MECHANISM UNDERLYING CONGENTIAL FVII VARIANT (FVIIR79Q) WITH VARIABLE FVII : c USING TISSUE THROMBOPLASTIN FROM DIFFERENT SOURCE

为了阐明FVIIR79Q利用不同来源的组织凝血活酶显示可变的FVII：C的机制，用野生型和突变型FVIcDNA对COS-1细胞进行瞬时转染，并在条件培养液中检测Fvii：C和Fvii：Ag。人Tf和兔Tf对细胞培养上清液中VII：C的测定值与人TF和兔TF的结果无差异，但用人TF和兔TF检测时，不同类型细胞的细胞培养上清液中的VII：C均低于野生型细胞。尽管重组FVIIR79G的VII：C没有不同水平的b…与人Tf相比，兔Tf测定的重组FVIIR79Q、FVIIR79W和FVIIR79P的VII：C均低于人Tf。人FVIIa/人Tf复合体的能量最小化模型结构表明，FVIIR79Q中的Q79和Tf中的e56之间的氢键消失。Tf和FVIIR79Q的E24和e56之间的距离估计为7~8A，推测重组FVIIR79Q的低VII：C是通过FVIIa/Tf复合体的微观构象变化而干扰的。推测FVIIR79G、FVIIR79W和FVIIR79P重组FVIIR79G、FVIIR79W和FVIIR79P的Vi：C降低是由于突变体Fvii在Tf附近的空间限制改变所致。FVIIR79Q的能量最小化模型结构表明，重组FVIIR79G的能量最小化模型结构在兔Tf和人Tf之间差异不大。FVIIR79G重组FVIIR79W和FVIIR79P的FVIIR79W和FVIIR79P的FVII：C在兔Tf和人Tf之间显示不同。通过比较FVIIa/兔Tf和FVIIa/人Tf复合体的能量最小模型结构很难简单地解释其机制。较少

To elucidate the mechanism by which FVIIR79Q shows variable FVII : c using tissue thromboplastin from different source, transient transfections were performed in COS-1 cells with wild type and mutant FVII cDNAs and FVII : c and FVII : ag in condition media were measured.The VII : ag in the media on cells transfected with pcDNAVIIwt contained 18ng/ml, pcDNAVIIR79Q ; 22ng, pcDNAVIIR79G ; 12ng, pcDNAVIIR79W ; 14ng, pcDNAVIIR79P ; 40ng. The VII : c in the media of cells transfected with the construct of pcDNAVIIwt did not have the difference between the result by the human TF and that by the rabbit TF.However the VII : c in the media on cells transfected with variant types were lower than that of wild type when assayed both with the human TF and the rabbit TF.The VII : c in the media on cells transfected with pcDNAVIIR79Q and pcDNAVIIR79W were 30% of that of wild type, and VIIc in pcDNAVIIR79W and pcDNAVIIR79P 10%. Although the VII : c of recombinant FVIIR79G did not have diffenent level b … More etween assayed with rabbit TF and with human TF, the VII : c of recombinant FVIIR79Q, FVIIR79W and FVIIR79P assayed with rabbit TF were lower than those of human TF.The energy minimised model structure of human FVIIa/human TF complex indicates that H-bond between Q79 in FVIIR79Q and E56 in TF disappears. And the distance between E24 and E56 of TF and Q79 of FVIIR79Q is estimated to be 7 to 8A.then it is speculated that the low VII : c of recombinant FVIIR79Q is interfering by the micro conformational change of FVIIa/TF complex. It is speculated that reduced VII : c of recombinant FVIIR79G, FVIIR79W and FVIIR79P are due to change of space restriction in mutant FVII close to TF.Modeling of human FVIIa/rabbitTF complex shows similar to human FVIIa/human TF complex. The energy minimized model structure of FVIIR79Q indicates little different complex between rabbit TF and human TF.VII : c of recombinant FVIIR79G showed no different between using by rabbit TF and using by human TF.FVII : c of recombinant FVIIR79W and FVIIR79P showed different between with rabbit TF and with human TF.It was difficult simply to explain the mechanism by comparison of the energy minimized model structure of FVIIa/rabbit TF complex with that of FVIIa/human TF complex. Less