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In Vitro Platelet Activation by an Echo Contrast Agent

Departments of Clinical Laboratory Medicine (K.S., N.T., K.O., K.I.) and Hematology (M.M., K.H.) and Division of Cell and Molecular Medicine, Center for Molecular Medicine (S.M., Y.S.), Jichi Medical School, Tochigi-ken, Japan.

Address correspondence and reprint requests to Kouichirou Shigeta, MD, Department of Clinical Laboratory Medicine, Jichi Medical School, 3311-1 Yakusiji, Minamikawachi-machi, Kawachi-gun, Tochigi-ken 329-0498, Japan.

Objective. We investigated whether an ultrasonic echo contrast agent containing microbubbles (Levovist [SH U 508A]; Schering AG, Berlin, Germany) could in routine use activate platelets. Methods. Levovist and its main component, galactose, were mixed with separate samples of whole blood (1.5–75 mg/mL) from 5 healthy volunteers to form a 1-mL suspension sample. After in vitro exposure to ultrasound emitted from a commercial ultrasonic scanner at a pulse frequency of 3.5 MHz with a mechanical index of 1.9 and an exposure duration of 5 minutes, 5 µL of the sample was incubated for 20 minutes with the fluorescein isothiocyanate-labeled CD61 antibody, which is a platelet-specific antigen, and the phycoerythrin-labeled CD62P (P-selectin) antibody, an activation-specific antigen, both on the platelet surface. After more than 30 minutes of fixing in 1% paraformaldehyde, flow cytometric analysis was performed. Results. The percentage of CD62P-expressing platelets increased according to the concentrations of Levovist and galactose, which showed almost equal effects. Ultrasound exposure did not enhance the effect except at the highest concentration of Levovist (75 mg/mL). Conclusions. In vitro, a galactose-based echo contrast agent could not activate the platelets at its routine concentration.

Key Words: CD62P • echo contrast agents • in vitro • platelet activation • ultrasound

Abbreviations: ADP, adenosine diphosphate • FITC, fluorescein isothiocyanate • GLUT, glucose transporter • MCV, mean cellular volume • MI, mechanical index • MPV, mean platelet volume • PE, phycoerythrin • RBC, red blood cell

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