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Technical Briefs |
1
Department of Medical Technology, School of Allied Medical Sciences, Shinshu University, Matsumoto 390-0820, Japan;
2
Department of Pediatrics, Saiseikai Tondabayashi Hospital, Tondabayashi 584-0082, Japan;
a author for correspondence: fax 81 236-37-2391, e-mail itosamu{at}gipac.shinshu-u.ac.jp
Inherited factor VII (FVII) deficiency is a rare autosomal recessive disorder that occurs in roughly 1 out of 500 000 people. Although some patients with homozygous FVII deficiency have a life-long tendency to bleed, a coincident thromboembolic complication has been reported (1) . In general, the bleeding tendency of FVII deficiency is not as severe as that of hemophilia. The clinical features are quite variable, with a rather poor correlation between measured coagulation activity and clinical bleeding tendency (2) . In laboratory testing, FVII deficiency has been divided into three types: cross-reactive material (CRM)- type, with decreased synthesis of the FVII molecule; CRM+, with decreased FVII activity but an antigen concentration within reference values; and CRMR, with reduced synthesis of FVII (3) . Specific FVII variants have been characterized as FVII molecules that give different procoagulant activities using tissue factor (TF) from various sources, such as human, rabbit, and bovine brain TF (4)(5)(6) . Some of the dysfunctional FVII variants possess either an Arg 79-to-Gln substitution in the first epidermal growth factor (EGF)-like domain or an Arg 353-to-Gln substitution in the catalytic domain of FVII (7)(8)(9)(10)(11) .
In the present study, using an ELISA with the monoclonal antibody that recognizes a specific epitope located in the three-dimensional structure near position 79 in the first EGF-like domain of human FVII (12) , we determined the mutated site of a dysfunctional FVII variant that possessed different FVII activity with human placental TF than with rabbit and ox brain TF.
The propositus was a 12-year-old girl with FVII deficiency. She was born at 36 weeks gestation, had no hemorrhagic problems, and showed no evidence of abnormal hepatic function either at birth or later. Her parents were not consanguineous, and the family history did not reveal any bleeding disease. Decreased Normotest values were noted at her health examination at the age of 1 month. She was admitted to our Department of Pediatrics because of a suspected deficiency of vitamin K-dependent coagulation factors. The Normotest did not increase after the administration of vitamin K2 (3.5 mg). Hemostatic examination revealed a prolonged prothrombin time (Simplastin, Organon Teknika), whereas the bleeding time, platelet count, activated partial thromboplastin time, and fibrinogen were within reference ranges. The coagulation factor activities were within health-related reference values except for FVII, and her plasma showed no inhibitors of coagulation factors. However, the levels of FVII activity (FVII:c) exhibited different activation patterns when TFs from different sources were used. Her FVII:c was 7.2% of the healthy control when rabbit TF was used, and 40% and 65% when human placenta and bovine brain TF, respectively, were used. Abnormal coagulation values were observed constantly over a period of 10 years.
The FVII:c and FVII:ag levels of the propositus and her family members
are shown in Table 1
. The FVII:c level using recombinant human TF was similar to
that using human placenta TF. Both the father and sister of the
propositus showed different activation patterns with human TF and
rabbit TF, although their FVII:c levels were not as low as that of the
propositus. The mother's activation pattern did not vary with the TFs
used. The FVII:ag level of the propositus was only 5% of healthy
controls when determined by ELISA-1 binding with hVII-B101/B1 as the
solid phase, although it was in the lower limit of the health-related
reference range when determined by ELISA-2 and ELISA-3, respectively.
As measured by quantitative immunoelectrophoresis (QIEP)
(13) , the FVII:ag level was 64% of healthy controls. The
ELISA/QIEP ratio was 0.078 in ELISA-1, 0.984 in ELISA-2, and 1.016 in
ELISA-3. The FVII:ag levels of the father and sister were below
reference values with ELISA-1, and within reference values with
ELISA-3, respectively. Those of her mother were within reference values
with ELISA-1 and ELISA-3. The ability of TF to bind to FVII
(14) of the propositus was 14% of healthy controls; the
values for the father, sister, and mother were 29%, 34%, and 72%,
respectively. These findings strongly suggested that the mutation in
the abnormal FVII of the propositus was in the structure near position
79 in human FVII. Therefore, a fragment spanning exons 3 and 4 and the
adjacent intron of the FVII genes of the propositus and her family
members were amplified by PCR. The PCR fragments were digested with the
restriction endonuclease MspI and analyzed by agarose gel
electrophoresis. The propositus, her father, and her sister had both a
310-bp band produced by the loss of the MspI restriction
site and a 245-bp band, indicating that they were heterozygous for the
mutation (Fig. 1
), which could be concluded to consist of base substitutions in
the CCGG from nucleotide position 6053 to 6056. Single-strand
conformational polymorphism analysis (15) of PCR products of
five exons and their adjacent intron junctions (exons 2–8) of the FVII
genes of the propositus revealed no difference in mobilities compared
with healthy controls analyzed in parallel except with PCR fragments
that spanned exons 3 and 4. The nucleotide sequence of exon 4 was
determined by directly sequencing the PCR fragments. A heterozygous
G-to-A point mutation was found at nucleotide position 6055, producing
the substitution of Arg 79 (CGG) by Gln (CAG).
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It is difficult to determine molecular defects in FVII by protein
analysis because FVII is present in plasma at extremely low
concentrations. Only a few studies have detected the sites of mutations
in coagulation factors by using monoclonal antibodies, although
monoclonal antibodies for coagulation factors have been useful for
studying the structure of coagulation proteins and developing sensitive
immunoassays. Fair et al. (16) reported that immunochemical
assays using a polyclonal antibody or 46 monoclonal antibodies against
Factor X failed to reveal any structural deviation for the
dysfunctional Factor X Friuli variant. Protein C Yonago, which contains
the substitution of Arg 15 by Gly, was nonreactive to monoclonal
antibodies JTC-1 and -3, which recognize only the calcium-dependent
conformation of the 
-carboxyglutamic acid domain (17) . We
know of no report that has determined the mutational site of a
dysfunctional FVII variant with monoclonal antibodies. It was
speculated that the FVII of the propositus, which has different
activation patterns for TFs from different sources, might possess
substitution of Arg 79 or Gln 304. The FVII-TF binding in the
propositus was lower than in healthy controls. The level of the FVII:ag
of the propositus was 65% of healthy controls in immunoelectrophoresis
using a polyclonal antibody as the probe for determining FVII. The
FVII:ag level determined by ELISA using a polyclonal antibody or a
mixture of monoclonal antibodies that recognize the light and heavy
chains of FVIIa was similar to that determined by
immunoelectrophoresis. However, the level of the FVII:ag, using the
monoclonal antibody recognizing the specific epitope located in the
three-dimensional structure near position 79, was remarkably low
compared with QIEP or ELISA using a polyclonal antibody or a mixture of
monoclonal antibodies that recognize the light and heavy chains of
FVIIa. The levels of FVII:ag of the father and sister revealed a
similar pattern to that of the propositus, although they were not as
low. These findings strongly suggested that the mutation in the
abnormal FVII of the propositus, her father, and her sister was in the
structure near position 79 in the first EGF-like domain of human FVII.
DNA sequencing revealed a G-to-A point mutation that was found at
nucleotide position 6055 in exon 4 of the FVII gene. This produces a
substitution in the CGG codon for Arg 79 in the first EGF-like domain
such that it is changed to CAG. The mutation in the propositus, her
father, and her sister was confirmed by restriction endonuclease
digestion.
Clark et al. (18) reported that the first EGF-like domain of FVII is essential for binding TF, as analyzed by the reaction of monoclonal antibodies with amino acid residues 51–88 of the first EGF-like domain of human FVII, which was mapped with fusion protein fragments. Interaction between FVIIa and TF involves direct contact between TF, the first EGF-like domain of FVIIa, and the catalytic domain (19) . O'Brien et al. ((20)) showed that the first EGF-like domain of FVII plays a key role in FVII complex formation with TF, as analyzed by surface plasmon resonance of the interaction between TF and recombinant FVII-R79Q. We reported (7) that the loss of charge associated with the substitution of Arg by Gln at position 79 in FVII Shinjo has a direct effect on the TF-binding site in this part of the first EGF-like domain of FVII. Recently, Banner et al. (21) determined the x-ray crystal structure of the complex of active site-inhibited FVIIa with subtilisin-treated soluble TF and showed that Arg 79 was close to Glu 24 and Glu 56 in the N-terminal domain of TF. The amino acid residues close to Arg 79 of FVIIa in the complex are conserved in human, rabbit, and bovine TFs. Why FVII with the substitution of Arg by Gln at position 79 gives different procoagulant activities using TF from various species is still unknown and will require additional studies. Our ELISA system can be used to check the abnormal FVII molecules that give different procoagulant activities using TF from various sources.
References
Trp). J Biol Chem 1994;269:7355-7363.
GGG). Deteriorated calcium-dependent conformation of the gamma-carboxyglutamic acid domain relevant to a thrombotic tendency. Int J Hematol 1993;57:9-14.
[Web of Science][Medline]
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