Synthesis of modified fragments of fibrinogen and their effect on the activity of proteolytic enzymes

Article abstract of DOI:10.1134/S106816200602004X

New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized: Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV), Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their eff

Full text of DOI:10.1134/S106816200602004X

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2006, Vol. 32, No. 2, pp. 129–135. © Pleiades Publishing, Inc., 2006.
Original Russian Text © V.N. Nikandrov, N.S. Pyzhova, V.P. Golubovich, O.V. Mel’nik, V.P. Martinovich, 2006, published in Bioorganicheskaya Khimiya, 2006, Vol. 32, No. 2,
pp. 144–150.
Synthesis of Modified Fragments of Fibrinogen and Their Effect
on the Activity of Proteolytic Enzymes
V. N. Nikandrova, N. S. Pyzhovaa, V. P. Golubovichb,
O. V. Mel’nikb, and V. P. Martinovichb, 1
a
Institute of Physiology, National Academy of Sciences of Belarus, ul. Akademicheskaya 28, Minsk, 220141 Belarus
b
Institute of Bioorganic Chemistry, National Academy of Sciences of Belarus,
ul. akademika Kuprevicha, 5/2, Minsk, 220141 Belarus
Received August 30, 2005; in final form, October, 27, 2005
Abstract—New analogues of the Gly-Pro-Arg and Arg-Gly-Asp fragments of fibrinogen were synthesized:
Gly-Pro-Arg-Pro (I), Gly-Pro-Arg-Pro-Met-OMe (II), Gly-Pro-Arg-Pro-Phe (III), Gly-Pro-Arg-Pro-Asp (IV),
Gly-Pro-Arg-Pro-Glu (V), and Arg-Asn-Trp-Asp (VI). Their effect on the activity of proteases of various types
was studied with the method of lysis of fibrin plates. All the peptides were found to inhibit plasmin activity (by
60–85%) and the γ-subunit of nerve growth factor (by 55–93%). Tetrapeptide (VI) proved to be an effective
inhibitor of tissue activator of plasminogen and the γ-subunit of nerve growth factor (by 96 and 93%, respec-
tively). The peptides exerted practically no effect on the activity of urokinase and moderately inhibited the
activity of streptokinase [(III), IV), and (VI)], papain [(I), (II), IV), and (VI)], subtilisin [(V) and (VI)],
α-chymotrypsin [(III), (V), and VI)], and Bacillus subtilis metalloprotease (VI). They inhibit trypsin [except
for (I) and (III)] when applied on fibrin plates at a concentration of 1 × 10–2 M, while, at the concentration of
1 × 10–3 M, (I) and (II) induced an increase in proteolytic activity by 35 and 47%, respectively.
Key words: fragments of fibrinogen, synthesis of analogues, effect on activity of proteases
DOI: 10.1134/S106816200602004X
INTRODUCTION
trum of anticoagulant or antithrombotic activities of
these peptides is determined by their interaction with
various proteases of blood plasma. Different levels of
anticoagulant activity of these compounds detected in
our experiments in the fibrinogen–thrombin model sys-
tem and directly in blood plasma supports this hypoth-
esis.
The goal of this research is the synthesis of Gly-Pro-
Arg-Pro analogues modified at N-end by the introduc-
tion of residues of dicarboxylic acids, Met, and Phe and
the synthesis of tetrapeptide Arg-Asn-Trp-Asp, an Arg-
Gly-Asp analogue, which possesses immunomodulat-
ing in addition to antithrombotic activity [11]. The
effect of all the analogues synthesized on the fibrin-
olytic activity of proteases of the various types was also
studied.
Peptide fragments of fibrinogen are known to pos-
sess their own biological activities, such as anticoagu-
lant, antithrombotic etc. [1, 2].2 Their modification
allows the obtaining of analogues, which often substan-
tially excel the natural peptides in activity, selectivity of
action, and enzymatic stability. The analogues based on
the sequence Arg-Gly-Asp, the binding site of fibrino-
gen and other adhesive glycoproteins with membrane
receptor complex GP IIIb/IIa [3–7], and those derived
from the Gly-Pro-Arg peptide, localized at the N-termi-
nus of fibrin [8–10], are the most studied.
The anticoagulant activity of Gly-Pro-Arg and its
analogue with Pro residue at C-terminus is known to be
due to their ability to selectively bind to specific sites of
the fibrinogen molecule and block the polymerization
process of fibrin monomer [9]. At the same time, the
structural peculiarities of the majority of Arg-Gly-Asp
and Gly-Pro-Arg analogues suggest that the total spec-
RESULTS AND DISCUSSION
The peptides were synthesized by classical methods
of peptide chemistry with the use of protonation for
blocking the guanidine group of arginine, which
allowed us to exclude the use of NG-derivatives of argi-
nine and the stage of cleavage of the NG-protection. The
N-terminal dicarboxylic acids were introduced into
1
Corresponding author; phone: +375 (17) 2648263; e-mail:
vermar@iboch.bas-net.by.
2
Abbreviations: DCC, N,N'-dicyclohexylcarbodiimide; HOBt,
1-hydroxybenzotriazole; NGF, nerve growth factor; NSu, N-suc-
cinimidyl; Pcp, pentachlorophenyl; Pfp, pentafluorophenyl; TAP,
tissue activator of plasminogen; and TEA, triethylamine.
129

130
NIKANDROV et al.
Boc–Pro–OH + HCl · Xaa(OMe)n–OMe
TEA, DCC, HOBt
Boc–Pro–ONSu + Arg–OH Boc–Pro–Xaa(OMe)n–OMe
HCl/EtOAc
Boc–Pro–Arg–OH + HCl · Pro–Xaa(OMe)n–OMe
DCC, HOBt
Boc–Pro–Arg–Pro–Xaa(OMe)n–OMe
HCl/EtOAc
HCl · Pro–Arg–Pro–Xaa(OMe)n–OMe
TEA, Boc–Gly–OPcp
Boc–Gly–Pro–Arg–Pro–Xaa(OMe)n–OMe
NaOH
Boc–Gly–Pro–Arg–Pro–Xaa–OH
HCl/EtOAc
2HCl · Gly–Pro–Arg–Pro–Xaa–OH
Scheme 1. Synthesis of pentapeptides of the general formula 2HCl · Gly-Pro-Arg-Pro-Xaa-OH, where Oaa is Met, Phe, Asp, or
Glu; n = 0, when Oaa is Met or Phe,and n = 1, when Oaa is Asp or Glu.
reaction as dimethyl esters. The peptides of the general given in Table 1. The structure and homogeneity of the
peptides were monitored by mass spectrometric,
HPLC, TLC and other methods.
formula Gly-Pro-Arg-Pro-Xaa were synthesized
according to Scheme 1. The tetrapeptide standard Gly-
Pro-Arg-Pro was synthesized according to a similar
scheme, with Pro-OMe being used as the N-terminal
fragment. The synthesis of the peptide Arg-Asn-Trp-
Asp is shown in Scheme 2, and the main physical and
chemical constants of the synthesized compounds are
Several groups of proteolytic enzymes were used in
studying the effect of the peptides on the activity of pro-
teases. The first group was the proteases with a narrow
specificity that possess an ability to activate plasmino-
gen. These were urokinase, streptokinase, γ-subunit of
NGF, and TAP.
The plasminogen-activating activity was deter-
mined by the action of the peptides on human plasmi-
nogen. The plasminogen specifically bound to fibrin
due to affine properties was used in these experiments;
this complex is always present in organism together
with fibrin and remains in fibrin preparations after iso-
lation, if no special procedures were used for its elimi-
nation.
Boc–Trp–OH + Asp(OMe)–OMe
DCC, HOBt
Boc–Trp–Asp(OMe)–OMe
HCl/EtOAc
HCl · Trp–Asp(OMe)–OMe
TEA, Boc–Asn–OPfp
The second group of proteolytic enzymes was rep-
resented by serine proteases. These were, first of all,
plasmin, and also trypsin, chymotrypsin, and subtilisin.
Of other families of proteases, we investigated papain,
a cysteine protease, pepsin, an aspartate protease, and
also metalloprotease from Bacillus subtilis. Fibrin
plates devoid of the functionally active plasminogen (it
was inactivated by UV-irradiation by a special tech-
nique [12]) were used as substrate at studying these
proteases.
Boc–Asn–Trp–Asp(OMe)–OMe
HCl/EtOAc
Boc–Asn–Trp–Asp(OMe)–OMe
HCl/EtOAc
Boc–Arg–Asn–Trp–Asp(OMe)–OMe
NaOH
Boc–Arg–Asn–Trp–Asp–OH
HCl/EtOAc
The synthesized peptides (I)–(VI) affected the
activity of the proteases in dependence on the protease
character and the peptide structure. The studies were
carried out at two working concentrations of the pep-
tides: 1 × 10–2 and 1 × 10–3 M. The inhibition of pro-
2HCl · Arg–Asn–Trp–Asp–OH
Scheme 2. Synthesis of the peptide 2HCl · Arg-Asn-Trp-
Asp-OH.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

SYNTHESIS OF MODIFIED FRAGMENTS OF FIBRINOGEN
Table 1. Physicochemical characteristics of the compounds synthesized
131
[α]D20
*
Peptide
Mp, °C
Rf (A)**
Rf (B)**
RT (C)***, min
Gly-Pro-Arg-Pro-OH · 2HCl (I)
–75
–112
–73
123–125
118–121
175–178
178–181
133–136
141–143
0.20
0.62
0.74
0.13
0.18
0.38
0.05
0.26
0.13
0.25
0.10
0.32
7.332
15.061
19.205
28.171
21.836
20.149
Gly-Pro-Arg-Pro-Met-OMe · 2HCl (II)
Gly-Pro-Arg-Pro-Phe-OH · 2HCl (III)
Gly-Pro-Arg-Pro-Asp-OH · 2HCl (IV)
Gly-Pro-Arg-Pro-Glu-OH · 2HCl (V)
Arg-Asn-Trp-Asp-OH · 2HCl (VI)
–42
–78
–56
Notes: * Concentration of the peptides 10 mg/ml; solvent, methanol.
** (A) 4 : 1 : 1 n-butanol–acetic acid–water, (B) 6 : 4 : 1 chloroform–methanol–20% ammonia.
*** RT, retention time (analytical HPLC was carried out as described in the Experimental section).
Table 2. Effect of the oligopeptides on plasminogen-activating capacity of plasminogen activators of urokinase, TAP, and
NGF γ-subunit
Peptide
C, M
Streptokinase
Urokinase
TAP
NGF γ-subunit
Gly-Pro-Arg-Pro-OH (I)
10–3 173 17 (0.87)
Not studied
462 15* (1.28)
491 20* (1.36)
324 11 (1.03)
113 14* (0.36)
10–2 157 21 (0.79)
282 12 (1.02)
Gly-Pro-Arg-Pro-Met-OMe (II) 10–3 173 14 (0.87)
Not studied
290 11 (1.05)
343 10 (0.95)
188 10* (0.52)
296 8 (0.94)
32 6* (0.10)
10–2 167 17 (0.84)
Gly-Pro-Arg-Pro-Phe-OH (III) 10–3 155 12* (0.78)
Not studied
301 13 (1.09)
350 11 (0.97)
188 17* (0.52)
296 20 (0.94)
138 9* (0.44)
10–2 175 14 (0.88)
Gly-Pro-Arg-Pro-Asp-OH (IV) 10–3 155 8* (0.78)
Not studied
282 7 (1.02)
332 0 (0.92)
108 10* (0.30)
305 10 (0.97)
139 13* (0.44)
10–2 221 11 (1.10)
Gly-Pro-Arg-Pro-Glu-OH (V)
Arg-Asn-Trp-Asp-OH (VI)
Control
10–3 161 16 (0.81)
Not studied
268 9 (0.97)
332 11 (0.92)
274 13* (0.76)
295 8 (0.94)
85 6* (0.27)
10–2 163 14 (0.82)
10–3 176 16 (0.88)
10–2 155 10* (0.78)
Not studied
257 8 (0.93)
332 24 (0.92)
14 4* (0.04)
280 15 (0.89)
22 7* (0.07)
–
199 10
276
9
361 17
315 13
2
Notes: * Experimental conditions: 0.06 M phosphate buffer, pH 7.4, n 5, 37°C, 20 h; area of the zones of lysis on fibrin plates (S , mm )
0
is shown; the ratio S /S is given in parentheses; S , the result of control experiment.
0
c
c
** p ≤ 0.05.
teolytic activity was mainly observed at the peptide
Particularly interesting effects of the compounds
studied were observed in the experiments with TAP:
tetrapeptide (VI) proved to be an effective inhibitor,
while tetrapeptide (I) exhibited a stimulating effect.
The substrate specificity of TAP is related to Arg resi-
due in substrate [13], and, therefore, the action of the
peptides to a certain degree may be due to the presence
of Arg residue in them. Note that the effect of the pep-
tides is caused by their influence on the protein activa-
tor rather than on the plasmin formed during the activa-
concentration of 10–2 M (Tables 2–4).
The results of studying the effect of the peptides on
plasminogen-activating activity are given in Table 2.
The compounds were practically inactive toward uroki-
nase: the effects did not exceed 10% at their concentra-
tion of 1 × 10–2 M. As to streptokinase, a moderate inhi-
bition of its activity by tetrapeptides (I) and (VI) was
observed. Note that a decrease in the peptide concentra-
tion by an order of magnitude led to an increased activ- tion process. This view is supported by a rather weak
action (or the complete absence of the action) of the
peptides on fibrinolysis promoted by either streptoki-
nase or urokinase.
ity of pentapeptide (IV) by approximately 30%
(p ≤ 0.05). Two other representatives of this group,
γ-subunit of NGF and TAP, were strongly effected by
the studied peptides at 1 × 10–2 M concentration: penta-
peptide (II) and tetrapeptide (VI) inhibited plasmino-
gen-activating activity of γ-subunit of NGF by 90–95%.
The effect disappeared at the reduction of concentration
by an order of magnitude.
All the studied peptides affected the activity of
serine proteases; the most pronounced effects were
observed at the peptide concentration of 10–2
M
(Table 3). At this concentration, the activity of trypsin
was inhibited by (II), (IV), (V), and (VI) by 22, 30, 54
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SYNTHESIS OF MODIFIED FRAGMENTS OF FIBRINOGEN
133
Table 4. Fibrinolytic activity of trypsin 2 h after the begin-
ning of incubation of fibrin gel plates in the presence of pep-
tides (10–2 M)
and 38%, respectively. At a tenfold lower concentra-
tion, there was observed even an increase in the fibrin-
olytic activity of trypsin: (I), (II), and (VI) increased it
by 35, 47, and 22% (in the latter case, no reliable
changes were revealed, only a tendency was observed).
At the concentration of 10–2 M, (I), (III), (IV), (V),
and (VI) inhibited the fibrinolytic activity of α-chymo-
trypsin by 20, 25, 22, 25, and 38%, respectively. The
peptides practically cause no effect on the activity of
this protease at a tenfold decreased concentration.
The peptides exerted the most apparent action on the
fibrinolytic activity of plasmin. The effectors' concen-
tration of 10–2 M inhibited this activity by 60–85%, the
strongest inhibition, by 83–85%, being caused by (IV)–
(VI). Such a strong effect, in comparison with those of
trypsin and α-chymotrypsin, might be caused by sev-
eral reasons. The presence of benzamidine-binding site
in the plasmin molecule may probably provide the
binding of the peptides studied. In addition, a particular
substrate specificity of this protease can cause a prefer-
able cleavage of peptide bonds formed by Arg and Lys
residues [14].
Only (V) and (VI) at a concentration of 10–2 M,
exerted a minor inhibitory action (by 19–25%, p ≤ 0.05)
on the activity of serine protease of another subfamily,
subtilisin.
A similar weak effect of the peptides was observed
on papain, a cysteine protease. Only at a concentra-
tions of 10–2 M, the peptides [except for (III) and (V)]
inhibited its activity by no more than 24–33%
(p ≤ 0.05).
The peptides exerted an unexpected and versatile
influence on the activity of pepsin. In all the cases, the
effects were also observed at the peptide concentration
of 10–2 M. The activity inhibition by (I)–(III) was 22–
30%, whereas (IV) and (VI) reduced activity practi-
cally by 50%. At the same time, (V) caused a small, but
distinct increase in proteolytic activity by 37%
(p ≤ 0.05).
Note that neither the presence of benzamidine-bind-
ing site, nor any plasmin-like substrate specificity can
be deduced from the literature data on the structure of
pepsin, although the peptides studied exerted the stron-
gest action on the activity of plasmin (probable reasons
for this phenomenon were mentioned above). However,
we study the effect of the peptides on the activity of
proteases using only one protein substrate, mainly bas-
ing on the relation to fibrinogen of the structures of the
peptides synthesized. It does not mean at all that, with
the use of other protein substrate, the whole described
picture would retain. Moreover, the nature of the pep-
Peptide
S0, mm2
69
35 4*
(I)
(II)
3
(III)
(IV)
(V)
(VI)
Control
73
71
2
0
0*
0*
71
4
Note: Experimental conditions : n 4, 37°C, 0.06 M phosphate
buffer.
* p ≤ 0.05.
(VI) (Arg-Asn-Trp-Asp) exhibited a practically com-
plete its inhibition.A comparison of these results makes
it obvious that, at initial stage, the peptides exert a more
noticeable effect. At the same time, originally, (IV)
(Gly-Pro-Arg-Pro-Asp-OH) did not cause any marked
change of fibrinolytic activity, whereas, after 20 h, a
small, but distinct decrease in this activity was
observed. This suggests that several different mecha-
nisms might underlie the effect of the peptides on the
activity of proteases.
Thus, the effects of the oligopeptides obtained on
the plasminogen-activating capacity of four plasmino-
gen activators and on the fibrinolytic activity of various
proteases are preliminarily characterized. The found
inhibitors of TAP and γ-subunit of NGF can be used for
the development of affinity chromatography of these
proteases. All in all, the compounds synthesized
induced a moderate effect on the fibrinolytic activity of
proteases. The effect was strong only in the case of
plasmin (60–85% of activity inhibition) and a rather
weak in the case of subtilisin and metalloprotease from
B. subtilis. Unexpectant was the inhibition of pepsin
activity by a number of the synthesized peptides, and
also an increase in the activity of trypsin at a lower con-
centrations of peptides (I) and (II). The discovery of
possible causes of such phenomena, including the
action of the peptides on the cleavage of other protein
and low-molecular substrates by proteases, is the sub-
ject of our future studies.
EXPERIMENTAL
L-Amino acids and their derivatives from Reanal
tides synthesized permits its cleavage by various pro- (Hungary), Fluka (Switzerland), and Sigma (United
teases to some or other extent. The effect of the peptides States) were used in the work. The processes of the syn-
on the fibrinolytic activity of trypsin at the initial phase thesis of substances and the removal of protective
of proteolysis, after 2 h, illustrates this statement (see groups were monitored by TLC on the plates with fixed
Table 4). For example, peptide (II) (Gly-Pro-Arg-Pro- silica gel (Sorbfil, Russia) layer in the following sys-
Met-OMe) caused the inhibition of the trypsin activity tems of solvents: (A) 60 : 40 : 10 chloroform–metha-
by 50%, while (V) (Gly-Pro-Arg-Pro-Glu-OH) and nol–25% ammonia, (B) 40 : 10 : 10 n-butanol–acetic
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 32 No. 2 2006

134
NIKANDROV et al.
acid–water, and (C) 40 : 10 : 10 isopropanol–acetic
Plasminogen-activating capacity of plasminogen
acid–water. The substance spots were detected on the activators was determined by a method of lysis of plas-
plates with a chlorine–benzidine reagent.
minogen-containing fibrin plates. For determination of
fibrinolytic activity of proteases, the fibrin plates sub-
jected to a UV-irradiation and not containing function-
ally active plasminogen, were used. We had previously
described in detail the determination of plasminogen-
activating and fibrinolytic activities (in mm2 of the zone
of lysis of fibrin gel) [12, 19, 20]. A 0.2 M acetate
buffer, pH 1.47, was used in the work with pepsin, and
0.06 M phosphate buffer, pH 7.4, with other proteases
and activators. Aliquots of aqueous solutions of the
peptides were added to enzyme solutions up to the final
concentration of 1 × 10–3 to 1 × 10–2 M, whereas the cor-
responding amount of deionized water, to control
probes. The plates with the samples applied were incu-
bated at 37°ë for 20 h.
The protein content in the solutions was estimated
by optical absorption at 280 nm using the following val-
ues of A11Ò%m: plasmin, 17.0 [21], streptokinase; 9.0 [22];
human fibrinogen, 16.0 [23]; bovine trypsin, 14.4 [24];
bovine chymotrypsin, 20.0 [25]; papain, 25.0 [26]; and
pig pepsin, 14.0 [27].
All the samples were measured no less than four-
fold, and the results were statistically processed using
the Student t criterion.
FAB mass spectra were recorded on a PE SCIEX
APl 150EX (Perkin Elmer, United States) instrument;
the ionization was carried out by an electron impact
with energy of 40 eV.
Analytical HPLC was carried out on a Waters
Millenium32 chromatograph equipped with a 966-pho-
todiode detector and a Vydac 201HS52 RP C18
(2.1 × 250 mm) column. A 10 to 40% gradient of aceto-
nitrile in 0.1% TFA was used for elution at a flow rate
of 1 ml/min.
Amino acid composition of peptides hydrolyzed in
sealed ampoules by 6 N HCl at 110°ë for 20 h, was
determined on an LMV automatic amino acid analyzer
(Sweden).
Melting points (noncorrected) were determined on a
Kofler instrument. Optical rotations of the compounds
studied were measured on a spectropolarimeter J-20
(Jasco, Japan).
Samples of trypsin (EC 3.4.21.4), α-chymotrypsin
(EC 3.4.21.1), and pepsin (EC 3.4.23.1) were from
Sigma (United States); and papain (EC 3.4.22.2), from
Fluka (Switzerland); urokinase (EC 3.4.21.73), from
J.C.R. (Japan); streptokinase, from OAO Belmed-
preparaty (Belarus); and B. subtilis subtilisin
(EC 3.4.21.62) and metalloprotease (EC 3.4.24.4),
from Diagnostikum (Moscow, RF).
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