AIMS: 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. METHODS AND 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. CONCLUSION: rTMD23 protein significantly reduces atherosclerosis and neointima formation through its thrombin-binding ability.
AIMS: 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. METHODS AND 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. CONCLUSION: rTMD23 protein significantly reduces atherosclerosis and neointima formation through its thrombin-binding ability.
Authors: Ronald Carnemolla; Carlos H Villa; Colin F Greineder; Sergei Zaitsev; Kruti R Patel; M Anna Kowalska; Dmitriy N Atochin; Douglas B Cines; Don L Siegel; Charles T Esmon; Vladimir R Muzykantov Journal: FASEB J Date: 2016-11-11 Impact factor: 5.191
Authors: Cláudia N Ferreira; Maria G Carvalho; Karina B Gomes; Helton J Reis; Ana-Paula Fernandes; András Palotás; Marinez O Sousa Journal: Exp Biol Med (Maywood) Date: 2014-07-29
Authors: Benjamin Valente-Acosta; Manuel Alfonso Baños-González; Marco Antonio Peña-Duque; Marco Antonio Martínez-Ríos; Leslie Quintanar-Trejo; Gad Aptilon-Duque; Mirthala Flores-García; David Cruz-Robles; Guillermo Cardoso-Saldaña; Aurora de la Peña-Díaz Journal: Cardiol Res Pract Date: 2016-08-11 Impact factor: 1.866
Authors: Julian I Borissoff; Jeroen J T Otten; Sylvia Heeneman; Peter Leenders; René van Oerle; Oliver Soehnlein; Sarah T B G Loubele; Karly Hamulyák; Tilman M Hackeng; Mat J A P Daemen; Jay L Degen; Hartmut Weiler; Charles T Esmon; Joanne van Ryn; Erik A L Biessen; Henri M H Spronk; Hugo ten Cate Journal: PLoS One Date: 2013-02-07 Impact factor: 3.240