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The interaction of C-type lectin receptor 2 (CLEC-2) on platelets with Podoplanin on lymphatic endothelial cells initiates platelet signaling events that are necessary for prevention of blood-lymph mixing during development. In the present study, we show that CLEC-2 signaling 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 center of the platelet to form a single structure. Fluorescence lifetime 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 immobilized Podoplanin using direct stochastic optical reconstruction microscopy. These findings provide mechanistic insight by which CLEC-2 signaling promotes adhesion to Podoplanin and regulation of Podoplanin signaling, thereby contributing to lymphatic vasculature development.

Original publication

DOI

10.1074/jbc.M114.584284

Type

Journal article

Journal

J Biol Chem

Publication Date

26/12/2014

Volume

289

Pages

35695 - 35710

Keywords

CLEC-2, Endothelial Cell, ITAM, Lipid Bilayer, Platelet, Podoplanin, Receptor, Src Family Kinase, Syk, Tyrosine-Protein Kinase (Tyrosine Kinase), Animals, Cell Adhesion, Cell Membrane, Cells, Cultured, Endothelial Cells, HEK293 Cells, Humans, Intracellular Signaling Peptides and Proteins, Lectins, C-Type, Lymphoid Tissue, Membrane Glycoproteins, Mice, Inbred C57BL, Mice, Transgenic, Platelet Adhesiveness, Protein Transport, Protein-Tyrosine Kinases, Signal Transduction, Syk Kinase, src-Family Kinases