InhIbItIon of fIbrIn polymerIzatIon by synthetic peptides corresponding to Аα 195-205 and γ 69-77 sItes of fIbrIn molecule

Using the idea of “proline brackets” we have found four sites in fibrin amino acid sequence, and appropriate peptides were synthesized: γ69NPDESSKPN77, Bβ228QPDSSVKPY236, Bβ455RPFFPQ460 and Aα195LPSRDRQHLPL205. Turbidity and electron-microscopy analyses have demonstrated that synthetic peptide Аα195-205 specifically inhibited the stage of fibrin protofibril formation and peptide γ69-77 – the stage of fibrin protofibril lateral association. The data obtained testify that there are the sites involved in these processes in the appropriate amino acid sequences of fibrin molecule.

O n activation of the blood coagulation system thrombin is formed.This enzyme cleaves two fibrinopeptides А (FpА) from fibrinogen and transforms it into fibrin desА which is able to polymerize spontaneously by the intermolecular pairing of the complementary polymerization sites "А" and "а" [1,2].As a result, the two-thread protofibrils are formed (the first step of polymerization), which associate laterally (the second step of polymerization), branch out and form the three-dimensional framework of thrombus [3].After protofibril formation thrombin cleaves fibrinopeptides B (FpВ) from fibrin desA, accelerating protofibrils lateral association.
There are data testifying to the existence of other functional sites participating in the fibrin polymerization process.Localization and elucidation of a functional role of these sites give a possibility to choose some of them for the synthesis of low-molecular weight peptides, which prevent the formation of three-dimensional fibrin network and thrombus creation.
The aim of this work is the localization of fibrin unknown functional sites, which participate in the process of its polymerization.
Identification of protein-protein interactions sites is one of the most complicated tasks in proteins chemistry.It has been known from the literature that two residues of proline, which are located at the distance of approximately 3-7 amino acid residues in a protein molecule, create so-called "proline brackets" and often encompass the sites of inter-protein interaction [4].A destruction of α-coil and a protein chain bend are observed in such a site.Thus, proline residues, perhaps, play a structural role, giving some conformation necessary for proteins interaction, to the sites they limit [5,6].Correspondingly, the sites of protein-protein interactions may be predicted in the presence of "proline brackets" [7].Basing on such approach, Budzynski et al. [8] have presumed existence of fibrin polymerization site in the amino acid sequence Аα541-574, where the "proline bracket" is located (Аα546-552).We have earlier localized the site of the fibrin polymerization in the fragment Bβ26APSLRPAPPPISGGGYRARPA46 [9].
We have applied such approach in this work to preliminary localization of functionally important sites of fibrin molecule.

matherials and methods
Human fibrinogen was isolated from human blood plasma by fractional salting out with natrium sulphate [10].Fibrin desА [11] and fibrin desАВ [12] were obtained in fibrinogen+thrombin reaction.
Turbidity analysis of fibrin polymerization.The effects of synthetic peptides at various concentrations on fibrin polymerization were studied spectrophotometrically at 350 nm.Polymerization of fibrin desA and desAB was studied at a final concentration 0.1 mg/mL in the polymerization medium containing 0.05 M ammonium acetate (pH 7.4) with 0.1 M NaCl and 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 mg/mL and a final concentration of thrombin of 0.4 NIH units/ mL in the same polymerization medium.The curve of increasing turbidity during fibrin clotting shows the following parameters: τ, the lag-time, which corresponds to the time of protofibril formation; V max , maximum rate of fibrin polymerization, which was defined by graphic calculation of the angle of the tangent to the turbidity increase curve at the point of maximum steepness; and Δh, final turbidity of fibrin clots.
electron microscopy.The samples of polymerizing fibrin desAB in the absence or presence of synthetic peptides Aα195-205 or γ69-77 were taken out of the reaction medium at various times, placed on a carbon-coated grid for 2 min, and then stained with 1% (w/v) uranyl acetate (Merck, Germany) for 1 min.Transmission electron microscopy was performed in a H-600 electron microscope (Hitachi, Chiyoda, Japan) operated at 75 kV.Electron micrographs were obtained at a magnification of 50 000 on Kodak SO-163 film.
Statistical analysis of the received data has been conducted by usage of software Prism 5 (GraphPad Software Inc., USA).To determine connection between studied definitions, Pirson correlation coefficients (characterize measure of linear relation between variables) have been used, validity has been estimates in compliance with Student's t-criterium.Maximal rate of polymerization (V max = (dЕ 350 /dt) t=τ , where τ -lag-period) has been determined by graphic differentiation of turbidity curve (tangent of pitch angle to the most steep initial section of light dispersion ).

results and discussions
Amino acid sequences of fibrin α-, βand γ-chains involving "proline brackets": γ69NPDESSKPN77, Bβ228QPDSSVKPY236, Bβ455RPFFPQ460 and Aα195LPSRDRQHLPL205 were chosen.Corresponding peptides were synthesized using solid-phase method (State Scientific Center "Institute of Immunology" of Federal Medical-Biology Agency of Russia) and their influence on fibrin polymerization was studied by turbidity analysis.
We found that two synthetic peptides, imitating the β-chain fragments of fibrin(ogen) Bβ228-236, Bβ455-460, did not affect the process of fibrin polymerization.Thus, do not all sites which structurally correspond to "proline brackets" and are involved into intermolecular associations of fibrin.These peptides have been used as a negative control.
The synthetic peptides, which imitate the fibrin(ogen) amino acid sequences Аα195-205 and γ69-77, specifically inhibit the process of fibrin polymerization.
Fig. 1 shows that synthetic peptide Aα195-205 significantly decreases maximal rate of fibrin polymerization, prolongs the time of protofibrils formation and does not practically change the final turbidity of the clot.The peptide concentration under which the maximal rate of the fibrin polymerization decreases two times (IC 50 ) was equal 2.38•10 -4 M at the molar ratio of the protein to the peptide 1 : 800.ЕкспЕримЕнтАльні роботи Peptide Aα195-205 inhibits polymerization of monomeric fibrins desА and desАВ as well.IC 50 value for fibrin desА polymerization is 1.34•10 -4 M at molar ratio of the fibrin to the peptide 1 : 400 and 2.2•10 -4 M -for fibrin desАВ, that coincides with IC 50 for fibrinogen+thrombin system.Thus, the inhibitory effect of the peptide is related to the process of the fibrin polymerization but not to the enzymatic step of fibrinogen to fibrin transformation.
To answer the question which stage of polymeri zation is inhibited by peptide Aα195-205, electron-microscopy study has been performed.
Transmission electron microscopy showed that peptide Aα195-205 inhibits the first stage of fibrin polymerization -protofibrils formation out of fibrinmonomers.It has been demonstrated that while in the absence of Aα195-205 peptide only cross-striated fibrils are observed, only fibrin oligomers are observed in the presence of the peptide (Fig. 2).
Thus, results of the turbidity and electron-microscopy studies demonstrate that synthetic peptide, which imitates amino acid sequence Aα195-205 of fibrin molecule, inhibits the first stage of the fibrin polymerization -protofibrils formation.Thus, we

Fig. 2. Electron micrographs of negatively contrasted structure formed during polymerization of fibrin produced in fibrinogen+thrombin reaction in the absence (А, B) and in the presence (C, D) of synthetic peptide
Аa195-205 in 60s and 180s from start of the reaction may presume that fibrin site Aα195-205 is involved in the process of protofibrils formation.
An analysis of fibrin(ogen) amino acid sequence Аα195-230 (Аα195 LPSRDRQHLPLIK-MKPVPDLVPGNFKSQLQKVPPEWK230) has demon strated the existence of the significant quantity of proline residues and thus so called "proline brackets" [4].This indicates non-structuring of this portion of fibrin(ogen) molecule and its increased ability to inter-protein connections.It is known that recombinant fibrinogen, α-chain of which consists of Аα1-251 amino acid residues, is polymerized by thrombin almost with the same rate as native fibrinogen [14].This fact and the inhibition of the intermolecular interaction of fibrin monomers by peptide Аα195-205 allows us to presume that there is a site in this section of fibrin molecule, which participates in the fibrin protofibrils formation.Thus, the inhibitory action of synthetic peptide Аα195-205 may be explained by the competitive inhibiting of interaction of the Аα195-205 site in fibrin molecule with a complementary site during intermolecular interaction of fibrin-monomers.
Turbidity analysis showed a more pronounced inhibitory effect of synthetic peptide γ69-77 on fibrin polymerization.IC 50 in the system of fibrinogen+thrombin was 1.5•10 -5 M at molar ratio of protein to peptide 1 : 50, that testifies to the high specificity of the effect (Fig. 3).As concentration of the peptide increases, its inhibitory action increases.This fact proves competitive type of inhibition.This permits us to suggest that fibrin site γ69-77 is involved in the fibrin polymerization process.
Synthetic peptide γ69-77 also inhibits polymerization of fibrins desА and desАВ monomers.IC 50 value for fibrin desА is a 1.25•10 -4 and 0.5•10 -4 Mfor fibrin desАВ.Thus the inhibition action of the peptide γ69-77 concerns the process of fibrin polymerization, but not the step of fibrinogen into fibrin transformation by thrombin.
Transmission electron microscopy showed that peptide γ69-77 inhibits the second step of fibrin polymerization -the protofibrils lateral association.Fig. 4 shows that mature fibrils were observed in the absence of peptide, but in the presence of γ69-77 only separate protofibrils were observed.
Amino acid sequence γ69-77 is the structural component of the part of the coiled-coil connector, which binds the central and the peripheral regions of fibrin(ogen).In this part of the coiled-coil γ69-77 α-coil interacts with Вβ126-135 and Аα91-103 α-coils.A possible reason of inhibiting effect of the synthetic peptide γ69-77 is its competitive interaction with Аα91-103 and Вβ126-135 constituents, and, as a result -disturbance of the whole structure in this part of coiled-coil.Synthetic peptide Bβ121-138 which includes the site Вβ126-135 also inhibits protofibrils lateral association [15].Such competitive mechanism of inhibition was proposed earlier by Jing et al. for the synthetic peptide, which imitates fibrinogen amino acid sequence γ23-57, which belongs to the coiled-coil region of fibrin(ogen) molecule [16].
Thus, two new functional sites Аα195-205 and γ69-77 have been localized in fibrinogen molecule.The former is involved in the process of protofibrils formation, and the latter -in the protofibrils lateral association.Interestingly that the sequence γ69-77 involves peptide bond γ72-73 which is cleaved by plasmin.The cleavage of this bond decreases ob-  It is known that proline amino acid residues which encompass sites of interprotein interactions form a stable structure in such regions of the molecule by decreasing of the number of possible conformations.Obviously, the stabilization of the structure by "proline brackets" provides a high affinity of protein-protein interactions [6].Thus, encompassing of a biologically active synthetic peptide by proline residues can significantly increase its physiological activity.The approach may be useful for the design of peptide medicines.Allowing for the high inhibitory activity of the synthetic peptide γ69-77 (IC 50 = 1.5•10 -5 M), it may be the basis for the creation of a new antithrombotic medicine.

Fig. 1 .
Fig. 1.The influence of the synthetic peptide Aα195-205 on polymerization of fibrin produced in fibrinogen+thrombin reaction in turbidity analysis.The dependence of the maximal rate of the fibrin polymerization (а), the lag time (b), and final turbidity of fibrin clots (c) on concentration of Aα195-205.The figure shows typical shapes of the curves illustrating the dependence of three parameters of fibrin polymerization on peptide concentration

Fig. 3 .
Fig. 3.The influence of the synthetic peptide γ69-77 on polymerization of fibrin produced in fibrinogen+thrombin reaction in turbidity analysis.The dependence of the maximal rate of the fibrin polymerization (а), the lag time (b), and final turbidity of fibrin clots (c) on concentration of γ69-77.The figure shows typical shapes of the curves illustrating the dependence of three parameters of fibrin polymerization on peptide concentration

Fig. 4 .
Fig. 4. Electron micrographs of negatively contrasted structure formed during polymerization of fibrin produced in fibrinogen+thrombin reaction in the presence of synthetic peptide g69-77 in 60 s and 180 s from the reaction start