Effect of warfarin on prothrombin synthesis and secretion in human Hep G2
cells
S Karpatkin, TH Finlay, AL Ballesteros and M Karpatkin
Prothrombin synthesis and secretion were studied in a human hepatoma cell
line (Hep G2) incubated with 35S-methionine for 2 to 24 hours at 37 degrees
C. Extracellular and intracellular prothrombin were detected by
immunoprecipitation with affinity-purified antiprothrombin antibody.
Incorporation of 35S-methionine into prothrombin was monitored by counting
specific bands excised from 10% sodium dodecyl sulfate- polyacrylamide gel
electrophoresis (SDS-PAGE). Prothrombin represented 0.3% to 0.7% of total
newly synthesized protein secreted into the media. Warfarin had no effect
on total prothrombin synthesis (extracellular plus intracellular). However,
warfarin inhibited secretion of newly synthesized prothrombin by 58% to 73%
over a 2 to 4 hour period. This was accompanied by the intracellular
accumulation of an immunoprecipitable species of prothrombin of 78 kd, 6 kd
less than extracellular prothrombin. At the end of the 4-hour incubation
with warfarin, intracellular prothrombin increased from 44% to 82%
(twofold) of total prothrombin, whereas extracellular prothrombin decreased
from 56% to 19% (threefold) of total prothrombin. After 24-hour incubation
with warfarin, intracellular and extracellular immunoprecipitable
prothrombin approached control values. Deglycosylation of extracellular and
intracellular prothrombin with hydrofluoric acid (HF) resulted in a
decrease in mol wt for both species to 66 kd. Endoglycosidase-H treatment,
which digests "early mannosyl" residues, resulted in a decrease in the mol
wt of the intracellular species of 8 kd with no effect on the extracellular
species. Thus, the lower mol wt intracellular species that accumulates
following early warfarin treatment is due to the presence of incompletely
processed carbohydrate chain. The data are compatible with the hypothesis
that optimum glycosylation and secretion require Vitamin K-dependent
carboxylation.
Volume 70,
Issue 3,
pp. 773-778,
09/01/1987
Copyright © 1987 by The American Society of Hematology
