The c-Src inhibitor dasatanib exhibits a low threshold for migration inhibition. (A) Dose-response curves of human platelets in migration, retraction, activation and in vitro thrombus formation assays to doses of dasatinib (10 μM – 1 nM), normalized to sham-treated samples (%). Supplemental Figure 4F-G for corresponding micrographs and statistical analyses. (B) Representative micrographs of confocal images of human platelets migrating on fibrinogen-albumin matrices treated with 10 nM dasatinib or PBS. Quantification of migrating platelets and the cleared area per cell (μm²) (n = 4 biological replicates). Student t test, 2-tailed, unpaired. Scale bar, 50 μm. (C) Cell-based shape analysis of cell size, circularity, aspect ratio and the number (#) of filopodia of migrating platelets exposed to sham treatment or dasatinib 10 nM. Student t test, 2-tailed, unpaired. (D) Representative micrographs of confocal images of whole blood pretreated with sham treatment or dasatinib 10 nM superfused over a collagen matrix (shear rate 1000/s). Scale bar, 50 μm. Quantification of CD41- and P-selectin (PSEL)/CD62P-positive areas. n = 4. Student t test, 2-tailed, unpaired. (E) Representative confocal images of migrating human platelets treated with dasatinib 10 nM or vehicle and stained for CD41 (white), total c-Src (blue) and phospho-c-Src (Tyr418, red). Scale bar, 10 μm. White arrowheads indicate punctual phospho-c-Src localization at the leading edge, white lines represent migration tracks. Right panels: cell-based quantification of MFI of > 50 platelets from n = 2 biological replicates. (F) Representative western blots of human platelet lysates (n = 3) treated with vehicle or dasatinib before activation with ADP and thromboxane. Lower panels: quantification of densitometry. 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.