Amano K, Michnick D A, Moussalli M, Kaufman R J
Howard Hughes Medical Institute, Department of Biological Chemistry, University of Michigan Medical Center, Ann Arbor 48109-0650, USA.
Thromb Haemost. 1998 Mar;79(3):557-63.
Activated protein C (APC)-mediated inactivation of factor VIII (FVIII) correlates with cleavage at either Arg336 and/or Arg562. To elucidate the APC cleavage requirements for inactivation of FVIII, APC cleavage site mutants in FVIII (R336I, R562K and R336I/R562K) were made by site-directed mutagenesis. Analysis of these FVIII mutants expressed in COS-1 monkey cells demonstrated the thrombin-cleaved mutant R562K was resistant to APC cleavage at residue 562 but not at Arg336 and the thrombin cleaved mutant R3361 was mostly resistant to APC cleavage at residue 336, but was sensitive to APC cleavage at Arg562. The double mutant R336I/R562K was mostly resistant to cleavage at residue 336 and completely resistant to cleavage at residue 562. Thus, APC cleavage of FVIII does not require a specific order of cleavage at either residue. The functional inactivation by APC was studied using partially purified preparations of FVIII expressed in Chinese hamster ovary cells. Both single mutants were inactivated at similar rates but slower than wild-type FVIII, whereas the double mutant R336I/R562K was resistant to inactivation. The ability of a commercially available APC-resistance assay kit to detect APC resistant FVIII was tested by reconstituting FVIII deficient plasma with the APC resistant mutants. Only the R336I/R562K demonstrated a reduced APC-resistance ratio, indicating that this assay can not detect the single APC cleavage site mutant of FVIII. These results suggest that APC-mediated cleavage at either Arg336 or Arg562 partially inactivate FVIII.
活化蛋白C（APC）介导的因子VIII（FVIII）失活与在精氨酸336和/或精氨酸562处的切割相关。为了阐明APC切割对FVIII失活的要求，通过定点诱变制备了FVIII中的APC切割位点突变体（R336I、R562K和R336I/R562K）。对在COS-1猴细胞中表达的这些FVIII突变体的分析表明，经凝血酶切割的突变体R562K在第562位残基处对APC切割具有抗性，但在精氨酸336处不具有抗性，而经凝血酶切割的突变体R336I在第336位残基处对APC切割大多具有抗性，但在精氨酸562处对APC切割敏感。双突变体R336I/R562K在第336位残基处大多对切割具有抗性，在第562位残基处完全对切割具有抗性。因此，FVIII的APC切割不需要在任一残基处按特定顺序进行切割。使用在中国仓鼠卵巢细胞中表达的部分纯化的FVIII制剂研究了APC的功能失活。两个单突变体以相似的速率失活，但比野生型FVIII慢，而双突变体R336I/R562K对失活具有抗性。通过用APC抗性突变体重组FVIII缺陷血浆，测试了市售的APC抗性检测试剂盒检测APC抗性FVIII的能力。只有R336I/R562K显示出降低的APC抗性比率，表明该检测方法无法检测FVIII的单个APC切割位点突变体。这些结果表明，APC在精氨酸336或精氨酸562处介导的切割会使FVIII部分失活。
