Alexander N Kapustin1, Michael Schoppet1, Leon J Schurgers1, Joanne L Reynolds1, Rosamund McNair1, Alexander Heiss1, Willi Jahnen-Dechent1, Tilman M Hackeng1, Georg Schlieper1, Paul Harrison1, Catherine M Shanahan2. 1. 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.). 2. 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.
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. APPROACH AND 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.
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. APPROACH AND 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: Hanuma Kumar Karnati; Joseph H Garcia; David Tweedie; Robert E Becker; Dimitrios Kapogiannis; Nigel H Greig Journal: J Neurotrauma Date: 2018-10-25 Impact factor: 5.269
Authors: Hong S Lu; Ann Marie Schmidt; Robert A Hegele; Nigel Mackman; Daniel J Rader; Christian Weber; Alan Daugherty Journal: Arterioscler Thromb Vasc Biol Date: 2018-10 Impact factor: 8.311