Thrombomodulin domains attenuate atherosclerosis by inhibiting thrombin-induced endothelial cell activation
Thrombomodulin domains attenuate atherosclerosis by inhibiting thrombin-induced endothelial cell activation
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DOI：
10.1093/cvr/cvr220
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发表时间：
2011-11-01
影响因子：
10.8
通讯作者：
Wu, Hua-Lin
中科院分区：
文献类型：
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作者：
Wei, Hsi-Ju;Li, Yi-Heng;Wu, Hua-Lin
目的凝血酶通过激活蛋白酶激活受体1（PAR-1）刺激多种细胞效应来调节动脉粥样硬化病变的形成。血栓调节蛋白（TM）通过其结构域2和3（TMD 23）结合凝血酶来抑制凝血酶效应。方法和结果在毕赤酵母表达系统中表达并纯化野生型小鼠rTMD 23和3个凝血酶结合位点改变的突变体rTMD 23（I425 A）、rTMD 23（D424 A/D426 A）和rTMD 23（D424 A/I425 A/D426 A）。野生型rTMD 23（D424 A/D426 A）和rTMD 23（I425 A）能有效地结合凝血酶，激活蛋白C，延长凝血酶凝固时间，而rTMD 23（D424 A/I425 A/D426 A）和rTMD 23（D424 A/I425 A/D426 A）则丧失了这些功能。野生型rTMD 23，而不是rTMD 23（I425 A），降低凝血酶诱导的表面PAR-1内化和内皮细胞（EC）胞浆Ca（2+）浓度的增加。野生型rTMD 23和rTMD 23（D424 A/D426 A）也抑制凝血酶诱导的粘附分子和单核细胞趋化蛋白-1的表达，并增加内皮细胞的通透性，而rTMD 23（I425 A）和rTMD 23（D424 A/I425 A/D426 A）没有这种作用。此外，野生型rTMD 23和rTMD 23（D424 A/D426 A）可有效减少C57 BL/6小鼠颈动脉结扎诱导的新生内膜形成和载脂蛋白E缺陷（ApoE-/-）小鼠的动脉粥样硬化病变形成，而I425 A突变的rTMD 23则显示该功能受损。野生型rTMD 23（I425 A）也能显著抑制PAR-1、粘附分子表达和巨噬细胞含量。结论rTMD 23蛋白通过其凝血酶结合能力显著抑制动脉粥样硬化和新生内膜形成。
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. Wildtype 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.