Previous work in our lab has demonstrated that human platelet Ca2+ signaling utilises a pericellular recycling system.1,2 This model predicts that Ca2+ release from intracellular Ca2+ stores occurs initially into a cytosolic nanodomain enclosed within the membrane complex (MC; a close apposition of an invagination section of the platelet plasma membrane and endoplasmic reticulum – analagous to the cardiac diad). From this cytosolic nanodomain, the Ca2+ is spread via removal by the Na+/Ca2+ exchanger (NCX) into the lumen of invaginated membrane system, where it can then accumulate and recycle back into the cytosol through Ca2+-permeable ion channels.2 This study aimed to test the hypothesis that Ca2+ release initially accumulates within an NCX-associated cytosolic nanodomain. If Ca2+ release occurs onto a close associated NCX within a nanojunction then the NCX-mediated Ca2+ removal should be largely unaffected by the presence of the fast Ca2+ chelator Dimethyl BAPTA (DM-BAPTA), which buffers the bulk cytosol but will not significantly affect Ca2+ signals within about 10 nm of its point of entry into the cytosol.3 Platelets were isolated from blood obtained by venepuncture of healthy volunteers under informed consent and with local ethical committee approval in accordance with the declaration of Helsinki. Thrombin-evoked changes in cytosolic- and extracellular Ca2+ concentration were monitored in Fura-2-loaded platelets and washed platelet suspensions containing 2.5 µM Fluo-4 salt respectively, using our previously published methodologies.2 Single cell imaging was performed in washed platelet suspension containing 5 µM Fluo-4 salt using a Fluoview FV1200 laser-scanning confocal microscope. Data are presented as mean % of control ± SEM of the number of samples (n) indicated. Statistical significance was tested by Student’s t-test and one way ANOVA followed by post-hoc Tukey test. DM-BAPTA-loading prevented any notable rise in the cytosolic Ca2+ concentration after stimulation with 0.5 U/mL Thrombin (1.0% ± 0.6%, P<0.05, n=4), yet in the absence of a rise in Ca2+ in the bulk cytosol it was possible to identify a Ca2+ removal in the extracellular fluid (69.7% ± 10.5% of untreated controls, P<0.05, n=7). This extracellular Ca2+ accumulation in DM-BAPTA-loaded cells was inhibited by pretreatment with the NCX inhibitor, KB-R7943 (35.4% ± 7.7%, P<0.05, n=7). Single cell imaging demonstrate the presence of a single hotspot in each cell, which were rarely observed in KB-7943-pretreated cells. In addition, Ca2+ removal from DM-BAPTA-loaded cells was significantly inhibited in cells in which the PMC was disrupted by treatment with nicergoline (50.4% ± 19.6%, P<0.05, n=9). These results suggest that NCX removes Ca2+ from an isolated cytosolic nanodomain which is not readily reported by Fura-2. Its sensitivity to nicergoline suggest this is likely to be enclosed within the MC.1