Heterogeneity of Single-platelet Calcium Responses to Activation

Cytosolic calcium oscillations play a central role in platelet activation. However, signaling heterogeneity between platelets of the same individual, or between individuals, is poorly characterized. We utilized total internal reflection fluorescence microscopy of calcium fluorophore-loaded, surface-attached human platelets to monitor single-platelet calcium responses to collagen, ADP, and thrombin. For all activation types in healthy adult donors, four types of platelet calcium dynamics ("activation groups) were distinguished: (I) isolated spikes; (II) oscillations with a period of 3-10 s; (III) clusters of spikes following each other with calcium levels never returning to baseline; and (IV) a sustained high calcium level. The activation Groups I and II were predominant in the immobilized platelets of healthy adults (46 ± 22% and 33 ± 10%, respectively), with 18 ± 13% of platelets in Group III. Stimulation with ADP shifted the activation pattern, with Group I fraction falling to 15 ± 9% and Group III fraction rising to 43 ± 13% instead. For stimulation with ADP plus thrombin or collagen, Group III was predominant (71 ± 11% for thrombin, 46 ± 18% for collagen). A combination of all three agonists mainly produced Level III (69 ± 15%) and Level IV (18 ± 12%) platelets. Confocal microscopy revealed a gradual increase in all activation markers (including integrin activation, granule secretion, and spreading type) when one goes from Group I to Group IV. In the triple-negative breast cancer patients samples before therapy, the responses were shifted toward Group I indicating that their platelets were refractory. These results indicate the importance of platelet heterogeneity analysis and suggest a novel technique to investigate minor populations of refractory or hyperactive platelets.