Mechanosensing Via a Gp2b3a-Src-Axis Regulates Platelet Migration in Vitro and In Vivo

Platelets are vascular first responders in thrombosis and hemostasis, but also central players in inflammation. Compared to platelets recruited to thrombi, immune responsive platelets use distinct effector functions, including Arp2/3 dependent migration along substrate gradients (haptotaxis), which prevents inflammatory bleeding and contributes to host defense. How platelet migration in this context is regulated on a cell biological level is incompletely understood. Here, we use in vitro assays to show that in contrast to retractile function, polarized actin polymerization at the leading edge in combination with Myosin II dependent retraction at the rear is necessary for initiation and maintenance of migratory behavior. In migrating platelets, myosin II function is regulated via ROCK and MLCK, but blocking these pathways does not abrogate platelet polarization preceding movement. Rather, Arp2/3 dependent actin nucleation is necessary to initiate polarization. Using morphodynamic analyses integrating shape as well as indicators of migration/retraction behavior, we performed a drug screen to identify relevant upstream regulators of actin nucleation. These experiments revealed coordinated integrin GPIIbIIIa outside-in signaling via Galpha13, that triggers tyrosinkinase c-Src dependent lamellipodium formation. This cascade functions independent of soluble agonists ADP and Thromboxane or chemotactic signals. Inhibitors of this signaling cascade interfere with the migratory ability of platelets. In particular, clinically employed Src inhibitor Dasatinib abrogates migration at nanomolar concentrations, notably below its threshold to impede other platelet functions like procoagulant activation or adhesion. In murine inflammation models, low dose Dasatinib treatment translated to reduced migration of platelets visualized by 4D intravital microscopy, as well as increased pulmonary hemorrhage in acute lung injury, with no effect on tail bleeding times and vascular occlusion in a Ferric Chloride induced carotid thrombosis assay. In summary, we define a distinct signaling pathway essential for platelet migration in inflammation and uncover a novel mechanism explaining Dasatinib-related platelet dysfunction and bleeding phenotype. This underscores the differential molecular regulation of platelets in inflammatory/mucosal hemostasis and could explain the occurrence of mucosa-associated bleeding in up to 40% of patients treated with Dasatinib.