Morphodynamic analysis reveals c-Src as central regulator of Arp2/3 in platelet migration. (A) Experimental scheme of morphodynamic inhibitor screening platform: inhibitor screening and quantification of substrate clearance on substrates leading to retraction (cross-linked fibrin layers) or migration (fibrin monomer layers) in combination with morphological analysis of platelet shape. (B) Quantification of cleared area per cell (μm2) of both migrating (left) and retracting human platelets (right panel) in the presence of the indicated inhibitors, with values being normalized to control in %. One-way ANOVA with post hoc Dunnett's testing. (C) Representative morphologies of human platelets treated with the indicated inhibitors. (D) Representative micrographs of human platelets costained for F-actin and myosin activity (pMLC) following incubation with the indicated treatments. (E) Shape analysis of platelet circularity (a.u., left) and platelet size (μm2, right panel) of migrating human platelets treated with the indicated antagonists. One-way ANOVA with post hoc Dunnett’s testing. (F) Quantification of 3D clot retraction of human PRP incubated with the indicated inhibitors. One-way ANOVA with post hoc Dunnett’s testing. (G) Longitudinal micrographs of live imaging of isolated murine LifeAct-eGFP platelets before (panels 0, 12 seconds) and after addition of the Src inhibitor PP2 (panels 162-492 seconds). Pink arrows mark actin nucleation at the leading edge; pink asterisks show filopodia formation. Right panel: Quantification of # of actin waves (per minute) moving along the leading edge. Student t test, 2-tailed, unpaired. (H) Colocalization plot of fibrinogen and human platelet of sham treatment (left) or after treatment with 10 μM PP2 (right panel). (I) Quantification of cleared area in μm2 upon treatment with indicated inhibitors/agonists (sham vs 10 μM PP2 in the presence or absence of 50 mM ADP and 1 mM TRAP). One-way ANOVA with post hoc Dunnett's testing. (J) Quantification of migrating platelets (%) and cleared area per platelet (μm2) (left panels) as well as the absolute number of recruited and relative amount of spreading platelets on fibrinogen (right panels) for human platelets treated with sham or the indicated inhibitors at the indicated concentrations. One-way ANOVA with post hoc Dunnett’s testing. Unless indicated with asterisks, post hoc testing revealed nonsignificant results (P ≥ .05). P values corresponding to asterisks: ∗P < .05, ∗∗P < .01, ∗∗∗P < .01, ∗∗∗∗P < .001. Scale bars, 5 μm.
