Functional characterizaion of coagulation factor IX heavy chain mutants and the FIX-X chimeric proteins

• Main Authors: Yu-Chiu Ysai, 蔡玉秋
• Other Authors: Shu-Wha Lin
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/36573416091275320815

碩士 === 國立臺灣大學 === 醫事技術學研究所 === 89 === Abstract To understand the function of surface amino acids of human coagulation factor IX, this study is focused on the N-terminal region of the heavy chain, which is thought to interact with cofactor, factor VIII. One strategy was to generate factor IX with mutations at position 248, 292 and 294. Recombinant factor IXR248A, D292A and E294A exhibited 115%, 54% and 192% of wild type FIX’s clotting activity. All mutants were readily activated by FVIIa-TF complex or by FXIa. The dissociation constant for FVIIIa binding were normal or better with the three mutants. The binding results cannot explain the observed clotting activities with individual mutants. In activating factor X, IXE294A has great cleavage activity in the present or absence of FVIIIa. IXR248A seems to affect binding to factor X, and IXD292A may affect the catalytic domain stability. With respect to inhibitor (ATIII) interactions, IXD292A forms ATIII-FIXa complex more easily than wild-type factor IX, but in slow binding assay, all these mutants displayed equal binding efficiency. The other strategy was to perform domain swapping. The N-terminal region of residues 193-289, 193-287 and 193-291 of factor IX were exchanged with corresponding areas of factor X (each named IX-X, IX-X9 and IX-X10). These chimeras were also expressed in 293 cells. None of chimeric proteins were secreted into the cultured media, supernatant in Fluorescent staining assay of transfected cell clones showed strong signals within the cytoplasm. This suggests that the heavy chain of factor IX is an integral domain and not interchangeable.