Warfarin causes the degradation of protein C precursor in the endoplasmic reticulum.
Warfarin, an antagonist of vitamin K, is known to disrupt the microsomal vitamin K cycle, which results in a decrease in the plasma level of protein C, an anticoagulant factor, as well as some other vitamin K-dependent coagulation factors. Here, we examined the effect of warfarin on the secretion of recombinant protein C expressed in human kidney 293 or BHK cells. In transient expression, warfarin caused a 2-4-fold decrease in the quantity of protein C secreted, compared to findings with vitamin K-treated cells. Pulse-chase experiments using stable cells showed that, although recombinant protein C was secreted in the presence of vitamin K, the decrease in the total amount of radioactivity in the warfarin-treated cells suggested intracellular degradation. This degradation depended on the concentration of warfarin and was not inhibited by an endoplasmic reticulum (ER)-Golgi transport inhibitor (brefeldin A) or by lysosomotropic inhibitors (chloroquine and NH4Cl). Thus, protein C synthesized in the presence of warfarin is probably selectively degraded, and this degradation occurs in a pre-Golgi, nonlysosomal compartment. Among the protease inhibitors tested, N-alpha-acetyl-Leu-Leu-methioninal and N-alpha-acetyl-Leu-Leu-norleucinal blocked the degradation of protein C precursor synthesized in the presence of warfarin, and the precursor accumulated intracellularly, in a dose-dependent manner. Both inhibitors, however, did not disturb the secretion of protein C precursor in the vitamin K-treated cells. Thus, a cysteine protease(s) appeared to be responsible for the degradation.(ABSTRACT TRUNCATED AT 250 WORDS)
Tokunaga
F
F
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo, Japan.
Wakabayashi
S
S
Koide
T
T
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002451:Cell Compartmentation / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D004792:Enzyme Precursors / Q000378:metabolism; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D011486:Protein C / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins; D014812:Vitamin K / Q000494:pharmacology; D014859:Warfarin / Q000494:pharmacology*
D004792:Enzyme Precursors; D011486:Protein C; D011994:Recombinant Proteins; D014812:Vitamin K; D014859:Warfarin; D006596:Hexosaminidases