Podoplanin expression and tumor cell–induced platelet aggregation and platelet activation in vitro. (A) Podoplanin expression on cancer cell lines “Gli16” and “AMCH” was investigated by flow cytometry. (B) The ability of the cell lines to induce platelet aggregation was investigated by LTA: A total number of 5 × 105 cancer cells were added to 225 µL of PRP,and maximal platelet aggregation (%) after 10 minutes was recorded. Gli16 cells induced significantly stronger platelet aggregation compared with control cell line AMCH (***P < .001). Platelet aggregation in response to Gli16 was abrogated by the antipodoplanin antibody NZ-1, but not by control antibody (***P < .001). ns, not significant. Box plots show data from 5 individual experiments (performed in multiple replicates; total n = 22). (C) Platelet activation and degranulation upon coincubation of 225 µL PRP with 5 × 105 cancer cells was determined by a PF4 ELISA. Gli16 cells induced marked release of PF4 from platelets, which was not observed for AMCH cells. The ability of Gli16 cells to induce PF4 release from platelets could be inhibited by the anti-podoplanin antibody NZ-1, but not by control antibody. The experiment was performed in duplicates.
