Alice Y. Pollitt, Natalie S. Poulter, Eelo Gitz, Leyre Navarro-Nuñez, Ying-Jie Wang, Craig E. Hughes, Steven G. Thomas, Bernhard Nieswandt, Michael R. Douglas, Dylan M. Owen, David G. Jackson, Michael L. Dustin, Steve P. Watson

• The interaction of CLEC-2 on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signalling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signalling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin.The interaction of CLEC-2 on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signalling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signalling via Src family and Syk tyrosine kinases promotes platelet adhesion to primary mouse lymphatic endothelial cells at low shear. Using supported lipid bilayers containing mobile Podoplanin, we further show that activation of Src and Syk in platelets promotes clustering of CLEC-2 and Podoplanin. Clusters of CLEC-2-bound Podoplanin migrate rapidly to the centre of the platelet to form a single structure. Fluorescence life-time imaging demonstrates that molecules within these clusters are within 10 nm of one another and that the clusters are disrupted by inhibition of Src and Syk family kinases. CLEC-2 clusters are also seen in platelets adhered to immobilised Podoplanin using direct stochastic optical reconstruction microscopy (dSTORM). These findings provide mechanistic insight by which CLEC-2 signalling promotes adhesion to Podoplanin and regulation of Podoplanin signalling thereby contributing to lymphatic vasculature development.…