Intracellular events determine the fate of antithrombin Utah
WP Sheffield, JE Castillo and MA Blajchman
Canadian Red Cross Society Blood Services, Hamilton, Ontario, Canada.
We sought to determine whether intracellular or extracellular events
contribute to the decrease in circulating antithrombin (AT) levels that is
seen in subjects with the Utah mutation (Pro 407 to Leu). Site- directed
mutagenesis was used to recreate this mutation within a previously
characterized rabbit AT cDNA. Cell-free expression of the mutated cDNA
yielded an AT protein that failed to react with thrombin. Expression 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 transfection efficiency, because AT
levels were normalized to the product of a cotransfected plasmid,
chloramphenicol acetyl transferase. Moreover, on Northern blot analysis, AT
mRNA levels were comparable in cells expressing either the rabbit AT-Utah
or wild-type recombinant rabbit AT. Immunoblots of conditioned media from
the two populations of transfected cells showed that the recombinant
AT-Utah protein was intact. The results obtained with Cos cells were
reproduced using permanently transfected Chinese hamster ovary (CHO) cells.
Pulse-chase experiments with the CHO lines showed that both initial levels
of rabbit AT-Utah after the pulse labeling and the rate of subsequent
secretion during the chase period were reduced compared with that seen with
cells expressing the wild-type AT. The observed reduction in AT secretion
was also observed for the AT-Oslo mutation (Ala 404 to Thr) when recreated
in the rabbit AT background, and expressed in Cos cells. In these
experiments, the media levels of mutant AT were reduced by 50%, compared
with wild-type. These results show that intracellular events, as opposed to
accelerated clearance or other extracellular causes, contribute to the
paucity of AT secretion seen in these strand 1C AT mutants.
Volume 86,
Issue 9,
pp. 3461-3467,
11/01/1995
Copyright © 1995 by The American Society of Hematology
