Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII

We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly(97)Cys or Gln(100)Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys(97) did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII-Arg(100). the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg(100) was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII-Arg(100) had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation. (C) 1999 by The American Society of Hematology.