Evidence of clinically relevant leucocyte-platelet interaction was reported in the early 1960s . Subsequent evaluation has demonstrated such interactions in acute coronary syndrome , adult respiratory distress syndrome (ARDS) and acute lung injury . 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.
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 . Jy and colleagues demonstrated that expression of P-selectin in platelets leads promptly to binding to leucocytes in whole blood  and showed that P-selectin is the main receptor involved in ADP-induced platelet-leucocyte aggregation . As we know, platelet-leucocyte aggregation is more dependent on platelet activation than on leucocyte activation . 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 . 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 . Such differences between leucocytes may explain why monocytes constituted the main proportion of platelet thrombus-bound leucocytes .
Platelet-leucocyte aggregation in inflammatory states can lead to leucocyte activation resulting in increased cytokine production, tissue factor expression  and reactive oxygen species production . Clinically, platelet-leucocyte aggregation may be found in patients suffering from sepsis or stable coronary disease , and also undergoing coronary by pass surgery or haemodialysis . 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  and IL-8 . 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 . In addition, midazolam was reported to inhibit the mRNA IL-6 response in human peripheral blood mononuclear cells , to decrease extracellular IL-8 accumulation from human polymorphonuclear leucocytes  and to suppress lipopolysacchride-induced TNF-α activity in mouse macrophages . 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 . 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 . 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 . 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 . In our study, we involved only ADP-induced platelet P-selectin secretion and platelet-leucocyte aggregation.
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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