title: Syk and Src family kinases regulate C-type lectin receptor 2 (CLEC-2)-mediated clustering of podoplanin and platelet adhesion to lymphatic endothelial cells.
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.
authors.affiliation: 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.
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Pollitt
authors.affiliation: 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.
authors.forename: Natalie S
authors.initials: NS
authors.lastname: Poulter
authors.affiliation: 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, the University Medical Center Utrecht, Department of Clinical Chemistry and Haematology, 3584 CX, Utrecht, The Netherlands.
authors.forename: Eelo
authors.initials: E
authors.lastname: Gitz
authors.affiliation: 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.
authors.forename: Leyre
authors.initials: L
authors.lastname: Navarro-Nuñez
authors.affiliation: the Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom.
authors.forename: Ying-Jie
authors.initials: YJ
authors.lastname: Wang
authors.affiliation: 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.
authors.forename: Craig E
authors.initials: CE
authors.lastname: Hughes
authors.affiliation: 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.
authors.forename: Steven G
authors.initials: SG
authors.lastname: Thomas
authors.affiliation: the Department of Experimental Biomedicine, University Hospital, University of Würzburg, Würzburg 97080, Germany.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Nieswandt
authors.affiliation: the School of Immunity and Infection, University of Birmingham, Birmingham B15 2TT, United Kingdom, the Department of Neurology, Dudley Group National Health Service Foundation Trust, Dudley DY1 2HQ, United Kingdom.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Douglas
authors.affiliation: the Randall Division of Cell and Molecular Biophysics, King's College London, Guy's Campus, London SE1 1UL, United Kingdom.
authors.forename: Dylan M
authors.initials: DM
authors.lastname: Owen
authors.affiliation: the Human Immunology Unit, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Headington, Oxford OX3 9DS, United Kingdom.
authors.forename: David G
authors.initials: DG
authors.lastname: Jackson
authors.affiliation: the Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Diseases, University of Oxford, Headington OX3 7FY, United Kingdom, and the 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.
authors.forename: Michael L
authors.initials: ML
authors.lastname: Dustin
authors.affiliation: 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, s.p.watson@bham.ac.uk.
authors.forename: Steve P
authors.initials: SP
authors.lastname: Watson
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000502:physiology*; D037181:Lectins, C-Type / Q000378:metabolism*; D008221:Lymphoid Tissue / Q000166:cytology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D010973:Platelet Adhesiveness; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D015398:Signal Transduction; D000072377:Syk Kinase; D019061:src-Family Kinases / Q000502:physiology*; D000098853:Podoplanin
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; D019061:src-Family Kinases; C529518:CLEC-2 protein, mouse; C490441:Pdpn protein, mouse; D000098853:Podoplanin; C000606896:SYK protein, human; C000606897:Syk protein, mouse
keywords: CLEC-2; Endothelial Cell; ITAM; Lipid Bilayer; Platelet; Podoplanin; Receptor; Src Family Kinase; Syk; Tyrosine-Protein Kinase (Tyrosine Kinase)