High-Speed Confocal Imaging Reveals Complex Calcium Transients in Plateles

Intracellular calcium transients have long been known to play an important role in platelet activation. The main processes involved are the IP3 mediated release of calcium from the endoplasmic reticulum and the consequent activation of the store-operated calcium entry (SOCE) mechanism, consisting of the STIM-Orai system and TRPC channels. Traditionally the following methods have been used to measure the calcium level in platelets: flow cytometry, spectrofluorimetry and fluorescent microscopy, with Fura-2-AM used as a calcium indicator. These methods, however, are either incapable of visualising the time course of the calcium transient for the same cell during the activation process or show only the composite calcium level for all the cells.Combining co-loading with Fluo-4-AM and Fura-Red-AM and high-speed confocal imaging (Zeiss LSM Live) we have been able to visualize the calcium transients in case of platelet activation. We investigated the effects of thrombin receptor activating peptide (TRAP), acting on the receptor involved in the physiological platelet activation.Our results indicate that cellular calcium response to TRAP was highly heterogeneous, with only 22.2% (95% CI: 15.1-30.8%) of the platelets exhibiting a calcium transient after activation by TRAP. The non-responding cells still showed an increase of intracellular calcium level when calcium ionophore A23187 was applied. By first using a calcium-free buffer and re-adding calcium after TRAP activation we were also able to distinctly measure SOCE on these cells. Also, by directly activating IP3 receptors with thimerosal, we have been able to measure the effects of inhibitors of the phospholipase C pathway on SOCE.We believe that the proposed imaging method is useful for studying the calcium homeostasis of platelets and possibly other blood cells. The observed heterogeneity of platelet responses may have important clinical and pharmacological consequences.