Interaction between GPVI and galectin-3 promotes tumor cell–induced platelet activation, degranulation, and transendothelial migration. (A-C) Representative SEM images of mouse platelets adhering to recombinant galectin-3. Washed WT mouse platelets were incubated for 10 minutes with 10 µmol/L of the Syk inhibitor Bay 61-3606 (A) and Src-kinase inhibitor PP1 and its nonfunctional analog PP3 (B) and was allowed to adhere to galectin-3–coated surfaces. (C) Similar experiments were performed with Syk^+/+ and Syk^−/− mouse platelets. The morphology of the adhering platelets was examined after 1 hour, and (D) the percentage of spread platelets was quantified for each condition. The bar represents 20 µm. Mean ± standard deviation (SD) of n = 4 mice per group; *P < .05, by Mann-Whitney test. (E) Relative levels of ATP released from platelets (Syk^+/+ and Syk^−/−) adhering to dalectin-3. Mean ± SD; n = 4 mice per group; *P < .05, by Mann-Whitney test. (F) Relative levels of ATP released from WT and Gp6^−/− platelets incubated with control MC38 (Ctrl) or Lgals3 KO tumor cells. Data are presented as the mean ± SD of 4 separate experiments. *P < .05, **P < .01, by 1-way analysis of variance followed by Tukey’s post hoc test. (G) bEnd.3 mouse endothelial cells (ECs) incubated with supernatants derived from platelets (Plts) tumor cell (TC) cocultures (WT Plts+MC38 ctrl or Lgals3 KO TC) and (Gp6^−/− Plts+MC38 Ctrl or Lgals3 KO TC), or with 10 µM ATPγS, alone or in combination with 20 U/mL apyrase, for 8 hours. Representative immunofluorescence images of ECs with an anti-VE-cadherin antibody (red). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue). The bar represents 10 µm. (H-I) MC38 and AT-3 ctrl or Lgals3 KO tumor cells were seeded on endothelial cells (ECs), and tumor cell transmigration was determined in the absence or presence of WT, Gp6^−/−, and Unc13d^−/− plts. Data are presented as the mean ± SD of 6 separate experiments. **P < .01, by Mann-Whitney test.