title: Thrombomodulin domains attenuate atherosclerosis by inhibiting thrombin-induced endothelial cell activation.
abstract: OBJECTIVE
  Thrombin modulates the formation of atherosclerotic lesions by stimulating a variety of cellular effects through protease-activated receptor-1 (PAR-1) activation. Thrombomodulin (TM) inhibits thrombin effects by binding thrombin through its domains 2 and 3 (TMD23). We investigated whether recombinant TMD23 (rTMD23) could inhibit atherosclerosis via its thrombin-binding ability.
  
  
  RESULTS
  Wild-type mouse rTMD23 and three mutants with altered thrombin-binding sites, rTMD23 (I425A), rTMD23 (D424A/D426A), and rTMD23 (D424A/I425A/D426A), were expressed and purified in the Pichia pastoris expression system. Wild-type rTMD23 and rTMD23 (D424A/D426A) could effectively bind thrombin, activate protein C, and prolong thrombin clotting time, whereas rTMD23 (I425A) and rTMD23 (D424A/I425A/D426A) lost these functions. Wild-type rTMD23, but not rTMD23 (I425A), decreased both the thrombin-induced surface PAR-1 internalization and the increase in cytoplasmic Ca(2+) concentrations in endothelial cells (ECs). Wild-type rTMD23 and rTMD23 (D424A/D426A) also inhibited thrombin-induced adhesion molecules and monocyte chemoattractant protein-1 expression and increased permeability in ECs, whereas rTMD23 (I425A) and rTMD23 (D424A/I425A/D426A) had no such effects. Furthermore, wild-type rTMD23 and rTMD23 (D424A/D426A) were effective in reducing carotid ligation-induced neointima formation in C57BL/6 mice and atherosclerotic lesion formation in apolipoprotein E-deficient (ApoE-/-) mice, whereas rTMD23 with the I425A mutation showed impairment of this function. Wild-type rTMD23, but not rTMD23 (I425A), also markedly suppressed the PAR-1, the adhesion molecules expression, and the macrophage content in the carotid ligation model and ApoE-/- mice.
  
  
  CONCLUSIONS
  rTMD23 protein significantly reduces atherosclerosis and neointima formation through its thrombin-binding ability.
authors.affiliation: Department of Biochemistry and Molecular Biology,  College of Medicine, National Cheng Kung University, Tainan 701,  Taiwan.
authors.forename: Hsi-Ju
authors.initials: HJ
authors.lastname: Wei
authors.forename: Yi-Heng
authors.initials: YH
authors.lastname: Li
authors.forename: Guey-Yueh
authors.initials: GY
authors.lastname: Shi
authors.forename: Shu-Lin
authors.initials: SL
authors.lastname: Liu
authors.forename: Po-Chiao
authors.initials: PC
authors.lastname: Chang
authors.forename: Cheng-Hsiang
authors.initials: CH
authors.lastname: Kuo
authors.forename: Hua-Lin
authors.initials: HL
authors.lastname: Wu
mesh_terms: D000818:Animals; D001057:Apolipoproteins E / Q000172:deficiency / Q000235:genetics; D050197:Atherosclerosis / Q000097:blood / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D001665:Binding Sites; D001777:Blood Coagulation; D002118:Calcium / Q000378:metabolism; D002340:Carotid Artery Diseases / Q000097:blood / Q000378:metabolism / Q000473:pathology / Q000517:prevention & control*; D018932:Chemokine CCL2 / Q000378:metabolism; D004195:Disease Models, Animal; D061307:Human Umbilical Vein Endothelial Cells / Q000378:metabolism*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D009154:Mutation; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D010539:Permeability; D054730:Protein Interaction Domains and Motifs; D044463:Receptor, PAR-1 / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism*; D018180:Thrombomodulin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism
chemical_list: D001057:Apolipoproteins E; C111835:CCL2 protein, human; D018932:Chemokine CCL2; D010446:Peptide Fragments; D044463:Receptor, PAR-1; D011994:Recombinant Proteins; D018180:Thrombomodulin; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D018799:Intercellular Adhesion Molecule-1; D013917:Thrombin; D002118:Calcium