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Midazolam attenuates adenosine diphosphate-induced P-selectin expression and platelet-leucocyte aggregation

Tsai, C. S.*; Hsu, P. C.*; Huang, G. S.; Lin, T. C.; Hong, G. J.*; Shih, C. M.; Li, C. Y.

European Journal of Anaesthesiology: November 2004 - Volume 21 - Issue 11 - p 871-876
Original Article

Background and objective: The expression of P-selectin on the surface of platelets and platelet-leucocyte conjugate formation are considered to be an indicator of platelet activation in thrombotic and inflammatory disease. Midazolam is a widely used sedative and anaesthetic induction agent. It may inhibit platelet aggregation and suppress interleukin-6 and -8 response in human leucocytes, but any effect on the adhesion of activated platelets to leucocytes remains obscure. We have examined the influence of midazolam on adenosine diphosphate (ADP)-induced platelet surface P-selectin expression and platelet-leucocyte aggregation in whole blood.

Methods: Human whole blood was stimulated with 2 × 10−5 M ADP in the presence of midazolam (3 × 10−4 to 3 × 10−6 M). Samples were stained with a fluorochrome-conjugated CD62P and CD41a antibody for detecting human platelet P-selectin antigens. The leucocyte subpopulations were separately gated and platelet-leucocyte aggregates were defined as cells found positive for CD45 and CD62P. All samples were analysed and were electronically separated into specific cell types (platelets, neutrophils, monocytes and lymphocytes) according to their typical forward/side scattering by flow cytometry.

Results: Midazolam significantly inhibited ADP-induced platelet P-selectin expression and attenuated platelet-leucocyte aggregation (mainly in neutrophils and monocytes) in a dose-dependent manner with a maximum inhibitory effect at 3 × 10−4 M (P < 0.01).

Conclusions: This study demonstrated that midazolam decreases the ADP-induced expression of platelet surface P-selectin and platelet-leucocyte aggregation.

Tri-Service General Hospital, *Departments of Surgery andAnesthesiology;Department of Parasitology and Tropical Medicine, National Defence Medical Centre, Taipei, Taiwan, ROC

Correspondence to: Chi-Yuan Li, Department of Anesthesiology, Tri-Service General Hospital, #325, Section 2, Cheng-Gung Road, Taipei, Taiwan, ROC. E-mail:; Tel: +886 2 8792 7128; Fax: +886 2 8792 7127

Accepted for publication May 2004 EJA 1744

P-selectin, an adhesion molecule found in the α-granule membrane protein of platelets, becomes externalized on the platelet surface following platelet activation and granule secretion [1]. Expression of P-selectin mediates adhesion of activated platelets to leucocytes and incorporation of leucocytes into thrombi [2]. P-selectin surface expression on platelets and platelet-leucocyte aggregation have been reported as potential markers of platelet activation in various diseases, such as acute coronary syndrome [3], atrial fibrillation [4] and rheumatic mitral stenosis [5].

Midazolam, a benzodiazepine γ-aminobutyric acid type A receptor agonist, is widely used as a sedative and anaesthetic induction agent [6]. Recently, midazolam was reported to inhibit platelet aggregation [7], to suppress interleukin (IL)-6 mRNA expression in human peripheral blood mononuclear cells [8] and to decrease extracellular IL-8 accumulation from human polymorphonuclear leucocytes [9]. However, the effect of midazolam on platelet membrane P-selectin and platelet-leucocyte interaction has not been investigated. Since binding of activated platelets to leucocytes may contribute to the regulation of neutrophil and monocyte function as well as the pathogenesis of inflammatory diseases, the aim of this study was to examine whether midazolam alters adenosine diphosphate (ADP)-induced platelet surface P-selectin expression and adhesion of activated platelets to leucocytes to gain further insight into the mechanism of anaesthetic-induced immunomodulation in whole blood.

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After approval of the local institutional Ethics Committees and the obtaining of informed consent, venous blood samples were taken from 10 healthy volunteers who had no history of coagulation disorders, systemic inflammatory diseases or had taken nonsteroidal anti-inflammatory drugs for at least 2 weeks prior to donation. Blood was collected without a tourniquet from the antecubital vein of one arm via a 19-G needle, using the two-syringe technique. The first 3 mL of blood was discarded, while the second sample was used for flow cytometric analyses. 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. Whole blood stimulation and flow cytometric analysis were modified as previously described by de Rossi and colleagues [10]. The following monoclonal antibodies (Mab) were used to measure platelet-leucocyte aggregation and expression of specific platelet surface P-selectin: anti-CD45-fluorescein-isothiocyanate (FITC) (clone HI30), Mab for leucocyte common antigen; anti-CD41a-FITC (clone HIP8), Mab recognizing platelet GPIIb/IIIa complex independent of activation; anti-CD62P-phycoerythrein (PE) (clone AC1.2), Mab directed against P-selectin expressed on platelet surface and negative IgG1-FITC and IgG1-PE antibodies (clone MOPC-21) for non-specific binding (all from Beckton Dickinson, San Jose, CA, USA). In brief, whole blood stimulation was performed within 10 min of blood collection and ADP (ADP; Sigma Chemicals, St. Louis, USA) was used at a final concentration of 2 × 10−5 M. Aliquots of samples were incubated with the desired concentration of either midazolam (from 3 × 10−4 to 3 × 10−6 M, i.e. in the range from clinically relevant blood concentrations to high pharmacological concentrations) or phosphate-buffered saline (PBS) at 37°C for 5 min which contained negligible soluble P-selectin in plasma [11] and then divided into two parts: one for examining platelet P-selectin and other for examining platelet-leucocyte aggregation, separately.

While measuring platelet P-selectin in whole blood, anti-CD62P-PE antibody and anti-CD41a-FITC antibody were added to the equal volume sample and the suspensions were incubated for 20 min in the dark. Samples were then fixed with 1% paraformaldehyde (Sigma-Aldrich) and assayed with flow cytometry.

While studying the effects of midazolam on platelet-leucocyte aggregation, blood samples were fixed by FACS Lysing Solution (Beckton Dickinson), centrifuged, and the remaining pellet resuspended in 0.3 mL PBS containing 0.1% bovine serum albumin. Saturating concentrations of anti-CD45-FITC and anti-CD62P-PE antibodies were added to samples and incubated for 10 min. All samples were then washed and analysed within 24 h.

Flow cytometric analyses were performed using a FACScan cytometor and CellQuest software (Beckton Dickinson), which detected the emitted fluorescence and light-scattering properties of each cell to electronically separate them into specific cell types (platelets, neutrophils, monocytes and lymphocytes). For determination of P-selectin expression on platelet surfaces in whole blood, the platelet population was defined by size and CD41a-FITC immunofluorescence. From each sample, 30 000 platelets were measured. The mean fluorescence intensities of CD62P from the gated platelet populations were displayed graphically. To determine platelet-leucocyte conjugate formation, the leucocyte subpopulations were separately gated and platelet-leucocyte aggregates were defined as cells found positive for CD45 and CD62P in these subpopulations. For each sample, 10 000 leucocytes were counted. ADP-stimulated samples were expressed as percentage of positive control tests, whereas unstimulated samples served as negative controls.

All data are presented as mean ± standard deviation. All statistical comparisons were made using one-way analysis of variance. For multiple comparisons, the Student-Newman-Keuls test was used. P < 0.05 was accepted as indicative of significant differences between the groups. Calculations were performed with the Prism program (GraphPad Software, San Diego, CA, USA).

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Differential counting was performed by flow cytometry based on forward/side scatter gating events. Figure 1a shows the dot plot of gating platelets (R1) in whole blood. The relative fluorescence intensity increased by PE-CD62P binding to ADP (2 × 10−5 M)-activated platelet P-selectin, whereas resting platelets showed negligible PE-CD62P binding (Fig. 1b). Midazolam significantly inhibited platelet P-selectin expression in a dose-dependent manner, with a maximum inhibitory effect of 47 ± 19% at a concentration of 3 × 10−4 M (P < 0.01, n = 3; Fig. 2).

Figure 1

Figure 1

Figure 2

Figure 2

We further investigated the effect of midazolam on ADP-induced platelet-leucocyte aggregation. Figure 3a shows the dot plot of leucocytes by gating subpopulations as neutrophils (R1), monocytes (R2) and lymphocytes (R3). Two colour analyses were performed for the discrimination of FITC-CD45 (FL-1) and PE-CD62P (FL-2)-positive leucocytes (Fig. 3b). The relative fluorescence intensity increased by PE-CD62P binding to ADP (2 × 10−5 M)-activated platelets on leucocytes (reflecting adherent platelets on leucocytes), whereas the non-stimulated group showed negligible CD62P binding to platelets on leucocytes (Fig. 3c). The binding of CD62P-positive platelets was found predominantly on monocytes (mean fluorescence intensity from 14.47 ± 4.45 to 162.54 ± 35.28), less on neutrophils (from 12.36 ± 2.35 to 64.83 ± 18.86) and very little on lymphocytes (from 7.58 ± 2.61 to 10.72 ± 0.74). Midazolam, at a concentration of 3 × 10−4 M, significantly reduced the binding of CD62P-positive platelets on leucocytes by 53 ± 2%, on monocytes by 59 ± 1%, on neutrophils by 57 ± 4% and on lymphocytes by 19 ± 0.2% (Fig. 4).

Figure 3

Figure 3

Figure 4

Figure 4

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Evidence of clinically relevant leucocyte-platelet interaction was reported in the early 1960s [12]. Subsequent evaluation has demonstrated such interactions in acute coronary syndrome [13], adult respiratory distress syndrome (ARDS) and acute lung injury [14]. The present study has demonstrated that midazolam inhibited ADP-induced platelet P-selectin expression. The effects of midazolam on aggregation of platelets with leucocyte subpopulations in human whole blood were also observed.

Recently, Sheu and colleagues reported that the anti-aggregation effect of midazolam is involved in the conformational changes in platelet membranes, activation of phospholipase C, thromboxane A2 formation and inhibition of intracellular Ca2+ mobilization [7]. Using collagen as a stimulator, they demonstrated that midazolam has an anti-aggregation effect. As we know, ADP is present in platelet-dense granules and is released by platelet stimulating agents, such as thrombin or collagen, hence reinforcing their aggregation [15]. The ADP P2Y12 receptor is responsible for potentiation of platelet secretion [16,17], P-selectin expression [18] and stabilization of platelet aggregates [19]. Thus, ADP-induced platelet activation first initiates P-selectin expression followed by the platelet aggregation process [20]. In our study, using ADP as a stimulator, we demonstrated that midazolam significantly inhibited platelet P-selectin expression.

Thrombosis and inflammation involve complex platelet-leucocyte aggregation. Platelets bind with leucocytes via P-selectin expressed on the surface of activated platelets to the leucocyte ligand, P-selectin glycoprotein ligand-1 [2]. Jy and colleagues demonstrated that expression of P-selectin in platelets leads promptly to binding to leucocytes in whole blood [21] and showed that P-selectin is the main receptor involved in ADP-induced platelet-leucocyte aggregation [18]. As we know, platelet-leucocyte aggregation is more dependent on platelet activation than on leucocyte activation [22]. In this study, we found that the binding of CD62P-positive platelets on leucocytes increased mainly on monocytes and neutrophils by adding platelet-specific agonist ADP (2 × 10−5 M) in whole blood. Midazolam inhibited ADP-induced platelet-leucocyte aggregation, an effect predominantly on monocytes and neutrophils, with no significant effect on lymphocytes. In the previous report, monocytes bind with activated platelets very rapidly and have a much higher affinity for platelet binding than neutrophils [23]. This rapid kinetic may be explained by differences in the degree of dissociation of the aggregates and by structural alterations of the receptor on monocytes [23]. Such differences between leucocytes may explain why monocytes constituted the main proportion of platelet thrombus-bound leucocytes [24].

Platelet-leucocyte aggregation in inflammatory states can lead to leucocyte activation resulting in increased cytokine production, tissue factor expression [25] and reactive oxygen species production [26]. Clinically, platelet-leucocyte aggregation may be found in patients suffering from sepsis or stable coronary disease [27], and also undergoing coronary by pass surgery or haemodialysis [28]. Exposure of monocytes to P-selectin mobilizes the transcription factor nuclear factor-κB and induces expression of tumour necrosis factor (TNF)-α, monocyte chemoattractant protein-1 [29] and IL-8 [30]. Midazolam is commonly used in the intensive care unit for sedation. In a similar pharmacological concentration to our study, midazolam may suppress ADP-induced oxygen consumption and oxidative phosphorylation [31]. In addition, midazolam was reported to inhibit the mRNA IL-6 response in human peripheral blood mononuclear cells [8], to decrease extracellular IL-8 accumulation from human polymorphonuclear leucocytes [9] and to suppress lipopolysacchride-induced TNF-α activity in mouse macrophages [32]. In our study, midazolam attenuated ADP-induced platelet P-selectin expression and platelet-leucocyte aggregation in whole blood. Thus, it might exert a suppressing effect on P-selectin mediated platelet-leucocyte aggregation, leading to immobilization of transcription factor, cytokine and reactive oxygen species production. Ramoska and colleagues demonstrated adequate sedation with effective intravenous (i.v.) doses of 1-3 mg midazolam [10]. Harper and colleagues reported that i.v. doses of 0.3 mg kg−1 (approximately 6.7 × 10−6 M) may be required when midazolam is used for sedation in surgical patients [33]. In this study, midazolam was used at concentrations from 3 × 10−4 to 3 × 10−6 M, a range which encompasses clinically relevant blood concentrations. Since the concentrations of midazolam in clinical use do not alter the adhesion of platelets to leucocytes in vitro, we suggest that during sedation of critically ill patients, midazolam may not inhibit leucocyte and platelet function. It reduces the formation of platelet-neutrophil and platelet-monocyte conjugates only at higher concentrations due to an inhibition of P-selectin expression on platelets.

P-selectin plays a major role as the receptor that mediates interaction with leucocytes and incorporation of leucocytes into thrombus [34]. However, P-selectin is not the only leucocyte receptor on platelets: fibrinogen expressed on the platelet surface might also function as a leucocyte-binding receptor [35]. In our study, we involved only ADP-induced platelet P-selectin secretion and platelet-leucocyte aggregation.

In conclusion, our current study demonstrated that midazolam decreases ADP-induced platelet-leucocyte aggregation. The mechanism is likely to involve an inhibition of P-selectin expression on platelets. However, only at supraphysiological concentrations did midazolam reduce the formation of platelet-neutrophil and platelet-monocyte aggregation. Its clinical relevance in critically ill patients remains to be further investigated.

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This work was supported by grants from Tri-Service General Hospital (TSGH-C92-42) and the National Science Council (NSC 92-2314-B-016-056), Taiwan, ROC.

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MIDAZOLAM; CELL ADHESION MOLECULES, P-selectin; PLATELET ACTIVATION, platelet aggregation, platelet adhesiveness; LEUCOCYTES, aggregation; ADENOSINE DIPHOSPHATE

© 2004 European Academy of Anaesthesiology