Introduction: Podoplanin, a transmembrane receptor expressed on lymphatic endothelial cells (LEC), is the only known endogenous ligand for the platelet receptor CLEC-21. Both of these proteins are thought to be involved in lymphatic development as mice deficient in either CLEC-2 or podoplanin develop a blood-lymphatic mixing phenotype. Work by others has also shown that podoplanin knockdown reduces LEC migration in response to vascular endothelial growth factor C (VEGF-C)2. Therefore, we aimed to assess whether the interaction between podoplanin and CLEC-2 on platelets regulates LEC migration, and to begin to uncover the signalling mechanism underlying this. Methods: Transfilter assays were used to assess human LEC and human microvascular endothelial cell (HMEC)-1 migration. VEGF-C solution or normal culture medium was placed in the lower chamber of the well before endothelial cells were seeded onto the filter. After an hour, washed human platelets, antibodies or a Rho inhibitor (CT04; Cytoskeleton Inc., CO) were added to the filter. Percentage transmigration was assessed after 12 or 24 hours. A paired t-test was used to determine significance of single treatments. To compare several conditions, an ANOVA was performed, followed by post hoc Dunnett test. Results: Platelets inhibited VEGF-C mediated increase in LEC migration in a count-dependent manner, but had no effect in the absence of VEGF-C (P<0.05; N=4). Similarly, platelets were able to inhibit VEGF-C mediated increase in migration of HMEC-1 cells (P<0.05; N=3), which are described as a microvascular endothelial cell line, but are known to express podoplanin and vascular endothelial growth factor receptor 3 (VEGFR3)3. Crosslinking podoplanin with an anti-human podoplanin antibody and an appropriate secondary also inhibited VEGF-C mediated LEC migration (P<0.01; N=3). Rho inhibitor CT04 inhibited migration but combining the inhibitor with crosslinking did not have an additive effect. Conclusions: Platelets have a strong inhibitory effect on LEC migration in response to VEGF-C, which may be due to the interaction of CLEC-2 and podoplanin. Platelets also inhibited VEGF-C mediated migration of HMEC-1, further supporting the hypothesis that this is due to the interaction of CLEC-2 and podoplanin. Antibody-mediated crosslinking also inhibited VEGF-C mediated LEC migration, suggesting that CLEC-2 may be clustering podoplanin. Combining crosslinking with the Rho inhibitor CT04 did not have an extra inhibitory effect, suggesting that the effects of crosslinking podoplanin may involve Rho signalling.