凝血酶調節素(thrombomodulin, TM)為表現於細胞膜上的第一型膜醣蛋白(type I cell surface glycoprotein)，是體內的抗凝血因子 (anticoagulant)之一，在生理止血機制過程中扮演相當重要的角色。在正常的血管壁，內皮細胞表現大量的凝血酶調節素；然而在病態的情況下，如發生動脈硬化的血管壁中，內皮細胞的表現量急遽下降，凝血酶調節素主要是由血管平滑肌細胞所表現。目前對於凝血酶調節素在血管平滑肌細胞中表現的調控與生理意義均不甚清楚。本研究的目的在於探討血管平滑肌細胞表現凝血酶調節素之分子機制。目前已知的蛋白質當中，對血管平滑肌細胞增生與移行刺激效果最大的是血小板衍生生長因子(Platlet-derived growth factor; PDGF)。在血小板衍生生長因子的刺激下，人類主動脈平滑肌細胞增加凝血酶調節素之表現最高可達7.5倍之多，且呈現時間依賴性(time-dependent)與濃度依賴性(dose-dependent)之關係，同時也增加了活化態的蛋白質C (activated protein C)。在體外培養，去內皮的小鼠主動脈，血小板衍生生長因子亦可增加凝血酶調節素之表現達2倍。以cycloheximide停止蛋白質的轉譯，完全抑制血小板衍生生長因子增加凝血酶調節素之訊息RNA (mRNA)的作用，顯示此刺激作用需要有新蛋白的合成。以src kinases, PI3-kinase, mTOR之抑制劑進行前處理，或是在細胞內過量表現不活化的Akt分子，皆可以有效的阻斷血小板衍生生長因子增加凝血酶調節素之作用。相反的，過量表現持續活化的Akt分子則增加凝血酶調節素之表現。在不同長度的啟動子活性分析上，發現血小板衍生生長因子顯著地增加凝血酶調節素啟動子之活性，且核心啟動子片段位於轉譯起始點前-394到-255與-111到-34 bp的位置中。轉錄因子Ets-1的表現會因為血小板衍生生長因子的刺激而增加，且受到PI3-kinase與mTOR分子之調控。利用RNA干擾 (RNA interference)的技術減少Ets-1的表現量有效地降低血小板衍生生長因子所增加之凝血酶調節素。反之，細胞內大量表現Ets-1的結果會增加凝血酶調節素之表現量。利用染色質免疫沉澱技術 (chromatin immunoprecipitation; CHIP)及膠體電泳位移分析法(gel electrophoretic mobility shift assay; EMSA)證實，血小板衍生生長因子顯著地增加Ets-1與凝血酶調節素啟動子之結合。進一步分析後發現，Ets-1與凝血酶調節素啟動子結合的位置主要在 -369 到 -345 bp之間。以上的實驗結果顯示，在血管平滑肌細胞中，血小板衍生生長因子透過src kinases/PI3-kinase/Akt/mTOR訊息傳遞路徑而增加凝血酶調節素之表現，且轉錄因子Ets-1是調控人類凝血酶調節素基因表現的必要因子。

Thrombomodulin (TM), a potent anticoagulant, is not expressed in quiescent vascular smooth muscle cells (VSMCs). During vascular remodeling, TM is expressed in VSMCs but the regulatory mechanisms remain unclear. This study examined molecular mechanisms for TM expression in VSMCs. Platelet-derived growth factor-BB (PDGF-BB) treatment upregulated TM protein levels by 7.5- fold in time- and dosage-dependent manner in cultured human aortic VSMCs. PDGF-induced TM is functional in activating protein C. In isolated endothelium-denuded aortae, TM expression was stimulated over 2-fold by PDGF treatment. Addition of cycloheximide abolished PDGF-stimulated TM mRNA expression, suggesting that the enhancement of TM transcription requires de novo protein synthesis. The induction was eliminated by pre-incubation with inhibitors of Src, phosphatidylinositol 3-kinase (PI3-kinase) and mammalian target of rapamycin (mTOR) and by overexpressing dominant-negative Akt while overexpressing constitutively active Akt stimulated TM expression. PDGF-BB treatment activated TM promoter and deletion of a sequence segment -394/-255 drastically reduced TM promoter activity whereas that of segment -111/-34 eliminated the remaining activity. Transcription factor E26 transformation-specific sequence-1 (Ets-1) was upregulated by PDGF-BB in PI3-kinase- and mTOR-dependent manner. RNA interference of Ets-1 inhibited PDGF induction of TM, in contrast, overexpressing Ets-1 increased TM expression. Chromatin immunoprecipitation and electrophoretic mobility shift assay detected increased Ets-1 binding to the region located between -369 and -345 bp of TM promoter after PDGF treatment. These results suggest that in VSMC, PDGF-BB stimulates TM expression that is mainly mediated by Ets-1 via Src kinase /PI3-kinase/Akt/mTOR signaling pathway.

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