibition. AA = arachidonic acid (AA); Akt = Protein Kinase B; cPLA2 = cytosolic phospholipase A2; DAG = diacylglycerol; FAK = Focal adhesion kinase; IP3 = Inositol 1,4,5-triphosphate; MAPK = mitogen-activated protein kinases; PI3K = Phosphoinositide 3-kinase; PIP2 = phosphatidylinositol 4,5 bisphosphate; PLC = phospholipase C; PKC = protein kinase C; TxA2 = thromboxane A2.

Acknowledgments

This work was supported by Chang Gung Memorial Hospital Research Grants CMRPG33136 and CMRPG360331-2, and National Science Council of the Republic of China Grant NSC-93-2314-B-182A-198.