title: Thrombin induced by the extrinsic pathway and PAR-1 regulated inflammation at the site of fracture repair.
abstract: Thrombin (coagulation factor IIa) is a serine protease encoded by the F2 gene. Pro-thrombin (coagulation factor II) is cut to generate thrombin in the coagulation cascade that results in a reduction of blood loss. Procoagulant states that lead to activation of thrombin are common in bone fracture sites. However, its physiological roles and relationship with osteoblasts in bone fractures are largely unknown. We herein report various effects of thrombin on mouse osteoblastic MC3T3-E1 cells. MC3T3-E1 cells expressed proteinase-activated receptor 1 (PAR1), also known as the coagulation factor II receptor. They also produced monocyte chemoattractant protein (MCP-1), tissue factor (TF), MCSF and IL-6 upon thrombin stimulation through the PI3K-Akt and MEK-Erk1/2 pathways. Furthermore, MCP-1 obtained from thrombin-stimulated MC3T3-E1 cells induced migration by macrophage RAW264 cells. All these effects of thrombin on MC3T3-E1 cells were abolished by the selective non-peptide thrombin receptor inhibitor SCH79797. We also found that thrombin, PAR-1, MCP-1, TF as well as phosphorylated AKT and p42/44 were significantly expressed at the fracture site of mouse femoral bone. Collectively, thrombin/PAR-1 interaction regulated MCP-1, TF, MCSF and IL-6 production by MC3T3-E1 cells. Furthermore, MCP-1 induced RAW264 cell migration. Thrombin may thus be a novel cytokine that regulates several aspects of osteoblast function and fracture healing.
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Sato
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Jiro
authors.initials: J
authors.lastname: Ichikawa
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Wako
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Ohba
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Saito
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Hironao
authors.initials: H
authors.lastname: Sato
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Kensuke
authors.initials: K
authors.lastname: Koyama
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan; The Sports Medicine and Knee Center, Kofu National Hospital, 11-35 Tenjincho, Kofu, Yamanashi 400-8533, Japan.
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Hagino
authors.affiliation: Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, 2200 Children's Way, Nashville, TN 37232-9565, United States; Department of Orthopaedics, Vanderbilt University Medical Center, 2200 Children's Way, Nashville, TN 37232-9565, United States; Department of Center for Bone Biology, Vanderbilt University Medical Center, 2200 Children's Way, Nashville, TN 37232-9565, United States; Department of Pharmacology, Vanderbilt University Medical Center, 2200 Children's Way, Nashville, TN 37232-9565, United States; Department of Pediatrics, Vanderbilt University Medical Center, 2200 Children's Way, Nashville, TN 37232-9565, United States.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Schoenecker
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan. Electronic address: tando@mtg.biglobe.ne.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Ando
authors.affiliation: Department of Orthopaedic Surgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokatou, Chuo, Yamanashi 409-3898, Japan.
authors.forename: Hirotaka
authors.initials: H
authors.lastname: Haro
mesh_terms: D000818:Animals; D002465:Cell Movement; D018932:Chemokine CCL2 / Q000378:metabolism; D004195:Disease Models, Animal; D017102:Fracture Healing; D006801:Humans; D007249:Inflammation / Q000378:metabolism* / Q000473:pathology*; D015850:Interleukin-6 / Q000378:metabolism; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008954:Models, Biological; D010006:Osteoblasts / Q000378:metabolism / Q000473:pathology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D000067996:RAW 264.7 Cells; D044463:Receptor, PAR-1 / Q000378:metabolism*; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D013925:Thromboplastin / Q000378:metabolism
chemical_list: C491768:Ccl2 protein, mouse; D018932:Chemokine CCL2; D015850:Interleukin-6; D044463:Receptor, PAR-1; D013925:Thromboplastin; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D013917:Thrombin
keywords: Fracture model; MCP-1; Migration; PAR-1; Thrombin