Classical soluble agonists show no effect on platelet migratory behavior once migration is initiated. (A) Quantification of cleared area per platelet (μm2) and representative micrographs of the indicated treatments following platelet migration on albumin/fibrinogen matrices. Continous act. = addition of U46119 and ADP to wash buffer. Wash = regular treatment. Shear = continuous shear stress (15/s). Apyrase and indomethacin were used at 2.9 U/mL and 10 μM, respectively. One-way ANOVA with post hoc Dunnett's testing. (B) Experimental scheme, representative micrograph and quantification of platelets recruited to an albumin/fibrinogen matrix following preincubation with the P2Y12 inhibitor cangrelor (250 nM) and terutroban (1 μg/mL, blocking thromboxane-mediated activation) or sham treatment. Student t test, 2-tailed, unpaired. Scale bar, 5 μm. (C-D) Experimental scheme, representative micrograph, and quantification of cleared area per platelet (μm2) after superfusion of migrating platelets with cangrelor and terutroban or sham treatment. Student t test, 2-tailed, unpaired. Scale bar, 5 μm. (E) Quantification of % migrating cells and the absolute number of adherent cells per field of view (FOV) in a migration assay with human platelets following activation or not with ADP and U46619. Student t test, 2-tailed, unpaired. (F) Quantification of % migrating cells and the absolute number of adherent cells per FOV for the indicated treatments following initiation of platelet migration. One-way ANOVA with post hoc Dunnett's testing. (G) Representative immunofluorescence costainings of migrating human platelets, stained for c-Src, GPIIb and 14-3-3ζ. Lower panels: enlarged depiction of c-Src and 14-3-3ζ colocalization (pink), corresponding to the white rectangle in the merged right upper panel. Scale bars: upper panel 5 μm, lower panel 1 μm. (H) Histogram depicting colocalization of c-Src, CD41/GPIIb and 14-3-3ζ across the leading edge, along the indicated red line in panel G. MFIs were normalized to the maximum intensity of each antigen. (I-J) Representative micrographs and quantification of % migrating platelets and the cleared area per platelet (μm2) of human platelets treated or not with 10 μM 3′,4′,7′-trihydroxyisoflavone (THO). Scale bar, 25 μm. Student t test, 2-tailed, unpaired. 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.
