Super-resolution mapping of platelet α-granules uncovers non-polarized secretion and differential retention of von Willebrand Factor and thrombospondin-1
Platelet -granules contain hundreds of bioactive proteins that regulate thrombosis, inflammation and tissue repair. The interplay between -granule secretion and platelet biomechanics is central to platelet function but remains incompletely understood. Here we combined complementary super-resolution microscopy methods, computational image simulations and quantitative single-cell analysis to investigate -granule nanoscale organization and secretion in tandem with cytoskeletal remodeling in spreading human platelets. We show that P-selectin, thrombospondin-1 (TSP1) and von Willebrand factor (VWF) occupy distinct subgranular domains and that apparent co-localization strongly depends on imaging resolution. We identify ring-like P-selectin staining as a robust signature of intact -granules and demonstrate that -granule secretion in spreading platelets was confined to the platelet cell body rather than the actively remodeling periphery. Quantitative assessment at the single-cell level showed that degranulation coincided with transitions from filopodia-rich to lamellipodial morphologies. Comparisons across adhesive substrates and perturbations by soluble agonists indicated that these processes are coordinated through overlapping signaling networks rather than direct local coupling. Analysis of secreted cargo revealed distinct extracellular fates despite no evidence for polarized secretion. TSP1 accumulated beneath platelets within diffusion barrier-restricted membrane pockets and around platelets following matrix binding, whereas VWF remained predominantly tethered to platelet membranes and formed short VWF strings. Together, our findings redefine the organization and release of platelet -granules, demonstrate an absence of secretion polarity, and establish cargo-specific retention mechanisms that may influence platelet signaling and thrombus architecture.