Spatially multiplexed stimulation reveals nonlinear calcium signaling in platelets

Platelet activation arises from the integration of multiple biochemical cues, but how platelets respond to spatially heterogeneous combinations of agonists and inhibitors remains poorly understood. Here we applied spatially multiplexed stimulation using two independently controlled light-sensitive compounds to generate localized regions enriched in ADP, epinephrine, or nitric oxide, as well as their combinations. Calcium imaging of platelet populations revealed that different spatial contexts produced qualitatively distinct activation dynamics. In regions with high ADP, responses were robust and were selectively suppressed by nitric oxide, whereas epinephrine had little additional effect. In regions with low ADP, epinephrine enhanced calcium signals, indicating increased sensitivity to weak stimulation. A distinct activation mode emerged when low ADP was combined with both epinephrine and nitric oxide: platelets exhibited delayed yet sustained calcium elevations, suggesting nonlinear integration of sensitizing and inhibitory inputs. These results show that spatial variation in multiple stimuli can give rise to diverse platelet activation patterns, highlighting the importance of local agonist composition in shaping platelet signaling dynamics.