凝血酶調節素(TM)是一個鑲鉗在細胞膜上的醣蛋白質分子，主要是由五個結構區組成，包括從胺基端的類lectin結構區(D1)，接續六重複的類上皮生長因子結構區(D2)，絲胺酸/纈胺酸富含結構區(D3)，細胞膜穿越區(D4)，及細胞質結構區(D5)。凝血酶調節素一開始在內皮細胞被發現，並且作為抗凝血因子。當凝血酶與凝血酶調節素結合時，凝血酶的促凝血活性經由蛋白質C的活化作用而轉為抗凝血活性。而後續的研究發現在很多不同的細胞型態也有廣泛的凝血酶調節素的表現。以上的研究暗示著凝血酶調節素除了抗凝血之外也許還扮演了其它的生理角色。之前的研究指出凝血酶調節素的類上皮生長因子結構區對瑞士3T3細胞( Swiss 3T3 cells)有促進生長的活性。最近在我們的活體外和活體內實驗發現重組的人類凝血酶節調素結構區2到3蛋白可作為血管新生因子，因此本研究的主要目的是在探討不同凝血酶調節素結構區對於血管新生的影響。我們以酵母菌表現系統來表現不同組合的凝血酶調節素結構區，包含類lectin結構區(TMD1)，類上皮生長因子結構區(TMD2)，類上皮生長因子結構區到絲胺酸/纈胺酸富含結構區(TMD23)，細胞膜外結構區(TMD123)，第一個類上皮生長因子結構區到第三個類上皮生長因子結構區(TMD2EGF1-3)，第四個類上皮生長因子結構區到第六個類上皮生長因子結構區(TMD2EGF4-6)。我們以鎳離子鉗合的親和性管柱來純化這些凝血酶調節素重組蛋白質。隨著在凝血酶調節素作為輔因子的活性測試，發現TMD23有高於TMD2百分之三十的活性。將TMD23與TMD2以100 ˚C煮沸三十分鐘後仍保有百分之五十的輔因子活性。以上說明凝血酶調節素結構區重組蛋白質與天然原型蛋白質一樣穩定。在腫瘤的血管新生模式實驗，腫瘤團塊和新生血管密度在TMD23處理組都有被提昇，但是在活體外的實驗TMD23並不會改變腫瘤細胞的生長速度。進一步比較不同凝血酶調節素結構區的血管新生活性;在DNA合成測試實驗，TMD23比TMD2和TMD123較會促進人類臍靜脈內皮細胞的DNA合成速率。在趨藥性實驗，TMD23可引發人類臍靜脈內皮細胞較強的移動性。在動物體的Matrigel分析，TMD23比TMD123更強地引起血管新生的現象。這隱含著TMD1似乎會干擾TMD23在活體內引起血管新生的可能性。隨及我們發現TMD1不僅會降低與抑制由上皮生長因子(EGF)對於人類臍靜脈內皮細胞所誘發的細胞質外訊息調控激酶(ERK)的活化及移動性;在小鼠的血管新生模式實驗中 ，TMD1亦減弱了由上皮生長因子所誘發的血管新生現象。而且在大鼠眼角膜微型口袋模式，我們也發現TMD1強烈的抑制由鹼性-纖維母細胞生長因子(bFGF)所誘發的血管新生象現。基於以上的結果我們認為TMD23相對於其它的凝血酶調節結構區有較強的誘發血管新生能力。再者，TMD1對於TMD23在血管新生的過程中扮演著相反的角色。

　Thrombomodulin (TM) is an integral membrane glycoprotein composed of five domains, including N-terminal lectin-like domain (D1), followed by six tandem repeated epidermal growth factor (EGF)-like domain (D2), serine/threonine-rich domain (D3), transmembrane domain (D4), and cytoplasmic domain (D5). TM is initially recognized on endothelium and serves as an anticoagulant. Procoagulant activity of thrombin was converted to anticoagulant via protein C activation when thrombin bound to TM. Subsequent studies have shown broad expression of TM in a variety of cell types. It implies that TM may play other roles beyond that of anticoagulation. Previous study shows that EGF-like domain of TM possesses mitogenic activity in Swiss 3T3 cells. Recently, recombinant human TM domain 2-3 (TMD23) was found to be an angiogenic factor in our in vitro and in vivo studies. Therefore, this study aims to explore the effects of various TM domains in angiogenesis. The Pichica pastoris protein expression system was used to express distinct domains of TM, including lectin-like domain (TMD1), EGF-like domain (TMD2), EGF-like domain to serine/threonine-rich domain (TMD23), extracellular domain (TMD123), EGF1 to EGF3 (TMD2EGF1~3), and EGF4 to EGF6 (TMD2EGF4~6). The recombinant TM domains were purified by affinity nickel-chelating column chromatography. In the TM cofactor activity assay, TMD23 was 30% higher than TMD2. TMD23 and TMD2 still possessed 50 % thrombin’s cofactor activity after heating at 100 °C for 30 min. It suggests that recombinant TM domains are stable as natural TM. In tumor neoangiogenesis assay, tumor masses and microvessel density were both enhanced in TMD23-supplemented group. But TMD23 did not alter growth rate of tumor in the in vitro assay. The angiogenic activities of TM domains were further compared. In the BrdU incorporation assay, TMD23 had a higher activity than TMD2 and TMD123 to enhance DNA synthesis in human umbilical vein endothelial cells (HUVECs). In the chemotactic assay, TMD23 induced HUVECs migration more potently. In the in vivo Matrigel analysis, TMD23 strongly induced angiogenesis than TMD123. It implies that TMD1 may interfere with TMD23 to induce angiogenesis in vivo. Subsequently, we found that TMD1 not only down-regulated extracellular-signal-regulated kinase (ERK) activation, but also inhibited migration induced by EGF in HUVECs. And in murine angiogenesis model, TMD1 diminished EGF-induced angiogenesis. Furthermore, TMD1 potently inhibited angiogenesis in rat corneal micropocket model elicited by basic fibroblast growth factor (bFGF). Based on these results we conclude that TMD23 is more potent than the other TM domains in inducing angiogenic activity. Moreover, TMD1 plays a contrary role to TMD23 in the process of angiogenesis.

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