INTRACELLULAR EVENTS DETERMINE THE FATE OF ANTITHROMBIN UTAH

We sought to determine whether intracellular or extracellular events c ontribute to the decrease in circulating antithrombin (AT) levels that is seen in subjects with the Utah mutation (Pro 407 to Leu), Site-dir ected mutagenesis was used to recreate this mutation within a previous ly characterized rabbit AT cDNA, Cell-free expression of the mutated c DNA yielded an AT protein that failed to react with thrombin. Expressi on of the rabbit AT-Utah protein in transiently transfected Cos cells resulted in a 10-fold decrease in the amount of AT antigen detected in the conditioned media, as compared with that seen with the wild-type recombinant AT. This effect was not caused by variations in transfecti on efficiency, because AT levels were normalized to the product of a c otransfected plasmid, chloramphenicol acetyl transferase. Moreover, on Northern blot analysis, AT mRNA levels were comparable in cells expre ssing either the rabbit AT-Utah or wild-type recombinant rabbit AT, Im munoblots of conditioned media from the two populations of transfected cells showed that the recombinant AT-Utah protein was intact. The res ults obtained with Cos cells were reproduced using permanently transfe cted Chinese hamster ovary (CHO) cells. Pulse-chase experiments with t he CHO lines showed that both initial levels of rabbit AT-Utah after t he pulse labeling and the rate of subsequent secretion during the chas e period were reduced compared with that seen with cells expressing th e wild-type AT. The observed reduction in AT secretion was also observ ed for the AT-Oslo mutation (Ala 404 to Thr) when recreated in the rab bit AT background, and expressed in Cos cells, In these experiments, t he media levels of mutant AT were reduced by 50%, compared with wild-t ype, These results show that intracellular events, as opposed to accel erated clearance or other extracellular causes, contribute to the pauc ity of AT secretion seen in these strand 1C AT mutants. (C) 1995 by Th e American Society of Hematology.