title: Platelet adhesion to podoplanin under flow is mediated by the receptor CLEC-2 and stabilised by Src/Syk-dependent platelet signalling.
abstract: Platelet-specific deletion of CLEC-2, which signals through Src and Syk kinases, or global deletion of its ligand podoplanin results in blood-filled lymphatics during mouse development. Platelet-specific Syk deficiency phenocopies this defect, indicating that platelet activation is required for lymphatic development. In the present study, we investigated whether CLEC-2-podoplanin interactions could support platelet arrest from blood flow and whether platelet signalling is required for stable platelet adhesion to lymphatic endothelial cells (LECs) and recombinant podoplanin under flow. Perfusion of human or mouse blood over human LEC monolayers led to platelet adhesion and aggregation. Following αIIbβ3 blockade, individual platelets still adhered. Platelet binding occurred at venous but not arterial shear rates. There was no adhesion using CLEC-2-deficient blood or to vascular endothelial cells (which lack podoplanin). Perfusion of human blood over human Fc-podoplanin (hFcPDPN) in the presence of monoclonal antibody IV.3 to block FcγRIIA receptors led to platelet arrest at similar shear rates to those used on LECs. Src and Syk inhibitors significantly reduced global adhesion of human or mouse platelets to LECs and hFcPDPN. A similar result was seen using Syk-deficient mouse platelets. Reduced platelet adhesion was due to a decrease in the stability of binding. In conclusion, our data reveal that CLEC-2 is an adhesive receptor that supports platelet arrest to podoplanin under venous shear. Src/Syk-dependent signalling stabilises platelet adhesion to podoplanin, providing a possible molecular mechanism contributing to the lymphatic defects of Syk-deficient mice.
authors.affiliation: Leyre Navarro-Núñez, Centre for Cardiovascular Sciences, Institute for Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, B15 2TT, United Kingdom, Tel.: +44 1214158678; Fax: +44 1214158817, E-mail: l.navarronunez@bham.ac.uk.
authors.forename: Leyre
authors.initials: L
authors.lastname: Navarro-Núñez
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Pollitt
authors.forename: Kate
authors.initials: K
authors.lastname: Lowe
authors.forename: Arusa
authors.initials: A
authors.lastname: Latif
authors.forename: Gerard B
authors.initials: GB
authors.lastname: Nash
authors.affiliation: Steve P. Watson, Centre for Cardiovascular Sciences, Institute for Biomedical Research, College of Medical and Dental Sciences, University of Birmingham, B15 2TT, United Kingdom, Tel.: +44 1214158678; Fax: +44 1214158817, E-mail: s. p.watson@bham.ac.uk.
authors.forename: Steve P
authors.initials: SP
authors.lastname: Watson
mesh_terms: D000483:Alleles; D000818:Animals; D001792:Blood Platelets / Q000378:metabolism*; D002448:Cell Adhesion; D042783:Endothelial Cells / Q000166:cytology; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D037181:Lectins, C-Type / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018715:Microscopy, Video; D015539:Platelet Activation / Q000502:physiology; D010973:Platelet Adhesiveness; D010974:Platelet Aggregation; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000378:metabolism; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D000072377:Syk Kinase; D019061:src-Family Kinases / Q000378:metabolism*; D000098853:Podoplanin
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011505:Protein-Tyrosine Kinases; D011994:Recombinant Proteins; D000072377:Syk Kinase; D019061:src-Family Kinases; C529518:CLEC-2 protein, mouse; C104778:CLEC2B protein, human; C490441:Pdpn protein, mouse; D000098853:Podoplanin; C117468:PDPN protein, human; C000606896:SYK protein, human; C000606897:Syk protein, mouse
keywords: Platelet physiology; adhesion molecules; lymphatics; rheology; signal transduction