A scalable high-throughput platform for the discovery of intracellular and extracellular regulators of platelet...

The migration of platelets is increasingly recognized for its roles in inflammation, infection, and malignancy. Platelet migration has been found to be an αIIbβ3 integrin-dependent process that occurs on fibrinogen deposited at endothelial junctions, but other integrins and extracellular matrix (ECM) proteins may also support this behavior. Here, we present a novel imaging and analysis pipeline for quantifying platelet migration, combining an improved coating/blocking method with convolutional neural network-based automated segmentation and tracking to enable scalable, unbiased assessment of migration phenotypes. Using this high-throughput platform, we systematically investigated how ECM ligand availability, integrin subtype engagement (β1 vs β3), and pharmacological perturbation of motility pathways influence platelet migration. In addition to fibrinogen, platelet migration was observed on fibronectin and vitronectin, mediated by both αIIbβ3 and β1 integrins. Migrating platelets formed retraction fibers and left behind migrasomes, cell fragments with potentially proinflammatory/procoagulant activity. Platelet migration on fibrinogen was sensitive to inhibitors of the actomyosin cytoskeleton and was significantly impaired by high ligand concentrations, lack of the γ-chain AGDV motif, preincubation with agents blocking fibrinogen-αIIbβ3 interaction, or deficiency in key components of the integrin activation complex. In summary, establishment of a novel high-throughput assay facilitated in-depth studies of platelet migration mechanisms, with findings that highlight integrin-ligand interactions as potential therapeutic targets to limit platelet-driven inflammatory tissue damage.