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RESEARCH PRODUCT

Flow cytometric kinetic assay of calcium mobilization in whole blood platelets using Fluo-3 and CD41

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Background: Platelet activation plays a major role in the physiology and pathology of hemostasis. Flow cytometry is a promising approach for the structural and functional analysis of platelets. However, the choice of adequate biological parameters and most technical issues are still under discussion. A rise in cytosolic free Ca 21 is a key early event that follows platelet stimulation and precedes several activation responses, including shape change, aggregation, secretion, and expression of procoagulant activity. Our objective was to set up a fast and sensitive flow cytometric method to determine the kinetics of intracellular Ca 21 mobilization in platelets, which could be performed with the least artifactual perturbation of platelet function. Methods: Anticoagulated blood was diluted in Tyrode’s buffer and incubated with Fluo-3-acetoxymethyl ester prior to staining with phycoerytrin-conjugated antiplatelet GPIIb/IIIa complex monoclonal antibody. Platelets were identified by a gate including only CD41 1 events. After the determination of baseline Fluo-3 green fluorescence on a flow cytometer (EPICS XL-MCL, Coulter Electronics, Hialeah, FL), adequate agonists were added and timedependent changes in Fluo-3 fluorescence were recorded on-line for up to 3 min. Results: In these conditions, a very fast and transient increase of cytosolic-free Ca 21 was observed following the addition of thrombin, a strong platelet agonist. Stimulation with adenosine diphosphate (ADP), a weak agonist, also resulted in evident increase of Ca 21 levels. Conclusions: Our results show that this flow cytometric kinetic method provides a simple and sensitive tool to assess in vitro the time course and intensity of signal transduction responses to different platelet agonists under near physiological conditions. In this way, it may be useful to evaluate the degree of platelet reactivity and thus to monitor antiplatelet therapy. Cytometry 35:302‐310, 1999. r 1999 Wiley-Liss, Inc. Key terms: Ca 21 ; platelet activation; Fluo-3; CD41; GPIIb/IIIa; whole blood