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Vol. 28. No. 1. 1984 (31)
-〔シンポジウム〕血液凝固関連物質の電気泳動的解析-
4. Fibrinogen Degradation Products (FDP) with
a Special Emphasis on Fragment A*
By
Tadashi Kawai** and Kimiteru Takagi**
I. Fibrinogen and Fibrinogen Degradation
Products (FDP)
1. Structure and properties of fibrinogen
Fibrinogen, also called factor I, is of primary
importance in the blood coagulation process, and it
forms fibrin with the action of thrombin. Fibrino-
gen is mainly synthesized in the liver cells, and the
normal adult plasma contains the fibrinogen in the
concentration ranging from 200 to 400mg/100ml. Its
biological half-life is 4-6 days. The molecular weight
of fibrinogen is approximately 340,000 with the
molecular structure schematically shown in Fig. 1.
It consists of 3 pairs of polypeptide chains, namely,
Aα, Bβ, and γ, which are bound together with many
inter-chain and intra-chain disulfide bonds. The
molecular weight of Aα chain is approximately
70,000, Bβ chain approximately 60,000 and γ chain
approximately 50,000 daltons.1) Aα chains seem to
exist close to the molecular surface, and they are
most sensitive to various proteolytic enzymes.
During the blood coagulation process, thrombin
first attacks the N terminus of the fibrinogen mole-
cule, specifically between arginine and glycine. With
thrombin, fibrinopeptide A (consisting of 16 amino
acid residues) is released from Aα chain, while
fibrinopeptide B (consisting of 14 amino acid resi-
Fig. 1. A schematic structure of fibrinogen.
Solid arrows indicate the site to be attacked
by thrombin, while dotted arrows by plasmin.
A, B: fibrinopeptide A, fibrinopeptide B
FA: fragment A, C: C terminus, N: N ter-
minus.
Fig. 2. A schematic diagram of fibrinogen
degradation by plasmin.
A, D, E: fragment A, fragment D, fragment E
*FDP (fibrinogen degradation products)-と くに Fragment Aを 中 心 に-.
**河 合 忠, 高 木 皇 輝, 自治 医 科 大学 臨 床病 理 学 教 室.
Key words: fibrinogen degradation product, fragment A.
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(32) 生物物理化学
dues) from Bβ chain. Fibrin monomers thus formed
will then aggregate to form fibrin clot for hemo-
stasis.
2. Fibrinogen degradation by plasmin
Fibrinogen is degraded by plasmin, a proteo-
lytic enzyme, to form various fibrinogen degrada-
tion products (FDP) (Fig. 2).2-4) This degradation
process is divided into three major steps.
At the first step, α chain forms fragment X
(molecular weight of about 260,000), and at the
same time fragment A (molecular weight of about
22,500) is released from the C terminus of the α
chain. At the second step, fragment Y (molecular
weight of about 150,000) and fragment D (molecular
weight of about 85,000-100,000) are formed. At
the third step, fragment Y is further degraded into
fragment D and fragment E (molecular weight of
about 50,000).
During the plasmin degradation process, many
different peptides of various sizes are released.5)
FDP are analyzed by various methods, inclu-
ding biochemical and immunological ones. Various
properties of FDP are summarized in Table 1.
II. Fragment A
1. Separation of fragment A
Human fibrinogen was purified by ethanol frac-
tionation6) and it had colttability of more than
97%, containing approximately 1% plasmin. One
percent fibrinogen solution was made with 0.15M
NaCl-0.05M Tris buffer (pH7.5) and then added
was 0.05 CTAU plasmin (AB, Kabi) per 1mg
fibrinogen or 10u/ml urokinase (Green Cross Co.).
After incubation at 37℃ for various time lengths,
Table 1. Various properties of fibrin (ogen) degradation products.
Xfp: Fragment X derived from fibrinogen, containing fibrinopeptides.
X0: Fragment X derived from fibrin, devoid of fibrinopeptides.
Fig. 3. SDS-polyacrylamide gel electrophore-
tic patterns of the plasmin digests of
human fibrinogen.
Gel columns 1 to 6 indicate the digests treated
for 0, 10, 20, 30, 60, and 120 minutes.
F: fibrinogen; X, Y, D, E, A: fragment X, Y.
D, E, A; α, β, γ: α, β, γ chain.
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Vol. 28. No. 1. 1984 (33)
8M urea containing 2% SDS (sodium dodecyl
sulfate) was added to stop the plasmin activity.
For the evaluation of reduced forms, added was
10μl/ml β-mercaptoethanol.
Plasmin-treated samples were then analyzed by
5% SDS-polyacrylamide gel electrophoresis (PAG
E).7) As shown in Fig. 3, fragment X and fragment
A appeared within 20-20 minutes after plasmin
treatment, and thereafter, fragment Y, D and E
appeared subsequently. With reduction, the Aα
chain gradually decreased in its content when frag-
ment X and fragment A were formed. Therefore,
in order to maximize the separation of fragment
A, the incubation of 30-60 minutes at 37℃ was
satisfactory.7)
After an adequate digestion of 500mg fibrino-
gen by plasmin as described above, the digestion
was stopped by addition of SBTI (soybean trypsin
inhibitor), and the mixture was heated at 90℃ for
10 minutes or at 60℃ for 30 minutes to remove
any clottable fibrinogen and heat-labile fragments.
After the heated mixture was centrifuged at 15,000
rpm for 30 minutes at 4℃, the supernatant was
recovered and lyophilized.
The lyophilized material was re-dissolved by a
small volume of distilled water, and was chroma-
tographed with Sephadex G-200 column (2.5×90
cm). The second major peak was found to contain
fragment A, and it was identified as a single pro-
tein band on SDS-PAGE in both reduced and non-
reduced forms. With this method, approximately
70mg of fragment A could be recovered from 500
mg of human fibrinogen.
2. Properties of fragment A
The molecular weight of purified fragment A
was obtained to be approximately 22,500 by 7.5%
SDS-PAGE, using the known protein molecules
such as fibrinogen, IgG, bovine albumin, trypsin,
cytochrome c and bovine hemoglobin (Fig. 4).7)
Fragment A was hydrolyzed with 6 N HCl at
110℃ for 20, 40 and 60 hours, and the digests
were analyzed by Spachman's method, using auto-
mated amino acid analyzer JLC-6AH. Its amino
acid composition is shown in Table 2. It is mainly
composed of serine, glycine, threonine and proline,
amounting to approximately 51%. In contrast, it
contains only a scanty amount of hydrophobic
amino acids.7)
A fragment A solution of 3mg/ml was injected
repeatedly into 6 rabbits with complete Freund's
adjuvant, and the specific anti-fragment A was
Fig. 4. Estimation of the molecular weightof fragment A by SDS-PAGE (7.5%
gel). The black dott indicates thefragment A.
Table 2. Amino acid composition of fragmentA.a)
a) Values are given as molar ratios. Determinedfrom average values obtained from 20-, 40-,and 60-hr hydrolyses.
b) Values obtained from 20-hr hydrolysis.c) Extrapolated value obtained from 20-, 40-,
and 60-hr hydrolyses.
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(34) 生物 物理化学
obtained successfully. By using this specific anti-
fragment A rabbit serum, Ouchterlony double
immunodiffusion method was performed to evaluate
immunological cross reactivity with fibrinogen. As
shown in Fig. 5, anti-fibrinogen did not react against
fragment A, while anti-fragment A did react against
both fragment A and fibrinogen. On immunoelectro-
phoresis, fragment A was identified at the β-region.
3. Radioimmunoassay of fragment A
Radioiodination of fragment A was performed
with Chloramine-T method of Hunter and Green-
wood. Thus, 59.3% of radioactivity was recovered
in association with fragment A, and its specific
radioactivity was 43μCi/μg.
Radioimmunoassay system of two-antibody
method was established as shown in Table 3.
125I-labeled fragment A was made to show a
radioactivity of approximately 10,000cpm, and the
anti-fragment A rabbit serum was diluted at 1:
500 for routine analysis. Normal rabbit serum and
anti-rabbit immunoglobulin goat serum were both
diluted at 1: 40 for routine use.
Immediately after 9ml of venous blood was
drawn, added was 1ml of the solution containing
13mg ε-aminocaproic acid and 500 KIU Trasylol.
After incubation at 37℃ for 4 hours, serum was
separated by centrifugation. Serum samples were
then heated at 60℃ for 30 minutes and then centri-
fuged at 15,000 G for 20 minutes. The supernatant
serum was used for fragment A assay. Heating was
performed to remove clottable and heat-labile frag-
ments, if any.
4. Serum concentration of fragment A
Normal adult male and female individuals (20
-30 years of age; 15 male and 5 female) contained
3.57±1.62μg/ml of serum fragment A.
As shown in Table 4 and Fig. 6, some of the
patients' sera were found to contain a significantly
increased amount of fragment A. It was signifi-
cantly increased in the patients with acute leuke-
mia, cerebrovascular diseases, myocardial infarction
and malignancies. An extremely high level was
demonstrated in the patients with sepsis, SLE and
renal insufficiency. Among leukemic patients, it was
extremely high in acute promyelocytic leukemia,
while it was almost normal in acute myelocytic
leukemia in remission. Among the patients with
cerebrovascular diseases, it tended to be higher in
cerebral thrombosis than in cerebral bleeding. It
was definitely high in the patients with malig-
nancies, especially so in the cases with advanced
gastric, pulmonary, pancreatic, hepatic, renal and
prostatic carcinomas. Thus, the measurement of
serum fragment A level might be useful to under-
stand pathophysiology of various clinical conditions,
especially in relation to in vivo fibrinogenolysis and
Fig. 5. Immunodiffusion pattern of the frag-ment A (A) and fibrinogen (F) aga-inst anti-fibrinogen (A-F) and anti-fragment A (A-A).
Table 3. Radioimmunoassay of fragment A (two-antibody method).
* 0.05M phosphate buffer, pH7.5 containing 0.1
% bovine serum albumin, 3.3% Dextran-T 40,0.01M ethylenediamine tetraacetic acid and 50KIU/ml Trasylol.
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Vol. 28. No. 1. 1984 (35)
Fig. 6. Serum concentration of fragment A in various clinical conditions.
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(36) 生物物理化学
/or fibrinolysis.
Table 4 Serum concentration of fragment A in various diseases.
* Serum concentration of fragment A in 2 cases.
References
1) Pizzo, S. V., et al.: J. Biol. Chem., 247: 636,1972.
2) Marder, V. J. and Schulman, N. R.: J. Biol.Chem., 244: 2111, 1969.
3) Marder, V. J. and Schulman, N. R.: J. Biol.
Chem., 244: 2120, 1969.4) Edgington, T. S. and Plow, E. F.: J. Biol.
Chem., 250: 3393, 1975.5) Takagi, T. and Doolittle, R. F.: Biochemistry,
14: 5149, 1975.6) Doolittle, R. F., et al.: Arch. Biochem. Bio-
phys., 118: 456, 1967.7) Takagi, K.: J. Jpn. Ass. Int. Med., 66: 621,
1977.
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