Human platelets use a cytosolic Ca nanodomain to activate Ca-dependent shape change independently of platelet aggregation.

Human platelets use a rise in cytosolic Ca concentration to activate all stages of thrombus formation, however, how they are able to decode cytosolic Ca signals to trigger each of these independently is unknown. Other cells create local Ca signals to activate Ca-sensitive effectors specifically localised to these subcellular regions. However, no previous study has demonstrated that agonist-stimulated human platelets can generate a local cytosolic Ca signal. Platelets possess a structure called the membrane complex (MC) where the main intracellular calcium store, the dense tubular system (DTS), is coupled tightly to an invaginated portion of the plasma membrane called the open canalicular system (OCS). Here we hypothesised that human platelets use a Ca nanodomain created within the MC to control the earliest phases of platelet activation. Dimethyl-BAPTA-loaded human platelets were stimulated with thrombin in the absence of extracellular Ca to isolate a cytosolic Ca nanodomain created by Ca release from the DTS. In the absence of any detectable rise in global cytosolic Ca concentration, thrombin stimulation triggered Na/Ca exchanger (NCX)-dependent Ca removal into the extracellular space, as well as Ca-dependent shape change in the absence of platelet aggregation. The NCX-mediated Ca removal was dependent on the normal localisation of the DTS, and immunofluorescent staining of NCX3 demonstrated its localisation to the OCS, consistent with this Ca nanodomain being formed within the MC. These results demonstrated that human platelets possess a functional Ca nanodomain contained within the MC that can control shape change independently of platelet aggregation.