Isolation and Characterization of a Hepatoma ... - Cancer Research

NPT
HAPT
10 5 2.5 1.25
CHARACTERIZATION OF A HEPATOMA ABNORMAL PROTHROMB1N
1.25
Fig. 3. Binding of 125I-concanavalin A to NPT and HAPT. Serial concentra
tions of proteins were applied to nitrocellulose paper and incubated with radiolabeled
concanavalin A. A, radiograph of the dot blot: B, densitometric scans of
HAPT (gray) and NPT (black).
different assay methods, other investigators have also reported
increased blood levels of APT in 63-74% of patients with
hepatocellular carcinoma (11-13). We have named the APT
found in the blood of these patients the hepatoma-associated
abnormal prothrombin antigen.
APT is found in the blood of patients with vitamin K defi
ciency, patients with acquired disorders of hepatic function,
and patients treated with coumarin anticoagulants (8, 9). In
patients with vitamin K deficiency and patients taking cou
marin, circulating APT rapidly disappears from the blood with
parenteral vitamin K. In patients with hepatocellular carci
noma, HAPT does not disappear with the administration of
parenteral vitamin K (10, 13). Therefore, these patients do not
have vitamin K deficiency. In fact, measurements of blood
vitamin K levels in two hepatoma patients found elevated levels
of the vitamin.3 HAPT, furthermore, is eliminated or reduced
with resection of the hepatoma (10, 13). Therefore, it could be
concluded that HAPT is synthesized by the malignant hepatocyte
and is characteristic of an acquired tumor defect in vitamin
K-dependent carboxylation.
HAPT has been detected in blood by immunochemical meth
ods. However, antigenic identity between HAPT and APT
found in the blood of patients taking vitamin K antagonists
does not exclude the possibility of significant structural differ
ences between these proteins. An example is Factor IX Cam
bridge (23). Factor IX Cambridge has antigenic characteristics
similar to those of abnormal (des-7-carboxy) Factor IX. How
ever, Factor IX Cambridge has a point mutation at the -1
residue of the propeptide resulting in a protein with an un-
3 H. Liebman. unpublished observations.
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processed 18-residue amino-terminal propeptide. Therefore,
the protein has a higher molecular weight than either native or
abnormal Factor IX.
In this report I have purified a HAPT from the ascites of a
patient with hepatocellular carcinoma and have compared this
protein to NPT and APT. Analysis of this protein shows
structural identity to APT and further supports the hypothesis
that HAPT results from a defect in the vitamin K-dependent
posttranslational carboxylation of the prothrombin precursor
by the malignant hepatocyte.
Purified HAPT has the same molecular weight, amino acid
analysis (exclusive of 7-carboxyglutamic acid content), and
amino-terminal structure as NPT and APT. Analysis of the 7carboxyglutamic
acid content of HAPT shows the protein to
be partially carboxylated with an average of 5 Gla residues/
molecule. Electrophoretic analysis, in the presence of Ca(II),
suggest that HAPT is more heterogeneous with regard to its
Gla content than either NPT or APT. However, this apparent
difference in Gla content between HAPT and APT is probably
secondary to the methods used to isolate these proteins. The
purification of APT (9) includes a barium citrate absorption
step resulting in the removal of partially carboxylated pro
thrombin species. The purification of HAPT does not include
the barium absorption and utilizes an anti-prothrombin:Ca(II)
column to remove contaminating native prothrombin. The immunoaffmity
removal of NPT would leave partially carboxyl
ated variants with less than 8 Gla residues in the HAPT
preparation. Other investigators have prepared APT using dif
ferent methods and this has resulted in an APT preparation
which is more heterogeneous with regard to its Gla content (25,
26).
Hepatocellular carcinomas are associated with production of
aberrant and ectopie proteins including «-fetoprotein (27), ab
normal vitamin B12 binders (24), erythroprotein (28), and
abnormal fibrinogens (29). The abnormal vitamin B12-binding
proteins and fibrinogens are believed to result from aberrant or
excessive glycosylation of these proteins. I studied the glycosylation
of HAPT using a concanavalin A-binding assay to deter
mine if this protein is also abnormally glycosylated. There were
minor differences between HAPT and NPT in the binding of
1251-labeled concanavalin. These studies suggest that HAPT is
/V-glycosylated to an equal or slightly lesser degree than NPT.
Also previous studies on the role of sugar residues on the
function of the vitamin K-dependent proteins find no evidence
that glycosylation is essential for biologic activity.
A rat model for the production of abnormal prothrombin by
hepatocellular carcinoma was recently reported by Shah et al.
(30). Their data are consistent with the "hypothesis that the
tumors that increase circulating abnormal prothrombin are
those that are capable of expressing the prothrombin gene, but
that have lost the ability to express significant levels of the
vitamin K-dependent carboxylase enzyme." The structural char
acterization of the HAPT is consistent with this model since
the only defect noted was a deficiency in 7-carboxyglutamic
acid content.
The expression of H APT by the malignant hepatocyte results
from different biochemical derangements than those responsi
ble for secretion of «-fetoprotein. APT is the product of a
defective posttranslational processing step in the malignant
hepatocyte. n-Fetoprotein production results from the reexpression
of a suppressed fetal gene. The poor correlation between
these two tumor markers supports the independence of these
two acquired cellular defects (10-13). When measurements of
blood HAPT are used in combination with the assav for the
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