Mechanosensing alters platelet migration

Platelets have long been established as a safeguard of our vascular system. Recently, haptotactic platelet migration has been discovered as a part of the immune response. In addition, platelets exhibit mechanosensing properties, changing their behavior in response to the stiffness of the underlying substrate. However, the influence of substrate stiffness on platelet migration behavior remains elusive. Here, we investigated the migration of platelets on fibrinogen-coated polydimethylsiloxane (PDMS) substrates with different stiffnesses. Using phase-contrast and fluorescence microscopy as well as a deep-learning neural network, we tracked single migrating platelets and measured their migration distance and velocity. We found that platelets migrated on stiff PDMS substrates (E = 2 MPa), while they did not migrate on soft PDMS substrates (E = 5 kPa). Platelets migrated also on PDMS substrates with intermediate stiffness (E = 100 kPa), but their velocity and the fraction of migrating platelets were diminished compared to platelets on stiff PDMS substrates. The straightness of platelet migration, however, was not significantly influenced by substrate stiffness. We used scanning ion conductance microscopy (SICM) to image the three-dimensional shape of migrating platelets, finding that platelets on soft substrates did not show the polarization and shape change associated with migration. Furthermore, the fibrinogen density gradient, which is generated by migrating platelets, was reduced for platelets on soft substrates. Our work demonstrates that substrate stiffness, and thus platelet mechanosensing, influences platelet migration. Substrate stiffness for optimal platelet migration is quite high (>100 kPa) in comparison to other cell types, with possible implications on platelet behavior in inflammatory and injured tissue. STATEMENT OF SIGNIFICANCE: Platelets can feel and react to the stiffness of their surroundings - a process called mechanosensation. Additionally, platelets migrate via substrate-bound fibrinogen as part of the innate immune response during injury or inflammation. It has been shown that the migration of immune cells is influenced by the stiffness of the underlying substrate, but the effect of substrate stiffness on the migration of platelets has not yet been investigated. Using differently stiff substrates made from PDMS, we show that substrate stiffness affects platelet migration. Stiff substrates facilitate fast and frequent platelet migration with a strong platelet shape anisotropy and a strong fibrinogen removal while soft substrates inhibit platelet migration. These findings highlight the influence of the stiffness of the surrounding tissue on the platelet immune response, possibly enhancing platelet migration in inflamed tissue.