|Budget Amount *help
¥6,500,000 (Direct Cost : ¥6,500,000)
Fiscal Year 1992 : ¥2,200,000 (Direct Cost : ¥2,200,000)
Fiscal Year 1991 : ¥4,300,000 (Direct Cost : ¥4,300,000)
Thrombomodulin (TM) is a receptor for thrombin on the en- dothelial cells and acts as a cofactor for thrombin-catalyzed protein C activation. Activated protein C proteolytically inac- tivates blood coagulation Factors Va and VIIIa, thus plays an important role in the regulation of blood coagulation. TM also inhibits the procoagulant activities of thrombin, thus TM acts to convert thrombin from a procoagulant into an anticoagulant. In our previous study, we determined the nucleotide sequence of complementary and genomic DNA of human TM, and found that TM contains six epidermal growth factor (EGF)-like domains in the middle of the extracellular region. In the present research project, we studied the structure-function relationship of TM in expression of the cofactor activity for thrombin-catalyzed acti- vation of protein C, and moreover determined the promoter region of TM gene to elucidate the endocytosis of TM into the endotheli- al cells. We revealed that, (1) of the six consecutive EGF-like domains, the fourth, fifth and sixth EGF-like domains from the NH_2-terminus are crucial for expression of the full cofactor activity of TM; (2) the fourth and fifth EGF-like domains are minimumly essential for expressing the cofactor activity; (3) gamma- carboxyglutamic acid domain of protein C is essential to be activated by thrombin bound to TM; (4) protein C interacts with Asp^<349> residue in the fourth EGF-like domain of TM via Ca^<2+> ions, (5) an atherogenic stimulus, homocysteine inhibits the cofactor activity of endothelial TM probably by chemical denatu- ration and thereafter promotes expression of TM mRNA in the endothelial cells which results in the expression of functionally abnormal TM; and 6) the 5'-flanking region of TM gene was iso- lated from EMBL-genomic DNA library and its sequence was analyzed to understand the endocytosis of TM.