Functional role of Bb-chain N-terminal fragment in the
fibrin polymerization process
E. V. Lugovskoy, P. G. Gritsenko, L. G. Kapustianenko, I. N. Kolesnikova, V. I. Chernishov
and S. V. Komisarenko
Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv
2
, Ukraine
Fibrinogen is a central protein of the blood coagula-
tion system. The human fibrinogen molecule (molecu-
lar mass  344 kDa) consists of two identical subunits
connected by disulfide bonds [1]. Each monomer
subunit is formed by three nonidentical polypeptide
chains, Aa,Bb and c. The N-terminal ends of all six
polypeptide chains are situated in the fibrinogen cen-
tral region, which is known as the E-domain. Two
Keywords
fibrin; monoclonal antibodies; peptides;
polymerization sites
Correspondence
E. Lugovskoy, Palladin Institute of
Biochemistry, National Academy of
Sciences of Ukraine, 9 Leontovicha Street,
01601, Kyiv, Ukraine
Fax: +38 044 2796365
Tel: +38 044 2343354
E-mail: [email protected]
(Received 15 May 2007, revised 3 July
2007, accepted 10 July 2007)
doi:10.1111/j.1742-4658.2007.05983.x
Four mAbs of the IgG
1

Bb12–46 took part in fibrin protofibril formation simultaneously with site
‘A’ (Aa17–19) prior to removal of fibrinopeptide B. A model of the inter-
molecular connection between fragment Bb12–46 of one fibrin desAA
molecule and the D-domain of another has been constructed.
Abbreviations
1
Bb12–26, BbSARGHRPLDKKREEA(12–26); Bb19–26, BbLDKKREEA(19–26); Bb26–36, BbAPSLRPAPPPI(26–36); Bb26–46,
BbAPSLRPAPPPISGGGYRARPA(26–46); Bb40–46, BbGYRARPA(40–46); NaCl ⁄ P
i
, 0.02 M potassium phosphate buffer (pH 7.4) with 0.13 M
NaCl; t-NDSK, thrombin-treated N-terminal disulfide knot of fibrin; TPBS, NaCl ⁄ P
i
with 0.05% Tween-20.
4540 FEBS Journal 274 (2007) 4540–4549 ª 2007 The Authors Journal compilation ª 2007 FEBS
peripheral regions of the fibrinogen molecule were his-
torically named D-domains. However, it was discov-
ered by X-ray analysis that there were two distinct
bC- and cC-domains in the peripheral D-region [2]. As
a result of the activation of the blood coagulation sys-
tem, thrombin is formed; this attacks fibrinogen and
splits off two fibrinopeptides A (Aa1–16). Fibrinogen
is transformed into fibrin desAA, which is able to
polymerize spontaneously, forming two-stranded pro-
tofibrils with half-staggered fibrin molecules [3,4]. Pro-
tofibrils associate laterally, producing fibrils. The fibrils
also associate laterally, branching and forming a three-
dimensional network, which is the framework of the
whole blood thrombus [5]. It is now widely accepted
that the initial step of fibrin polymerization (protofibril
formation) is carried out by the intermolecular pairing

epitope involves BbArg14, specifically inhibited fibrin
desAA polymerization [15]. It was also shown that
synthetic peptide Bb40–54 dissociated the (DD)E com-
plex at a 5000 molar ratio of peptide to complex [16].
The Bb28–30 and Bb36–44 regions are framed by pro-
line brackets, which usually indicates that such pep-
tides are involved in protein–protein interactions [17].
Three mAbs to t-NDSK that inhibit fibrin polymeriza-
tion are described in this article. The inhibitory effects
of synthetic peptides Bb12–26 and Bb26–46 on fibrin
polymerization were also studied. The results suggest
that an unknown site (not ‘B’), important for fibrin
polymerization, is situated at fibrin fragment Bb12–46.
This site seems to take part in protofibril formation,
and is operational without fibrinopeptide B being
removed by thrombin.
Results
We have obtained 35 hybridomas producing different
antibodies of IgG
1
class to human fibrin fragment
thrombin-treated N-terminal disulfide knot of fibrin
(t-NDSK) with the molecular structure (Aa17–51,
Bb15–118, c1–78)
2
. Three of these hybridomas secret-
ing mAbs I-5G, I-3B and III-10D were chosen on the
basis of the strong and specific inhibition by these
mAbs of fibrin desAABB polymerization. Turbidity
analysis showed that these mAbs and their Fab-frag-

ELISA may be explained by the contamination of the
c1–78 preparation with Bb15–118. ELISA and immu-
noblot results indicate that epitopes for all four mAbs
are situated in the Bb15–53 fibrin fragment. K
D
values
of the binding of mAbs I-5G, I-3B, III-10D and
I-3C to t-NDSK were 1.0 · 10
)8
m, 7.4 · 10
)9
m,
E. V. Lugovskoy et al. Functional role of Bb-chain N-terminal fragment
FEBS Journal 274 (2007) 4540–4549 ª 2007 The Authors Journal compilation ª 2007 FEBS 4541
2.3 · 10
)8
m, and 3.7 · 10
)9
m, respectively. The com-
petitive ELISA showed that mAb I-5G competed
with I-3B and mAb III-10D competed with I-3C in
fibrinogen binding. mAbs I-5G and I-3B did not com-
pete with mAbs III-10D and I-3C.
To localize epitopes for these mAbs, the following
peptides comprising human fibrinogen sequences in
Bb12–46 were synthesized: B bSARGHRPLDKKRE
EA(12–26), BbLDKKREEA(19–26), BbAPSLRPAPP
PI(26–36), BbAPSLRPAPPPISGGGYRARPA(26–46),
and BbGYRARPA(40–46). Competitive ELISA was
used to study the binding of mAbs to fibrinogen in the

) on the molar ratios of mAbs I-5G (A), 1-3B (B), and
III-10D (C), and their Fab-fragments, to fibrin.
Fig. 2. Binding curves of mAb I-5G to the antigens fibrin fragment
Bb15–118, t-NDSK, fibrinogen (F), fibrin desAABB, fibrin fragment
c1–78 and fibrin fragment E
3
adsorbed to microtiter plates (ELISA).
Fig. 3. Immunoblot analysis of reduced t-NDSK using mAbs I-5G,
I-3B, and III-10D. Lane 1: molecular mass markers. Lanes 1–3:
SDS ⁄ PAGE stained with Coomassie R250. Lanes 4 and 5: immuno-
staining with mAb III-10D. Lanes 6 and 7: immunostaining with
mAb I-3B. Lanes 8 and 9: immunostaining with mAb I-5G. Lanes 2,
4, 6, and 8: fibrin fragment Bb15–118. Lanes 3, 5, 7, and 9:
t-NDSK + b-mercaptoethanol.
Functional role of Bb-chain N-terminal fragment E. V. Lugovskoy et al.
4542 FEBS Journal 274 (2007) 4540–4549 ª 2007 The Authors Journal compilation ª 2007 FEBS
synthesized peptides on fibrin polymerization. Turbi-
dity analysis showed that peptides Bb12–26 and Bb26–
46 increased lag-time and decreased the maximum
rate of polymerization of both fibrin desAABB and
fibrin produced in the fibrinogen + thrombin reaction
(Fig. 6A,B). The values of IC
50
(the concentrations of
the peptides at which 50% of inhibition of the fibrin
polymerization maximum rate were observed) for
Bb12–26 and Bb26–46 were 2.0 · 10
)4
m and
2.19 · 10

The peptide concentrations varied from 0.39 lgÆmL
)1
to 50 lgÆmL
)1
, and the mAb concentration was constant at 1 lgÆmL
)1
. Fibrinogen was
adsorbed to microtiter plates.
Fig. 5. The amino acid sequence of human fibrin fragment Bb12–
46. The epitope localization for mAbs I-5G, I-3B, III-10D and I-3C is
indicated by arrows.
16
Fig. 6. The influence of the synthetic peptides Bb12–26 and Bb26–46 on fibrin desAABB polymerization in turbidity analysis. The depen-
dence of the lag-time s (A), the maximum rate of fibrin polymerization V
max
(B) and final turbidity Dh (C) of fibrin clots on the molar ratio of
the synthetic peptides to fibrin.
E. V. Lugovskoy et al. Functional role of Bb-chain N-terminal fragment
FEBS Journal 274 (2007) 4540–4549 ª 2007 The Authors Journal compilation ª 2007 FEBS 4543
ratio to fibrin had no inhibitory effect on fibrin poly-
merization.
To determine which stage of fibrin polymerization is
affected by these peptides, we performed transmission
electron microscopy at different stages of the fibrin
polymerization process in the presence of peptides
Bb12–26 and Bb26–46. Electron microscopy showed
(Fig. 7B,C) that fibrin stayed in monomeric form when
peptides Bb12–26 and Bb26–46 were present in poly-
merization media at a molar ratio to fibrin of 1500.
Without these peptides, fibrin formed protofibrils and

merization of fibrin desAA. These results show that
mAbs block a previously undescribed polymerization
site, which is not a short peptide fragment like site ‘A’
(Aa17–19) or ‘B’ (Bb15–17), but comprises a longitudi-
nal amino acid sequence in Bb15–36. As mAbs retard
both fibrin desAABB and fibrin desAA polymeriza-
tion, one can conclude that the blocked polymerization
site does not coincide with polymerization site ‘B’
(Bb15–17).
mAb 2d)2a targets an epitope encompassing the
peptide bond BbArg14-Gly15, which is cleaved by
thrombin [15]. This mAb inhibited the maximum rate
of fibrin desAA polymerization by 60%, whereas its
Fab-fragment inhibited it by 100%, at molar ratios of
antibody to fibrin of 1 and Fab to fibrin of 2, respec-
tively. Moen et al. [14] found impaired polymerization
of fibrin obtained from recombinant fibrinogen with
histidine substituted for arginine at Bb14. Turbidity
analysis showed an increase in lag-time and a decrease
in maximum polymerization rate of this fibrinogen in
the desAA fibrin form. The final turbidity of these
fibrin clots proved to be decreased, and electron
microscopy showed that fibrin fibrils were thinner than
ABC
DEF
Fig. 7. Electron micrographs of negatively contrasted structures formed during fibrin desAABB polymerization in 80 s (A,B,C) and 180 s
(D,E,F) from the start of the process: (A,D) in the absence the synthetic peptides; (B,E) in the presence of Bb12–26 peptide; and (C,F) in the
presence of Bb26–46 peptide. Initial protofibril lateral associates are indicated by arrows. The bars represent 100 nm.
Functional role of Bb-chain N-terminal fragment E. V. Lugovskoy et al.
4544 FEBS Journal 274 (2007) 4540–4549 ª 2007 The Authors Journal compilation ª 2007 FEBS

the whole longitudinal site Bb12–46 (we have named it
‘C’), which takes part in fibrin intermolecular binding
during the construction of two-stranded protofibrils
simultaneously with site ‘A’ (Aa17–19). This site ‘C’ is
operational without fibrinopeptide B (Bb1–14) splitting
off; that is, it does not coincide with polymerization
site ‘B’ (Bb15–17). Siebenist et al. found that fibrino-
gen lacking fragment Bb1–42 was clotted by thrombin
but with an essential delay of protofibril formation
[12]. Pandya et al. [13] showed an inhibitory effect of
peptide Bb15–42 on fibrin polymerization, and this
effect was not determined by polymerization site ‘B’
(Bb15–18). Our results obtained with mAb 2d)2a [15]
and with peptide Bb12–26, and the findings of Moen
et al. [14], show that the polymerization site situated at
the N-terminus of the Bb-chain comprises the amino
acid residues localized to the left of the peptide bond
Bb14–15. Pandya et al. [13] did not find an inhibitory
activity for peptide Bb40–54 up to a molar ratio with
fibrin of 1000. However, Moskowitz & Budzynski dis-
covered that this peptide dissociated the (DD)E com-
plex at a molar ratio of 5000 [16]. The latter result and
our data obtained with peptide Bb26–46 show that
polymerization site ‘C’ comprises amino acid residues
to the right of the peptide bond Bb42–43.
Fibrin protofibril formation is carried out by inter-
molecular pairing of ‘A’ and ‘a’ polymerization sites
localized in fibrin central E- and peripheral
D-domains, respectively [3,4]. As polymerization site
‘C’ (Bb12–46) is situated in the E-domain of the fibrin

γC
γC
βC
BβArg14
D
E
D
βC
‘‘A’’-‘‘a’’
17
Fig. 8. The model (in two projections) of the intermolecular connec-
tion between the D-domain of one fibrin desAA molecule (blue) and
Bb12–46 (magenta) of another. The model was prepared with
PYMOL [24] on the basis of the X-ray analysis data of chicken
fibrinogen [25] and human D-dimer bound with synthetic peptide
GPRP [7].
E. V. Lugovskoy et al. Functional role of Bb-chain N-terminal fragment
FEBS Journal 274 (2007) 4540–4549 ª 2007 The Authors Journal compilation ª 2007 FEBS 4545
belonging to another strand within the protofibril.
After fibrinopeptide B is split off the site, ‘B’ is
formed, and the latter interacts intermolecularly with
the complementary site ‘b’ situated in the bC-domain
in a ‘knob’–‘hole’ type of interaction [7,9]. The remain-
ing part of site ‘C’ (Bb19–46) probably remains bound
to the fibrin D-domain. To determine the localization
of a complementary site ‘c’, it is necessary to perform
X-ray analysis of the complex between D-dimer and
peptide Bb12–46. Recently, Pechik et al. [27] found an
interaction of recombinant peptide (Bb1–66)
2

known that fibrinogen is able to polymerize under
special conditions without splitting off fibrinopeptides
A and B, forming cross-striated fibrils [30]. This poly-
meric interaction of fibrinogen molecules may be
explained by the participation of the ‘C’–‘c’ pairing
sites, as both sites are probably exposed in the fibrin-
ogen molecule.
Experimental procedures
Preparation of fibrinogen, fibrin desAA, fibrin
desAABB and t-NDSK
Human fibrinogen was prepared by sodium sulfate precipi-
tation from human plasma [31]. DesAABB fibrin monomer
was prepared as described by Belitser et al. [32]
3
. DesAA
fibrin monomer was prepared by our original method as
described previously [33]. t-NDSK was obtained as des-
cribed by Timpl & Gollwitzer [34]
4
.
Preparation and purification of mAbs
Hybridomas were obtained essentially as described by Ko
¨
h-
ler & Milstein [35]. mAbs were isolated from hybridoma
culture medium by affinity chromatography on fibrinogen
Sepharose 4B, as described elsewhere [36]. The determina-
tion of IgG class and subclass was performed by ELISA
using an Isotyping kit (Clinical Credential; ICN Immunobio-
logicals, Lisle, IL, USA)

Bb15–118 and c1–78 fragments of t-NDSK, and E
3
-frag-
ment. Coating was achieved by adding to the wells 110 lL
of solutions (10 lgÆmL
)1
) of antigens (fibrinogen in 0.2 m
ammonium acetate buffer, pH 8.5; fibrin desAABB, Bb15–
118 and c1–78 fragments of t-NDSK in 0.2 m ammonium
acetate buffer, pH 8.5 with 3.0 m urea; t-NDSK and
E
3
-fragment in 0.02 m sodium bicarbonate buffer, pH 9.5),
with subsequent incubation for 18 h at 4 °C. The plates
were washed three times with NaCl ⁄ P
i
containing 0.05%
Tween-20 (TPBS), and 100 lL of mAbs solutions in
NaCl ⁄ P
i
were added to the wells and incubated for 60 min
at 37 °C. After washing of the plate, 100 lL aliquots of a
1 : 1000 solution in TPBS of the rabbit anti-(mouse IgG)
conjugated with horseradish peroxidase (Sigma
7
-Aldrich,
St Louis, MO, USA) were added to each well. After subse-
quent incubation (60 min, 37 °C) and washing with
NaCl ⁄ P
i

described above.
Determination of dissociation constants (K
D
)
K
D
values were determined by indirect competitive ELISA
as described by Friguet et al. [37]
9
. In brief, microtiter wells
were coated with either t-NDSK or fibrinogen, and mix-
tures of mAbs with relevant antigen (fibrinogen, t-NDSK,
etc.) were added to the wells. The concentration of mAb
was kept constant, and the concentration of competing
antigen was varied. The plates were incubated for 1 h at
37 °C, and washed three times with TPBS. Quantification
of the mAbs bound was performed with rabbit anti-(mouse
IgG) (Sigma-Aldrich) conjugated with horseradish peroxi-
dase as described above.
Immunoblot analysis
Immunoblot analysis was used to examine the reactivity of
mAbs obtained to t-NDSK and to its Bb15–118 peptide.
Briefly, 4 lg
10
of t-NDSK reduced by 5% b-mercaptoethanol
and 1.3 lg of fragment Bb15–118 were separated by
SDS ⁄ PAGE in 15% polyacrylamide gel, and the proteins
were electrophoretically transferred to nitrocellulose mem-
branes (Hybond ECL; Amersham, Uppsala, Sweden)
11

curve of increasing turbidity during fibrin clotting shows
the following parameters: s, the lag-time, which corre-
sponds to the time of protofibril formation; V
max
, maxi-
mum rate of fibrin polymerization, which was defined by
graphic calculation of the angle of the tangent to the tur-
bidity increase curve at the point of maximum steepness;
and Dh, final turbidity of fibrin clots. Polymerization of
fibrin desAA and desAABB was studied at a final concen-
tration 0.1 mgÆmL
)1
in the polymerization medium contain-
ing 0.05 m ammonium acetate (pH 7.4) with 0.1 m NaCl
and 1 · 10
)4
m CaCl
2
. Polymerization of fibrin formed in
the fibrinogen + thrombin reaction was investigated at a
final concentration of fibrinogen of 0.1 mgÆmL
)1
and a final
concentration of thrombin of 0.4 NIH unitsÆmL
)1
in the
same polymerization medium.
Electron microscopy
The samples of polymerizing fibrin desAABB in the absence
or presence of synthetic peptides Bb12–26 or Bb26–46 were

E. V. Lugovskoy et al. Functional role of Bb-chain N-terminal fragment
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