Syk and Src family kinases regulate C-type lectin receptor 2 (clec-2)-mediated clustering of podoplanin and platelet adhesion to lymphatic endothelial cells

Research output: Contribution to journalArticle

External organisations

  • University Medical Center Utrecht, Department of Clinical Chemistry and Haematology, 3584 CX, Utrecht, The Netherlands
  • Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom
  • Department of Neurology, Dudley Group National Health Service Foundation Trust, Dudley DY1 2HQ, United Kingdom
  • Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Diseases, University of Oxford, Headington OX3 7FY, United Kingdom
  • Department of Molecular Pathogenesis, New York University, Skirball Institute of Biomolecular Medicine, School of Medicine, New York University Langone Medical Center, New York, New York 10016
  • Department of Experimental Biomedicine; University Hospital, University of Würzburg; Würzburg Germany

Abstract

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.

Details

Original languageEnglish
Pages (from-to)35695-35710
Number of pages16
JournalJournal of Biological Chemistry
Volume289
Issue number52
Early online date3 Nov 2014
Publication statusPublished - 26 Dec 2014

Keywords

  • Endothelial Cell, Lipid Bilayer, Platelet, Receptor, Tyrosine-Protein Kinase (Tyrosine Kinase), CLEC-2, ITAM, Podoplanin, Src Family Kinase, Syk