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

The migration of platelets is increasingly recognized for its roles in inflammation, infection, and malignancy. Platelet migration was shown to be an ⍺IIbβ3 integrin dependent process that occurs on fibrinogen deposited at endothelial junctions, but other integrins and extracellular matrix proteins (ECM) 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 ⍺IIbβ3 and β1 integrins. Migrating platelets formed retraction fibers and left behind migrasomes, cell fragments with potentially pro-inflammatory/-coagulant activity. Platelet migration on fibrinogen was sensitive to inhibitors of the acto-myosin cytoskeleton and significantly impaired by high ligand concentrations, lack of the g-chain AGDV motif, preincubation with agents blocking fibrinogen-⍺IIbβ3 interaction, or deficiency in key components of the integrin activation complex. In summary, establishment of a novel high-throughput assay facilitated in-depth studies on platelet migration mechanisms, findings that highlight integrin-ligand interactions as potential therapeutic targets to limit platelet-driven inflammatory tissue damage.