凝血酶調節素 (thrombomodulin, TM) 是一種表現於血管內皮細胞表面的醣蛋白，是生理上重要的抗凝血因子，其蛋白質結構從氨基端依序被分為類外源凝集素(lectin-like)結構區(TMD1), EGF-like 結構區(TMD2), serine/thrtonine-rich 結構區(TMD3), transmembrane 結構區(TMD4) 及碳端的cytoplamic結構區(D5)。凝血酶調節素在不同組織中扮演著:抑制轉移、血管新生調節素、細胞間的連結並具有在不同組織間的抗發炎反應。在之前的研究中，我們發現TMD23重組蛋白，不論是在體內或是體外實驗，都能促進細胞的增生，移動，以及血管的新生。TMD23 重組蛋白，有效改善了K5-Cre/TMLox/Lox老鼠的傷口癒合情形。至於是哪一個EGF結構區或是因為不同凝血酶調節素，在醣化程度功能上的影響與否，仍然有待研究。在這個研究中，我們利用哺乳類表達系統呈現不同的D2以及D3結構區重組蛋白，分別為TMD2 (EGF1-6) D3, TMD2 (EGF2-6) D3, TMD2 (EGF3-6) D3, TMD2 (EGF4-6) D3, TMD2 (EGF5-6) D3 以及TMD2 (EGF6) D3。每一個重組蛋白都可以純化出兩種不同的醣化型：高分子量以及低分子量重組蛋白。高分子量重組蛋白經過chondroitinase ABC酵素作用後，其分子量即轉變成低分子量重組蛋白。接著我們更進一步證實，高分子量TMD23重組蛋白具有與低分子量TMD23重組蛋白相似的生物活性。高分子量重組蛋白不論在與凝血酶(thrombin)結合、活化蛋白質C或是延長凝血酶引起的凝血時間，對人類臍靜脈內皮細胞(human umbilical vein endothelial cell) 的生長，移動和類似毛細血管生成的調節也比低分子量重組蛋白來的有效。除此之外，高分子量重組蛋白在刮傷癒合(scratch wound healing assay)以及老鼠傷口癒合的分析中，其促進傷口癒合的速度也比低分子量重組蛋白來的迅速。在敗血症研究的動物模式中，我們也發現高分子量重組蛋白可以抑制LPS所引起的TNF-α 及IL-6的釋放。綜合以上的實驗結果，顯示高度醣類修飾的凝血酶調節素重組蛋白，在抗凝血與傷口癒合上具有較強的生物活性，也降低敗血症引起的死亡率，這意味著在臨床藥物上的開發，是具有潛在的實用價值。

Thrombomodulin (TM) is an endothelial cell surface anticoagulant glycoprotein is composed of five distinct domains contain a N-terminal lectin-like domain (D1), six epidermal growth factor (EGF)-like repeats (D2), a serine/threonine-rich region (D3), a transmembrane domain (D4) and a short cytoplasmic tail (D5) that perform antimetastatic, angiogenic, adhesive, and anti-inflammatory functions in various tissues. In our previous study, the recombinant soluble human TM domains 2 and 3 (TMD23) promoted cell proliferation, migration and angiogenesis. The TMD23 significantly improved wound closure in the K5-Cre/TM Lox/Lox mice. However, which epidermal growth factor (EGF)-like structure and whether the presences of carbohydrate were critical for TM functions remains unclear. Human embryonic kidney 293 mammalian expression system was used to prepare various TM domain 2 (D2) plus domain 3 (D3) including TMD2 (EGF1-6) D3, TMD2 (EGF2-6) D3, TMD2 (EGF3-6) D3, TMD2 (EGF4-6) D3, TMD2 (EGF5-6) D3 and TMD2 (EGF6) D3. Each TM mutant contains two glycoforms [high molecular weight (high-Mw) and low molecular weight (low- Mw)]. The high-Mw TMDs were converted into low-Mw TMDs by chondroitinase ABC digestion. The high-Mw TMDs had higher ability to bind thrombin, to activate protein C, prolonged clotting time in a dose-dependent fashion, and had higher activity to promote human umbilical vein endothelial cell (HUVEC) proliferation and migration than low-Mw TMDs. High-Mw TMDs enhanced HaCaT wound healing in vitro and re-epithelialization during wound healing in vivo. The high-Mw proteins were administrated intravenously in mice 30 min before infusing LPS (lipopolysaccharide). The result showed that high-Mw protein dramatically inhibited LPS-induced serum TNF-α脉 and IL-6 elevation. In conclusion, these studies suggest that the extent of glycosylation had profound effects on the anticoagulant and skin wound healing properties of human TM. High-Mw TM also reduced mortality in sepsis animal model. We suggest that high-Mw TM proteins have potential to become useful therapeutic agents in the future.

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