Abstract

Background

Activation of platelets, which plays an important role in inflammation, has recently been reported to enhance platelet P-selectin expression and form platelet-leukocyte aggregation (PLA). Platelet P-selectin expression and PLA formation have been reported to be potential markers of inflammatory diseases such as sepsis, thrombosis, myocardial ischemic disorders and stroke. Lidocaine, one of the most commonly used anesthetics, is known to inhibit platelet function, but its effect on platelet P-selectin expression and PLA remains unclear.

Methods

To determine the effect of lidocaine on platelet activation, and on platelet activation-related septic condition (lipopolysaccharide-induced), we treated platelets with lidocaine (0.03–3 mM) and then measured platelet P-selectin expression and PLA. Whole blood for in vitro study was obtained from healthy men aged 27 to 33 years who had not taken any medication for at least 15 days.

Results

All samples were analyzed by flow cytometry. We found that lidocaine produced a concentration-dependent inhibition of P-selectin expression and PLA. Moreover, in lipopolysaccharide-challenged samples, lidocaine at concentrations of 1–3 mM inhibited PLA.

Conclusion

Our findings may help to elucidate the inhibitory role of lidocaine on platelet P-selectin expression and PLA and infer possible therapeutic targets in the treatment of inflammatory diseases. However, further investigations are needed to determine whether the observed attenuation of excessive inflammatory responses has clinical implications. These results suggest that lidocaine might have potential clinical application in the modulation of excessive platelet activation, inflammatory response and septic condition.

Keywords

blood platelets; cell aggregation; leukocytes; P-selectin;

1. Introduction

Activated platelets play an important role in sepsis,¹ inflammation, hemostasis, and thrombosis,² and have recently been reported to enhance platelet P-selectin expression.³ Platelet P-selectin is present in the α-granules of platelets and translocates rapidly to the cell surface after platelet activation.⁴ Increased levels of P-selectin have been found in patients with sepsis,¹ peripheral artery disease,⁵ acute coronary syndromes treated with angioplasty or thrombolysis⁶ and stroke.⁷ Platelet P-selectin may bind to leukocytes and form platelet−leukocyte aggregation (PLA), mainly through coherence of platelet P-selectin with leukocyte P-selectin glycoprotein ligand-1 (PSGL-1), which is constitutively expressed on the leukocyte surface.⁸ PLA has been reported to be a potential marker of sepsis,⁹ disorder of cardiopulmonary bypass,¹⁰ thrombosis¹¹ and unstable angina.¹²

Lidocaine is commonly used to provide local or re gional anesthesia, and to treat and/or prevent ventricular arrhythmia in critically ill patients. It also has a variety of actions. Of particular interest are reports indicating that local anesthetics modulate the inflammatory response,¹³ and platelet aggre gation.¹⁴ In vivo, they prevent or reduce inflammatory disorders such as reperfusion injury in heart, lung, and brain, as well as endotoxin- or hypoxia-in duced pulmonary injury.¹⁵ In vitro, lidocaine inhibits signaling actions of macrophages and granulo cytes, which mediate early steps of the inflammatory response.¹⁶

In this study, we hypothesized that preconditioning with lidocaine would impair platelet signaling by decreasing platelet P-selectin expression and PLA. To test this hypothesis, we assessed the effect of lidocaine on platelet P-selectin expression and PLA using flow cytometry.

2. Methods

This study was approved by the institutional review board of Tri-Service General Hospital. Informed consent was obtained from all healthy volunteers before enrolment. Whole blood for this in vitro study was sampled from six examinees with age ranging from 27 to 33 years, who had not taken any medication for at least 15 days. Using a twosyringe technique, blood was collected from an antecubital vein with an 18-gauge needle without tourniqueting. The first sample of 2 mL was discarded to avoid tissue contamination, and the second sample was used for experiment. All samples were anticoagulated with 1:9 volume of 3.8% sodium citrate solution and then immediately processed for stimulation procedures and flow cytometric analyses.

The following reagents were used: anti-CD41a-PE or -FITC antibody (Becton Dickinson, San Jose, CA, USA), a platelet-specific monoclonal antibody conjugated with phycoerythrin (PE) or fluoresceinisothiocyanate (FITC) which recognizes platelet GPIIb/IIIa complex independent of activation; antiCD62P-PE antibody (Becton Dickinson), a monoclonal antibody conjugated with PE that is directed against P-selectin expressed on the platelet surface; and anti-CD45-FITC (Becton Dickinson), a monoclonal antibody for the leukocyte common antigen.
Dulbecco’s phosphate-buffered saline (PBS), bovine serum albumin, adenosine 5-diphosphate (ADP), lipopolysaccharides (LPS) and paraformaldehyde were obtained from Sigma Chemicals (St. Louis, MO, USA). Lidocaine was purchased from AstraZeneca Pharmaceuticals LP (Westborough, MA, USA).

We used our previously described protocol to study the effects of lidocaine on platelet activation.¹⁷ Briefly, the anticoagulated blood samples were divided into two parts: one part was used for whole blood assays, and the other part was centrifuged (100g for 10 minutes) to obtain platelet rich plasma (PRP). One portion of the whole blood or PRP was diluted with PBS to serve as control. Another portion was preincubated with the desired concentration of lidocaine (30 μM to 3 mM) for 15 minutes at 37ºC. For investigating the effect of LPS on PLA, a third portion of the blood sample was preincubated with LPS (1 μg/mL) for 10 minutes and the desired concentration of lidocaine (30 μM to 3 mM) for 5 minutes. Platelet agonist with and without ADP was used at a final concentration of 20 μM in whole blood and 8 μM in PRP for 5 minutes’ stimulation at room temperature.

To determine platelet P-selectin expression, blood samples were mixed with saturated concentrations of anti-CD62p-PE monoclonal antibody and anti-CD41a-FITC monoclonal antibody. To determine PLA, the blood samples were mixed with saturated concentrations of anti-CD45-FITC monoclonal antibody and anti-CD41a-PE monoclonal antibody. After staining with antibodies, both the whole blood and PRP samples were incubated for 20 minutes in the dark. Both samples were then fixed with 1% paraformaldehyde and maintained at 4ºC. After fixation, blood samples were immediately processed for flow cytometric analysis in a FACS Calibur flow cytometer (Becton Dickinson), having recourse to CELLQuest cell analysis software (Becton Dickinson). To determine platelet CD62P expression both in whole blood and PRP, individual platelets were identified by size (forward scatter) and anti-CD41aFITC immunofluorescence using a logarithmic scaled dot plot. P-selectin expression on the surface of platelets was defined as positive for anti-CD62P-PE (FL2). Results are expressed as mean fluorescence intensity (MFI). The anti-CD62P MFI reflects the number of P-selectin epitopes expressed on the plate let surface membrane. For each sample, 10,000 platelets were collected. PLA in whole blood was measured as positive for CD41a and CD45. Leukocytes were identified by their anti-CD45-FITC fluorescence and differentiated into subgroups based on cell size and granularity in the forward and side scatter. The two-color analysis enabled discrimination of platelet-coupled and platelet-free leukocytes, and calculation of the percentage of platelet-coupled leukocytes in the leukocyte population. Similarly, the percentages of platelet-conjugated lymphocytes, monocytes, and neutrophils could be measured by analysis of the individual leukocytes populations. For each sample, 10,000 leukocytes were measured.

2.1. Statistical analysis

All data are expressed as the mean ± standard error of the mean. Each diluted sample was compared with the corresponding control sample. Analysis of differences between test solutions was performed using one-way ANOVA and Fisher’s least-significant difference test as a post hoc test. A p value < 0.05 was considered significant.

3. Results

Lidocaine reduced ADP-stimulated P-selectin expression in a concentration-dependent manner (30 μM to 3 mM), with 32 ± 6.5% of maximal inhibition demonstrated at 3 mM in whole blood (Figure 1A). Lidocaine also inhibited ADP stimulated P-selectin expression in PRP. Partial inhibition of P-selectin expression was observed at concentrations below 1 mM, but significant inhibition was seen at concentrations of 1 mM and 3 mM (Figure 1B). Further investigation of the effect of lidocaine on ADP-induced PLA in whole blood revealed a significant inhibitory effect on total PLA at the concentration of 3 mM, while platelet− neutrophil and platelet−lymphocyte aggregates were both significantly inhibited at concentrations of 1 mM and 3 mM (Figure 2).

LPS was used as a model to challenge leukocytes in the acute phase response which leads to sepsis and septic shock. To determinate if lidocaine could inhibit the inflammatory response, LPS-challenged whole blood was incubated with lidocaine. We found that LPS could induce PLA significantly. PLA was inhibited at the concentrations of 1 mM and 3 mM in whole blood (Figure 3). This effect was predominantly seen in platelet−monocyte aggregates, and secondarily sig nificantly in platelet−neutrophil aggregates, but was insignificant in platelet−lymphocyte aggregates.

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Figure 1 Lidocaine inhibition of ADP-induced platelet P-selectin expression in: (A) whole blood (n = 6); and (B) plate- let rich plasma (n = 6). Results are presented as mean ± standard error. *p < 0.05; †p < 0.01.

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Figure 2 Lidocaine inhibition of ADP-induced plate let− leukocyte aggregation (n = 6), which was defined as the percentage of platelet-coupled leukocytes in the leukocyte population. Heterotypic aggregates of total leukocytes, neutrophils, monocytes and lymphocytes are illustrated. Results are presented as mean ± standard error. *p < 0.05.

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Figure 3 Inhibition of ADP-induced platelet−leukocyte aggregation with lidocaine after preincubation with lipopolysaccharides (LPS; n= 6). Platelet−leukocyte aggregation was defined as the percentage of platelet-coupled leukocytes in the leukocyte population. Heterotypic aggregates with total leukocytes, neutrophils, monocytes and lymphocytes are illustrated. Results are presented as mean ± standard error. *p< 0.05; † p< 0.01.

4. Discussion

Activated platelets with P-selectin expression and PLA have been reported to be sensitive markers of life-threatening thrombic diseases such as sepsis^1,9 and coronary syndrome.^6,12 In this study, it was revealed that lidocaine could attenuate ADP-induced P-selectin expression in a dose-dependent manner in both PRP and whole blood. The mechanism by which lidocaine inhibits platelet P-selectin expression might be due to its being an inhibitor of inositol trisphosphate-induced Ca²⁺ release from isolated platelet membrane vesicles.¹⁸ As Ca²⁺ is a potent stimulus of platelet granule secretion, the inhibition of Ca²⁺ release may lead to inhibition of platelet granule secretion with P-selectin expression. Furthermore, lidocaine depresses platelet membrane lipid fluidity, which is thought to play a crucial role in signal transduction and is believed to affect platelet P-selectin expression.¹⁹

This study also showed that lidocaine could attenuate ADP-induced PLA in a dose-dependent manner in whole blood. The formation of leukocyte− platelet conjugates is largely mediated by the binding of P-selectin expressed on activated platelets to PSGL-1 on leukocytes. In the present study, we demonstrated that lidocaine inhibits P-selection expression, which may play a partial role in the inhibition of PLA by lidocaine. Another contributing mechanism of PLA in hibition is fibrinogen, which cross-links activated GP IIb/IIIa receptors on platelets and integrin MAC-1 (CD11b/CD18) on leukocytes.⁸ Local anesthetics have been known to reduce CD11b20 and CD18²¹ expressions on leukocytes. They also reduce adhesion of leu kocytes to the microvascular endothelia²² and to nonbiologic surfaces.²³ In addition, lidocaine inhibits chemotaxis in human monocytes.²⁴ Therefore, the inhibitory effect of lidocaine on PLA may also be attributable to the effect on leukocytes.²⁵

In our findings, in addition to lidocaine being able to inhibit P-selectin expression and PLA, it also possesses several properties to inhibit platelet function. First, lidocaine can serve as a nonspecific platelet activation factor antagonist that leads to suppression of platelet aggregation.²⁶ Second, lidocaine may interact with thromboxane A2-induced platelet aggregation.¹⁴ Finally, lidocaine can potentiate the antiaggregatory effect of dibutyryl-cyclic AMP on the ADP-induced aggregation. In addition, lidocaine can also potentiate the effect of the adenylate cyclase stimulator prostaglandin E1.²⁷

LPS from the outer membrane of Gram-negative bacteria is the primary trigger of the systemic inflammatory response in sepsis. The induction of an acute phase response by LPS can be used as a model for sepsis and septic shock. Several studies have shown that bacterial LPS could trigger platelet and/or leukocyte activation and induce inflammatory events.²⁵ The results of the present study are consistent with those of previous reports,²⁸ showing that PLA increased after LPS challenge, and lidocaine could reduce this effect. Therefore, the inhibitory effect of lidocaine on LPS-induced immune responses may be a contributing effect in some clinical settings, in particular through the activation of platelets and/or leukocytes.²⁹

In agreement with our findings, a clinically relevant antithrombotic effect of regional anesthesia has been reported.³⁰ For example, the incidence of postoperative deep vein thrombosis following hip surgery was significantly reduced with regional anesthesia (relative risk reduction of 46−55%).³¹ Furthermore, in a previous study, it was stated that by clinical observation, a concentration of lidocaine lower than what was required in vitro may have some antithrombotic effects.³²

There are some limitations to this study. First, the dosages of lidocaine employed were clinically relevant to local application but not to intravenous application. Second, we could not determine whether the observed alterations in CD40L expression have functional effects on the inflammatory response.

In conclusion, this study showed that lidocaine inhibits platelet P-selectin expression and PLA. These effects appear to be attributable to modification of the cellular interaction between platelets and leukocytes. After challenge with LPS, the enhancement of PLA was also attenuated by lidocaine. These findings suggest that lidocaine could have potential clinical application in the modulation of excessive platelet activation, inflammatory response and sepsis.

Acknowledgments

This work was supported by grants from the Buddhist Tzu-Chi General Hospital, Taipei (TCRD-TPE-96-C2-3) and Tri-Service General Hospital (TSGH-C97-8-S03), Taiwan, R.O.C.