title: Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis.
abstract: The platelet receptor CLEC-2 is involved in thrombosis/hemostasis, but its ligand, podoplanin, is expressed only in advanced atherosclerotic lesions. We investigated CLEC-2 ligands in vessel walls. Recombinant CLEC-2 bound to early atherosclerotic lesions and normal arterial walls, co-localizing with vascular smooth muscle cells (VSMCs). Flow cytometry and immunocytochemistry showed that recombinant CLEC-2, but not an anti-podoplanin antibody, bound to VSMCs, suggesting that CLEC-2 ligands other than podoplanin are present in VSMCs. VSMCs stimulated platelet granule release and supported thrombus formation under flow, dependent on CLEC-2. The time to occlusion in a FeCl3-induced animal thrombosis model was significantly prolonged in the absence of CLEC-2. Because the internal elastic lamina was lacerated in our FeCl3-induced model, we assume that the interaction between CLEC-2 and its ligands in VSMCs induces thrombus formation. Protein arrays and Biacore analysis were used to identify S100A13 as a CLEC-2 ligand in VSMCs. However, S100A13 is not responsible for the above-described VSMC-induced platelet activation, because S100A13 is not expressed on the surface of normal VSMCs. S100A13 was released upon oxidative stress and expressed in the luminal area of atherosclerotic lesions. Suspended S100A13 did not activate platelets, but immobilized S100A13 significantly increased thrombus formation on collagen-coated surfaces. Taken together, we proposed that VSMCs stimulate platelets through CLEC-2, possibly leading to thrombus formation after plaque erosion and stent implantation, where VSMCs are exposed to blood flow. Furthermore, we identified S100A13 as one of the ligands on VSMCs.
authors.affiliation: Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Osamu
authors.initials: O
authors.lastname: Inoue
authors.affiliation: Department of Pharmacology, Hamamatsu University School of Medicine, Shizuoka, Japan.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Hokamura
authors.affiliation: Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Toshiaki
authors.initials: T
authors.lastname: Shirai
authors.affiliation: Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Osada
authors.affiliation: Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Nagaharu
authors.initials: N
authors.lastname: Tsukiji
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
authors.forename: Kinta
authors.initials: K
authors.lastname: Hatakeyama
authors.affiliation: Department of Pharmacology, Hamamatsu University School of Medicine, Shizuoka, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Umemura
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan.
authors.forename: Yujiro
authors.initials: Y
authors.lastname: Asada
authors.affiliation: Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Katsue
authors.initials: K
authors.lastname: Suzuki-Inoue
authors.affiliation: Department of Clinical and Laboratory Medicine, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Yukio
authors.initials: Y
authors.lastname: Ozaki
mesh_terms: D000818:Animals; D001161:Arteriosclerosis / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D015153:Blotting, Western; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D037181:Lectins, C-Type / Q000235:genetics / Q000378:metabolism*; D008024:Ligands; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010974:Platelet Aggregation; D011485:Protein Binding; D009418:S100 Proteins / Q000378:metabolism; D013927:Thrombosis / Q000378:metabolism*; D000098853:Podoplanin
chemical_list: D037181:Lectins, C-Type; D008024:Ligands; D008562:Membrane Glycoproteins; D009418:S100 Proteins; C529518:CLEC-2 protein, mouse; C104778:CLEC2B protein, human; C117468:PDPN protein, human; D000098853:Podoplanin; C511523:S100A13 protein, human