title: CLEC-2 expression is maintained on activated platelets and on platelet microparticles.
abstract: The C-type lectin-like receptor CLEC-2 mediates platelet activation through a hem-immunoreceptor tyrosine-based activation motif (hemITAM). CLEC-2 initiates a Src- and Syk-dependent signaling cascade that is closely related to that of the 2 platelet ITAM receptors: glycoprotein (GP)VI and FcγRIIa. Activation of either of the ITAM receptors induces shedding of GPVI and proteolysis of the ITAM domain in FcγRIIa. In the present study, we generated monoclonal antibodies against human CLEC-2 and used these to measure CLEC-2 expression on resting and stimulated platelets and on other hematopoietic cells. We show that CLEC-2 is restricted to platelets with an average copy number of ∼2000 per cell and that activation of CLEC-2 induces proteolytic cleavage of GPVI and FcγRIIa but not of itself. We further show that CLEC-2 and GPVI are expressed on CD41+ microparticles in megakaryocyte cultures and in platelet-rich plasma, which are predominantly derived from megakaryocytes in healthy donors, whereas microparticles derived from activated platelets only express CLEC-2. Patients with rheumatoid arthritis, an inflammatory disease associated with increased microparticle production, had raised plasma levels of microparticles that expressed CLEC-2 but not GPVI. Thus, CLEC-2, unlike platelet ITAM receptors, is not regulated by proteolysis and can be used to monitor platelet-derived microparticles.
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences.
authors.forename: Eelo
authors.initials: E
authors.lastname: Gitz
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences.
authors.forename: Alice Y
authors.initials: AY
authors.lastname: Pollitt
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences.
authors.forename: Jerney J
authors.initials: JJ
authors.lastname: Gitz-Francois
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences.
authors.forename: Osama
authors.initials: O
authors.lastname: Alshehri
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences.
authors.forename: Jun
authors.initials: J
authors.lastname: Mori
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences, National Institute for Health Research Surgical Reconstruction and Microbiology Research Centre, and.
authors.forename: Samantha
authors.initials: S
authors.lastname: Montague
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences, National Institute for Health Research Surgical Reconstruction and Microbiology Research Centre, and.
authors.forename: Gerard B
authors.initials: GB
authors.lastname: Nash
authors.affiliation: School of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom; Department of Neurology, Dudley Group National Health Service Foundation Trust, Dudley, United Kingdom; and.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Douglas
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors.forename: Elizabeth E
authors.initials: EE
authors.lastname: Gardiner
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Melbourne, Australia.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Andrews
authors.affiliation: School of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom;
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Buckley
authors.affiliation: School of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom;
authors.forename: Paul
authors.initials: P
authors.lastname: Harrison
authors.affiliation: Centre for Cardiovascular Sciences, College of Medical and Dental Sciences, National Institute for Health Research Surgical Reconstruction and Microbiology Research Centre, and.
authors.forename: Steve P
authors.initials: SP
authors.lastname: Watson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D001172:Arthritis, Rheumatoid / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism*; D006801:Humans; D007249:Inflammation; D037181:Lectins, C-Type / Q000378:metabolism*; D008533:Megakaryocytes / Q000166:cytology; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D015539:Platelet Activation; D040201:Platelet Membrane Glycoprotein IIb / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D017452:Receptors, IgG / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; C104778:CLEC2B protein, human; C080568:Fc gamma receptor IIA; D037181:Lectins, C-Type; D008562:Membrane Glycoproteins; D040201:Platelet Membrane Glycoprotein IIb; D010980:Platelet Membrane Glycoproteins; D017452:Receptors, IgG; D011994:Recombinant Proteins; C098341:platelet membrane glycoprotein VI