title: Exogenous heparin binds and inhibits bone morphogenetic protein 6 biological activity.
abstract: OBJECTIVE
  The purpose of this study was to explore the effect of heparin on bone morphogenetic protein 6 (BMP6) osteogenic activity.
  
  
  METHODS
  Western blot analysis was used to confirm the binding of BMP6 to heparin and to observe its effect on BMP6 signaling in C2C12-BRE-Luc myoblasts. Real-time RT-PCR was performed for the expression analysis of alkaline phosphatase (ALP) and osteocalcin (OC) in C2C12 myoblasts treated with BMP6 and heparin for 72 hours. Rat ectopic bone formation assay was performed to explore the effect of heparin on BMP6 osteogenic activity. Two weeks following implantation the implants were analysed morphologically and histologically. A mouse osteoporotic model was used to test the ability of BMP6 to improve the bone quality in vivo in the presence of heparin, followed by DEXA and μCT analyses. Blood coagulation was tested in rats previously treated with BMP6.
  
  
  RESULTS
  BMP6 specifically bound to heparin and induced Smad1/5/8 phosphorylation which was inhibited by heparin. After 48 and 72 hours of treatment, heparin inhibited BMP6-induced ALP and OC expression in C2C12 cells. Heparin dose dependently inhibited BMP6-induced new bone and cartilage formation in the rat ectopic bone formation assay, while in osteoporotic mice heparin inhibited the BMP6 potential to improve the bone quality as evidenced by decreased bone mineral density and trabecular bone parameters. Interestingly, BMP6 prevented the effect of heparin on the blood coagulation parameters.
  
  
  CONCLUSIONS
  The interaction of BMP6 with heparin might contribute to the heparin-induced osteoporosis and blood coagulation.
authors.affiliation: Laboratory for Mineralized Tissues, Center for Translational and Clinical Research, School of Medicine, University of Zagreb, Salata 11, 10000, Zagreb, Croatia. jelena.brkljacic@mef.hr
authors.forename: Jelena
authors.initials: J
authors.lastname: Brkljacic
authors.forename: Martina
authors.initials: M
authors.lastname: Pauk
authors.forename: Igor
authors.initials: I
authors.lastname: Erjavec
authors.forename: Antonio
authors.initials: A
authors.lastname: Cipcic
authors.forename: Lovorka
authors.initials: L
authors.lastname: Grgurevic
authors.forename: Renata
authors.initials: R
authors.lastname: Zadro
authors.forename: Gareth J
authors.initials: GJ
authors.lastname: Inman
authors.forename: Slobodan
authors.initials: S
authors.lastname: Vukicevic
mesh_terms: D000818:Animals; D000925:Anticoagulants / Q000494:pharmacology*; D055418:Bone Morphogenetic Protein 6 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D004195:Disease Models, Animal; D006493:Heparin / Q000494:pharmacology*; D051379:Mice; D010012:Osteogenesis / Q000187:drug effects*; D010024:Osteoporosis / Q000503:physiopathology; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D000925:Anticoagulants; D055418:Bone Morphogenetic Protein 6; D006493:Heparin