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

Isolation and Characterization of a Hepatoma-associated
Abnormal (Des- γ-carboxy)prothrombin
Howard A. Liebman
Cancer Res 1989;49:6493-6497.
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[CANCER RESEARCH 49, 6493-6497, December I, 1989]
Isolation and Characterization of a Hepatoma-associated Abnormal (Des-7carboxy)prothrombin1
Howard A. Liebman
William B. Cosile llematology Research Laboratory, Division of Hematology-Oncology, Boston City Hospital, and Boston University School of Medicine,
Boston, Massachusetts 02118
ABSTRACT
Hepatoma-associated abnormal (des-7-carboxy)prothrombin (HAPT)
is a newly described tumor marker for hepatocellular carcinoma. 11Al' I
has been measured in the blood of patients with hepatoma by immunoassay
but has not been isolated or characterized. This paper describes the
quantitative isolation and structural characterization of HAPT. Purified
HAPT has the same molecular weight, amino-terminal sequence, and
amino acid analysis (exclusive of 7-carboxyglutamic acid) as native
prothrombin and abnormal prothrombin isolated from the blood of pa
tients taking sodium warfarin. 11API' is heterogeneous in 7-carboxyglu
tamic acid (Gla) content with an average of 5 Gla residues/molecule
compared to 10 Cla residues for native prothrombin and 2 Gla residues
for abnormal prothrombin. HUM is glycosylated in a manner equivalent
to that for native prothrombin when evaluated by a concanavalin Abinding
assay. These studies find structural identity between 11\ P'l and
abnormal prothrombin. Therefore the findings support the hypothesis
that HAPT results from an acquired defect in the posttranslational
vitamin K-dependent carboxylation of the prothrombin precursor and not
an intrinsic defect in the prothrombin precursor molecule.
INTRODUCTION
Prothrombin is the major vitamin K-dependent blood coag
ulation protein synthesized by the liver (1). In the presence of
sufficient quantities of vitamin K, 10 NH2-terminal glutamic
acid residues undergo a posttranslational 7-carboxylation (2-
4). The resulting Gla2 residues confer metal-binding properties
essential for functional activity (5). In the absence of vitamin K
or in the presence of vitamin K antagonists, the vitamin Kdependent
carboxylase activity in the liver is inhibited and des•y-carboxyforms
of prothrombin (abnormal prothrombin) are
released into the blood (6, 7). The abnormal prothrombin
cannot bind metal ions and is devoid of functional activity.
Conformation-specific antibodies against these abnormal forms
of prothrombin have been developed and can be used to quantitate
the levels of abnormal prothrombin antigen in blood (8,
9).
We have reported that abnormal prothrombin antigen is
increased in the blood of patients with hepatocellular carcinoma
(10). Abnormal prothrombin antigen in these patients is not
due to vitamin K deficiency since it did not disappear with
parenteral vitamin K. The abnormal prothrombin antigen was
eliminated or reduced with tumor resection or with chemother
apy supporting the primary role of the hepatoma in the synthe
sis of this antigen. These findings suggest that abnormal pro
thrombin antigen may be a useful new serum marker of primary
hepatocellular carcinoma. Subsequently, other investigators
have confirmed these findings using different assay systems
(11-13).
Received 5/2/89; revised 8/28/89: accepted 9/6/89.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked adverlisenn'nìin
accordance with 18 U.S.C. Section 17.14solely to indicate this fact.
1This work was supported by grants from the American Cancer Society (PDT-
256) and the NIH (I R29 HL .Ì9C65).
J The abbreviations used are: Gla. vcarboxyglutamicacid; NPT. human name
prothrombin; APT. abnormal prothrombin; TBS. 0.05 M Tris-0.15 M NaCI. pH
7.4; HAPT. hepatoma-associated abnormal (des-vcarboxy) prothrombin: HPLC.
high performance liquid Chromatograph)-.
6493
While the abnormal prothrombin antigen in these patients is
assumed to be similar to abnormal prothrombin in the plasma
of patients treated with warfarin, the hepatoma-associated ab
normal prothrombin antigen has not been purified from the
blood of patients with hepatoma to demonstrate a structural
relationship. I have now quantitatively isolated and character
ized a hepatoma-associated abnormal prothrombin from the
malignant ascites of a patient. Analysis of this protein demon
strates structural identity with the partially carboxylated non
functional prothrombin species isolated from the blood of pa
tients taking sodium warfarin. These studies further support
the hypothesis that abnormal prothrombin in the blood of
patients with hepatocellular carcinoma is secondary to a defect
in the vitamin K-dependent carboxylation system in the pa
tient's tumor.
MATERIALS AND METHODS
Protein Preparation. NPT was purified by barium citrate absorption,
DEAE-Sephacel chromatography, and dcxtran sulfate chromatography
using standard methods (14. 15). In later experiments, human native
prothrombin was isolated directly from plasma on a column of antiprothrombin:Ca(Il)
antibodies by modification of methods published
previously (16, 17). APT was prepared by the method of Blanchard et
al. (9).
HAPT was purified from patient ascites fluid by ion-exchange chro
matography and immunoaffinity chromatography (17). Approximately
500 ml of ascites fluid collected in 3 mM EDTA was made 1 m.M
diisopropylphosphofluoride and 1 mM benzamidine. The ascites fluid
was then dialyzed at 4°Covernight against 0.1 M potassium phosphate,
pH 7.5, containing 1 mM benzamidine. Prothrombin species were
purified by ion-exchange chromatography using a DEAE-Sephacel A-
50 column (3x8 cm) equilibrated with 0.1 M potassium phosphate,
pH 7.5. containing 1 mM benzamidine. Dialyzed ascites fluid, after
centrifugation. was applied to the column, and the column was washed
exhaustively with the equilibration buffer. Bound protein was eluted
with 0.5 M potassium phosphate. pH 7.5, containing 1 mM benzami
dine. The eluate was pooled, made 1 mM in diisopropylphosphofluoride.
and dialyzed at 4°Covernight against 0.1 M boric acid-1 M NaCI-1 mM
benzamidine-0.1% Tween 20, pH 8.5.
The prothrombin species were purified by immunoafTinity chroma
tography. The eluate from the DEAE column was applied to an antiprothrombin
antibody-Sepharose column (1x5 cm) and the column
was washed with 0.1 M boric acid-1 M NaCl-1 mM benzamidine-0.1%
Tween 20. pH 8.5. Bound prothrombin was eluted with 4 M guanidine-
HC1 and then immediately pooled and dialyzed at 4°Cagainst 0.05 M
Tris-0.5 M NaCI-1 HIMbenzamidine-HCl-0.01% Tween 20, pH 7.5. An
anti-prothrombin:Ca(II) antibody-Sepharose affinity column was then
used to remove contaminating fully carboxylated native prothrombin
(16, 17). After dialysis, the prothrombin species were made 5 mM
CaC12 and applied to the anti-prothrombin:Ca(II) antibody-Sepharose
column equilibrated with TBS-5 mM CaCI2. The HAPT did not bind
to the column and was collected in the flow-through. The small quan
tities of contaminating native prothrombin were eluted from the column
with 10 mM EDTA.
Immunoassays and Coagulation Assays. Total prothrombin antigen,
fully carboxylated native prothrombin antigen, and abnormal pro
thrombin antigen were measured by radioimmunoassays as described
previously (8, 9). The calcium-dependent prothrombin coagulant activ-
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CHARACTERIZATION OF A HEPATOMA ABNORMAL PROTHROMBIN
ity was assayed by measuring the acceleration of the clotting of prothrombin-deficient
plasma using Stypven to initiate coagulation (9).
The activation of prothrombin and HAPT to thrombin in the absence
of calcium was performed using the Echis carinólas venom assay (7).
Protein concentrations of prothrombin and HAPT were determined
spectrophotometrically at 280 nm using an E^,inm„mof 14.4 (16).
Analytical Electrophoresis. Polyacrylamide gel electrophoresis in the
presence of dodecyl sulfate was performed on prothrombin, abnormal
prothrombin, and HAPT using 10% acrylamide gels (18). Proteins were
reduced using 2-mercaptoethanol. Electrophoretic analysis of the het
erogeneity of the partially carboxylated prothrombin species in the
HAPT was performed using 1251 radiolabeled proteins and disc gel
electrophoresis (19) in the presence of 8 mM CaC12 as described by
Borowski (17). Native prothrombin, abnormal prothrombin, and
HAPT were radiolabeled with Nal251 by the lactoperoxidase method
(20). Electrophoresis was performed on the tube gels with a 7.6%
acrylamide, 0.3% bisacrylamide resolving gel and a 1.8% acrylamide-
0.22% bisacrylamide stacking gel. Gels, buffers, and samples were made
8 mM in CaCl2. After electrophoresis the gels were sliced into 1-mm
fragments and counted for 1251 in a Beckman Gamma 8000 scintilla
tion spectrometer.
Ammo-terminal Sequence and Amino Acid Analysis. The amino
terminal sequence of HAPT was determined by automated Edman
degradation using a Beckman 8900 spinning cut sequenator equipped
with a cold trap. The phenylthiohydantoin derivatives were analyzed
on a Waters HPLC using a Waters C18-/iBondapak column with an
ethanol gradient in ammonium acetate, pH 5.1 (21).
Amino acid analysis was performed on a Beckman model 119CL
amino acid analyzer equipped with a Beckman model 126 data system.
Proteins were hydrolyzed in 6 MHC1 at 110°Cfor 24 h. For quantitation
of Gla, the proteins were hydrolyzed in 2 M KOH for 22 h at 110°C
(17, 22). The Gla composition was quantitated after alkaline hydrolysis
by HPLC using a fluorescence detection procedure (17, 22).
Lectin Blotting Studies. A comparison of the level of glycosylation of
HAPT and NPT was performed by the binding of 1251-labeled concanavalin
A to these proteins immobilized on nitrocellulose paper.
Serial dilutions of HAPT and NPT in TBS were applied to the nitro
cellulose paper. The paper was then incubated for l h in 3% bovine
serum albumin in TBS. After the blot was washed 4 times with TBS, it
was incubated for 3 h at 37°Cwith '25I-labeled concanavalin A in 3%
bovine serum albumin-TBS. The blot was then washed 5 times in TBS,
dried, and exposed to Kodak X-O mat R Film for 2-6 days. Quantita
tion of the binding of the concanavalin A was performed by densitometric
scanning of the autoradiographs using an EDC densitometer
(Helena Laboratories).
RESULTS
Purification of HAPT. HAPT was purified from the ascites
fluid of a patient with widely disseminated hepatocellular car
cinoma. Approximately 2.8 liters of ascitic fluid were used for
the isolation of HAPT. The patient had markedly elevated
blood levels of HAPT and had previously been reported (Ref.
10; Table 2, Patient 3). Listed in Table 1 are the concentrations
of NPT and HAPT in the blood and ascites fluid of this patient
as measured by radioimmunoassay. The ascites fluid concentra
tion of HAPT was equivalent to the levels found in the patient's
blood, while NPT was only 35% of the blood levels. After
purification, 4.2 mg of HAPT were obtained from the ascites
fluid. This was a final preparative yield of 38% of the estimated
total concentration of HAPT in the ascites fluid (Table 1).
The concentrations of total prothrombin antigen, native pro
thrombin antigen, and abnormal prothrombin antigen were
measured in the purified HAPT preparation by competition
radioimmunoassays (Table 2). The concentrations of these an
tigens were compared with the estimated concentration of
HAPT as determined spectroscopically. Approximately 92% of
the purified HAPT competed with NPT in the radioimmuno-
Table 1 Purification of hepawma-associated abnormal prothrombin
AmountSerum
(//g/ml")
NPT (Kg/ml0)
Ug/ml")Ascites
HAPT
NPT (Mg/nila)
HAPT („g/ml")
(liters)Total Volume
HAPT (mg)
Isolated HAPT (mg*)
HAPT yield ("¿)603.921
°Determined by competition radioimmunoassay.
* Determined spectroscopically.
3.8810.864.2
Table 2 Immunochemical characterisation of purified HAPT
HAPT"
TPT*NPT*
APT*Concentration
(jig/ml)640
588
11
511%
38
ofHAPT100
92
2
80
*Concentration determined spectroscopically.
*Concentration determined by competition radioimmunoassay.
assay for total prothrombin antigen. The native prothrombin
antigen was measured in the HAPT preparation using the antiprothrombin:Ca(II)
antibodies. Anti-prothrombin:Ca(ll) anti
bodies bind only fully carboxylated native prothrombin species
capable of undergoing a metal-induced conformational transi
tion. Less than 2% of the purified HAPT competed with NPT
in the radioimmunoassay for native prothrombin antigen. Antiabnormal
prothrombin antibodies bind abnormal prothrombin
but not fully carboxylated native prothrombin (9). In the com
petition radioimmunoassay for abnormal prothrombin antigen,
80% of the HAPT competed with APT. Approximately 10%
of HAPT as measured by the immunoassay for total prothrom
bin antigen failed to bind to either the anti-prothrombin:Ca(II)
or anti-abnormal prothrombin antibodies suggesting the pres
ence of a population of partially carboxylated prothrombin
species (17).
Structural Characterization of HAPT. The isolated HAPT
was studied by dodecyl sulfate-gel electrophoresis. The protein
appeared >95% pure and migrated as a single band in the
presence of 2-mercaptoethanol with the same mobility as NPT
(Fig. 1). The amino-terminal sequence of the HAPT was com
pared to that of NPT in order to determine whether the varia
tion in Gla content could be attributed to differences in the
primary structure of this region. The first 14 amino acid resi
dues of the HAPT were identical to those of NPT and the
published known sequence of prothrombin. These results indi
cate that there are no differences in the amino-terminal se
quences of HAPT and NPT between residues 1 and 14, nor
does HAPT possess an unprocessed leader sequence similar to
Factor IX Cambridge (23). However, I cannot exclude the
possibility of a mutation involving other Gla residues at posi
tions 16, 19, 20, 25, 26, and 32 of the prothrombin aminoterminal
region. Specific identification of Gla content in NPT
and HAPT cannot be obtained by this technique since the
formation of the phenylthiohydantoin derivative of Gla results
in its conversion into phenylthiohydantoylglutamic acid.
The amino acid composition of the acid hydrolysate of HAPT
showed no significant difference from NPT. The Gla compo
sition was quantitated after alkaline hydrolysis by HPLC and
fluorescence detection. The Gla content for NPT, APT, and
HAPT is presented in Table 3. These results indicate that
6494
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NPT HAPT
Fig. 1. Electrophoretic analysis on 10% polyacrylamide gels of purified NPT
and HAPT. The samples (25

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.
6496
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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fetoprotein over 80% of patients with hepatomas can be iden
tified with high certainty (10). HAPT, measured by immunoassay,
will provide an important new tool to survey populations
at risk for hepatocellular carcinoma and to monitor treatment.
ACKNOWLEDGMENTS
I am grateful for the support and helpful discussion of Dr. Bruce and
Barbara Furie. I wish to thank Dr. Myron Tong for providing patient
blood and ascites fluid. I also thank John Toomey for his excellent
technical assistance.
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