title: Prothrombin Loading of Vascular Smooth Muscle Cell-Derived Exosomes Regulates Coagulation and Calcification.
abstract: OBJECTIVE
  The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification.
  
  
  RESULTS
  Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleation site formation on the exosomal surface. PT was deposited in the calcified vasculature, and there was a negative correlation between vascular calcification and the levels of circulating PT. In addition, we found that VSMC exosomes induced thrombogenesis in a tissue factor-dependent and PS-dependent manner.
  
  
  CONCLUSIONS
  Gamma-carboxylated coagulation proteins are potent inhibitors of vascular calcification suggesting warfarin action on these factors also contributes to accelerated calcification in patients receiving this drug. VSMC exosomes link calcification and coagulation acting as novel activators of the extrinsic coagulation pathway and inducers of calcification in the absence of Gla-containing inhibitors.
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Alexander N
authors.initials: AN
authors.lastname: Kapustin
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Michael
authors.initials: M
authors.lastname: Schoppet
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Leon J
authors.initials: LJ
authors.lastname: Schurgers
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Joanne L
authors.initials: JL
authors.lastname: Reynolds
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Rosamund
authors.initials: R
authors.lastname: McNair
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Alexander
authors.initials: A
authors.lastname: Heiss
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Willi
authors.initials: W
authors.lastname: Jahnen-Dechent
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Tilman M
authors.initials: TM
authors.lastname: Hackeng
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Georg
authors.initials: G
authors.lastname: Schlieper
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.).
authors.forename: Paul
authors.initials: P
authors.lastname: Harrison
authors.affiliation: From the BHF Centre of Research Excellence, Department of Cardiology, Cardiovascular Division, King's College London, United Kingdom (A.N.K., J.L.R., R.M.N., C.M.S.); Department of Internal Medicine and Cardiology, Philipps-University, Marburg, Germany (M.S.); Department of Biochemistry, Cardiovascular Research Institute CARIM, University of Maastricht, The Netherlands (L.J.S., T.M.H.); Department of Biomedical Engineering (A.H., W.J.-D.) and Department of Nephrology and Clinical Immunology (G.S.), RWTH Aachen University, Germany; and Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, United Kingdom (P.H.). cathy.shanahan@kcl.ac.uk.
authors.forename: Catherine M
authors.initials: CM
authors.lastname: Shanahan
mesh_terms: D000368:Aged; D000925:Anticoagulants / Q000009:adverse effects; D001777:Blood Coagulation / Q000187:drug effects; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002478:Cells, Cultured; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D055354:Exosomes / Q000187:drug effects / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D010455:Peptides / Q000494:pharmacology; D010718:Phosphatidylserines / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D021381:Protein Transport; D011516:Prothrombin / Q000378:metabolism*; D015398:Signal Transduction; D061205:Vascular Calcification / Q000139:chemically induced / Q000378:metabolism* / Q000473:pathology / Q000517:prevention & control; D014859:Warfarin / Q000009:adverse effects; D000097568:Matrix Gla Protein
chemical_list: D000925:Anticoagulants; D002135:Calcium-Binding Proteins; D016326:Extracellular Matrix Proteins; D010455:Peptides; D010718:Phosphatidylserines; D011516:Prothrombin; D014859:Warfarin; D000097568:Matrix Gla Protein
keywords: anticoagulants; exosomes; myocytes, smooth muscle; prothrombin; vascular calcification