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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

The Journal of biological chemistry

Publication Date

12/2014

Volume

289

Pages

35695 - 35710

Addresses

From the University of Birmingham, Centre for Cardiovascular Sciences, Institute of Biomedical Research, College of Medical and Dental Sciences, Edgbaston, Birmingham B15 2TT, United Kingdom, a.y.pollitt@bham.ac.uk.

Keywords

Lymphoid Tissue, Cells, Cultured, Cell Membrane, Endothelial Cells, Animals, Mice, Inbred C57BL, Mice, Transgenic, Humans, src-Family Kinases, Intracellular Signaling Peptides and Proteins, Membrane Glycoproteins, Lectins, C-Type, Cell Adhesion, Signal Transduction, Protein Transport, Platelet Adhesiveness, Protein-Tyrosine Kinases, HEK293 Cells, Syk Kinase