凝血酶調節素(Thrombomodulin, TM)是血管內皮細胞表面的一種醣蛋白，並且是一種為人所知的抗凝血分子。在有功能性的心血管系統尚未建立之前，將凝血酶調節素的基因剔除掉，會造成早期胚胎的死亡。然而，凝血酶調節素在抗凝血之外的其他生理功能仍然尚未被研究的很清楚。在本實驗室最近的研究發現TM可以當作一個細胞黏著分子去媒介細胞與細胞之間的聯結，而且lectin-like結構區在此過程中是重要的。而細胞連結的作用，主要是藉由細胞表面的蛋白與鄰近細胞表面（細胞對細胞）或細胞基質(細胞對細胞基質)上的蛋白結合，以及調節細胞內的訊息傳遞路徑來調控細胞連結所造成的細胞生理機能的改變。
做為一個有效的細胞黏著分子，TM的胞內區域可能與細胞骨架做直接的聯結或是間接的透過一些聯繫蛋白的幫助，像是ERM家族中的其中一員，ezrin。為了進一步探討是否有其他的分子參與相關的連結或作用，我們利用μMAC的磁珠去找尋和TM可能有互相結合的蛋白。在本研究之中，由我們的結論顯示α-actinin，一種和actin結合的蛋白，被認為可能是與TM胞內區域有結合的候選分子，並且扮演著連繫蛋白的角色幫助細胞表面黏著蛋白與細胞骨架之間的連結。
在螢光免疫染色的觀察方面，我們發現TM和α-actinin在細胞連接處有同樣的分佈情形，不論是在TM過度表現的外生性系統(A2058-TMG黑色素瘤細胞)或是內生性TM表現的系統(HaCaT上皮細胞)都有一致的結果；而在A2058-TMG細胞中，我們也可以發現兩個分子在細胞前端(leading edge)有部分的相同分佈(partially colocalized)。另外利用細胞內共同免疫沉澱(Co-immunoprecipitation)以及細胞外的蛋白結合測試(in vitro binding assay)證實了TM和α-actinin之間的直接結合。為了找尋TM胞內區域中α-actinin的結合位置，我們進一步利用截短片段以及點突變的TM胞內區域去進行細胞外的蛋白結合測試並且加以分析。實驗結果發現，在TM胞內區域的前九個胺基酸中，靠近細胞膜附近的帶正電胺基酸群RKK和RAK被認為與α-actinin的結合有關。總結以上實驗的結果，並且比較α-actinin和ezrin在細胞裡面不同的分佈，我們認為α-actinin在TM所調節細胞與細胞連結的生理現象中扮演著重要的角色，當作是一個聯繫蛋白負責與細胞骨架做連結，而ezrin可能參與TM其他功能的調節。

Thrombomodulin (TM) is an integral membrane glycoprotein that is originally found on vascular endothelium and is a well-established potent anticoagulant factor. Ablation of the TM gene causes early postimplantation embryonic lethality that precedes the establishment of a functional cardiovascular system. Until recently the physiological function of TM beyond its anticoagulation activity has not been well investigated. Recently our lab reported that TM may function as a cell adhesion molecule to mediate cell-cell adhesion through its lectin-like domain. These adhesive interactions, mediated by cell surface proteins that bind to others on adjacent cells (cell-to-cell) or in the extracellular matrix (cell-to-extracellular matrix), also regulate intracellular signal transduction pathways that control adhesion-induced changes in cell physiology.
To be an effective adhesion molecule, cytoplasmic domain of TM may associate with actin cytoskeleton directly or indirectly through some linkage proteins such as ezrin, the member of ezrin/radixin/moesin (ERM) family. To further investigate molecules involved in this interaction, we used μMAC magnetic beads to pull down the proteins interacted with TM. In the study, our result indicated that α-actinin, an actin binding protein, which functions as a linker between cell surface adhesion molecules and the actin-containing cytoskeleton was indentified as a potential candidate to interact with TM cytoplasmic domain.
In the immunofluorescence study, we found that exogenous (A2058-TMG melanoma cells) and endogenous (HaCaT keratinocytes) systems showed a consistent result that TM and α-actinin were colocalized in cell-cell junctions, whereas there was partially colocalized in the leading edge of TMG stable expressed A2058 cells. Co-immunoprecipitation analysis in vivo and in vitro binding assay showed a direct interaction between α-actinin and TM. In vitro binding assay was further performed by truncated and mutated forms of TM cytoplsamic domain to study the α-actinin interaction sites in TM cytoplasmic domain. Based on these results, we conclued that α-actinin required the juxta-membrane positively charged residue clusters RKK and RAK within the first nine amino acids of the TM cytoplasmic domain for its binding. Together, comparing the different subcellular distributions between α-actinin and ezrin, we hypothesize that α-actinin rather than ezrin seems to be an important cytoskeletal linkage protein involved in TM-mediated cell-cell adhesion.

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