Development and validation of a platelet calcium flux assay using a fluorescent imaging plate reader

Calcium signaling in platelets is an important physiological response to various aggregation stimuli. Loading platelets with various fluorescent dyes and measuring the change in calcium concentration using a spectrofluorometer has been the traditional approach to studying calcium signaling. This method suffers from the need for large platelet samples and a decrease in total fluorescence signal with time due to photobleaching. Therefore, it is rarely used to measure the quantitative effect of an agonist or antagonist on calcium signaling. Adaptation of these measurements to a fluorescent imaging plate reader (FLIPR) format allows the sample size to be reduced by 5- to 10-fold, and the microplate format allows a significant increase in throughput. Addition of the agonists to all wells simultaneously serves to normalize the total response. This article describes the first use of a FLIPR to study the calcium flux in human platelets. The IC50 values showed a linear correlation with the Ki for receptor binding in washed platelets. The generality of the methodology was shown by measuring EC50 values for agonists and IC50 values for antagonists of the platelet G protein-coupled receptor P2Y1 and for the ion channel P2X1.