The Biological Effects of Human Recombinant Thrombomodulin Proteins Independent of Protein C Activation Pathway

• Main Authors: Meng-Chen Sung, 宋孟真
• Other Authors: Hua-Lin Wu
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/30446275161382588406

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.