| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 95 === Thrombomodulin (TM) is a vascular endothelial cell receptor and cofactor in the clinically important protein C anticoagulant system. TM contains five structure domains: N-termianal lectin-like domain (TMD1), EGF-like domain (TMD2), serine and threonine-rich domain (TMD3), transmembrane domain (TMD4), and C-terminal cytoplasmic domain (TMD5). In recent studies, TM domains were shown to have several biological functions beyond anticoagulation including mitogenic effect on various cells, angiogenic activity and the possible participation in the embryogenesis. In our previous studies, the novel angiogenic effects of TM domains 2 and 3 (TMD23) were discovered both in vitro and in vivo. However, the detailed mechanism of TMD23 modulating angiogenesis still remained to be solved. In this study, the Pichica pastoris protein expression system was used to express the recombinant TMD23 and three protein C activation-defected TMD23 mutant proteins using site-direct mutagenesis. The recombinant TMD23 proteins were purified by affinity nickel-chelating column chromatography. TM cofactor activity assay showed that these site-direct mutated proteins lost their protein C activation activity. We further demonstrated that the biological function of the three mutated proteins was similar to that of the wild type TMD23. These three mutants also stimulated proliferation, migration and tube formation of human umbilical vein endothelial cell (HUVEC) in vitro and induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. We showed that the angiogenic activity of TM is independent of protein C activation pathway. In addition, the function of many candidate mediators was investigated. By modifying far Western blotting assay and immunoprecipitation - Western blotting analysis, we discovered that the recombinant TMD23 protein may interact with fibroblast growth factor receptor 1 (FGFR1; Flg) in HUVECs. These results suggested that TMD23 might act through tyrosyl kinase-like receptors such as FGFR1 to modulate angiogenesis.
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