title: Targeted downregulation of platelet CLEC-2 occurs through Syk-independent internalization.
abstract: Platelet aggregation at sites of vascular injury is not only essential for hemostasis, but may also cause acute ischemic disease states such as myocardial infarction or stroke. The hemi-immunoreceptor tyrosine-based activation motif-containing C-type lectinlike receptor 2 (CLEC-2) mediates powerful platelet activation through a Src- and spleen tyrosine kinase (Syk)-dependent tyrosine phosphorylation cascade. Thereby, CLEC-2 not only contributes to thrombus formation and stabilization but also plays a central role in blood-lymphatic vessel development, tumor metastasis, and prevention of inflammatory bleeding, making it a potential pharmacologic target to modulate these processes. We have previously shown that injection of the anti-CLEC-2 antibody, INU1, results in virtually complete immunodepletion of platelet CLEC-2 in mice, which is, however, preceded by a severe transient thrombocytopenia thereby limiting its potential therapeutic use. The mechanisms underlying this targeted CLEC-2 downregulation have remained elusive. Here, we show that INU1-induced CLEC-2 immunodepletion occurs through Src-family kinase-dependent receptor internalization in vitro and in vivo, presumably followed by intracellular degradation. In mice with platelet-specific Syk deficiency, INU1-induced CLEC-2 internalization/degradation was fully preserved whereas the associated thrombocytopenia was largely prevented. These results show for the first time that CLEC-2 can be downregulated from the platelet surface through internalization in vitro and in vivo and that this can be mechanistically uncoupled from the associated antibody-induced thrombocytopenia.
authors: Department of Experimental Biomedicine, University Hospital of Würzburg and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany;
authors: Viola
authors: V
authors: Lorenz
authors: Department of Experimental Biomedicine, University Hospital of Würzburg and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany;
authors: David
authors: D
authors: Stegner
authors: Department of Experimental Biomedicine, University Hospital of Würzburg and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany;
authors: Simon
authors: S
authors: Stritt
authors: Department of Experimental Biomedicine, University Hospital of Würzburg and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany;
authors: Timo
authors: T
authors: Vögtle
authors: Mammalian Cell Signaling Laboratory, Department of Vascular Cell Biology, Max Planck Institute for Molecular Biomedicine, Münster, Germany;
authors: Friedemann
authors: F
authors: Kiefer
authors: Institute of Genetics, University of Bonn, Bonn, Germany;
authors: Walter
authors: W
authors: Witke
authors: Walter-Brendel-Centre of Experimental Medicine, Department of Cardiovascular Physiology and Pathophysiology, Ludwig-Maximilians-University München, München, Germany; and.
authors: Jürgen
authors: J
authors: Schymeinsky
authors: Centre for Cardiovascular Sciences, Institute of Biomedical Research, School of Clinical and Experimental Medicine, College of Medical and Dental Sciences, University of Birmingham, Birmingham, United Kingdom.
authors: Steve P
authors: SP
authors: Watson
authors: Walter-Brendel-Centre of Experimental Medicine, Department of Cardiovascular Physiology and Pathophysiology, Ludwig-Maximilians-University München, München, Germany; and.
authors: Barbara
authors: B
authors: Walzog
authors: Department of Experimental Biomedicine, University Hospital of Würzburg and Rudolf Virchow Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany;
authors: Bernhard
authors: B
authors: Nieswandt
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D015536:Down-Regulation; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D037181:Lectins, C-Type / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015539:Platelet Activation / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D000072377:Syk Kinase; D013921:Thrombocytopenia / Q000139:chemically induced
chemical_list: D000911:Antibodies, Monoclonal; C529518:CLEC-2 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D037181:Lectins, C-Type; D011505:Protein-Tyrosine Kinases; D000072377:Syk Kinase; C000606897:Syk protein, mouse