Inhibition of fibrin polymerization by synthetic peptides corresponding to Aα195-205 and γ69-77 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, Bß228QPDSSVKPY236, Bß455RPFFPQ460 and Aα195LPSRDRQHLPL205. Turbidity and electron-microscopy analyses have demonstrated that synthetic peptide Aα195-205 specifically inhibited the stage of fibrin protofibril formation and peptide γ69-77 - the stage offibrin 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.

[Molecular mechanisms of the polymerization of fibrin and the formation of its three-dimensional network].

The results of biochemical, immunochemical, and X-ray studies of the structures of fibrinogen and fibrin molecules were analyzed. The mechanisms of the successive formation of the fibrin three-dimensional network were described: the polymerization of monomeric molecules with the formation of bifilar protofibrils, the lateral association of protofibrils, and the embranchment of the forming fibrils. Data on the electron and confocal microscopy of the polymeric fibrin were considered. The role of the known polymerization centers of fibrin which participated in the formation of protofibrils and their lateral association was discussed. Data on the existence of the previously unknown polymerization centers were given. In particular, the experimental results demonstrated that one of such centers which participated in the formation of protofibrils was located in the Bbeta12-46 fragment, and did not require the cleavage of fibrinopeptide B for its functioning. The results of the computer modeling of the spatial structure of the fibrin(ogen) molecule and the intermolecular interactions in the course of the fibrin polymerization were presented. The location of the alphaC domains in the fibrin(ogen) molecule and their role in the polymerization process were discussed. Information on the structure of the calcium-binding sites of fibrin(ogen) and the functional role of Ca2+ in fibrin polymerization was published. The structure of factor XIII(a) and the mechanisms of fibrin stabilization by this factor were briefly described.

A neoantigenic determinant in coiled coil region of human fibrin beta-chain.

INTRODUCTION: The transformation of fibrinogen into fibrin by thrombin exposes neoantigenic determinants that are buried in fibrinogen. Fibrin-specific monoclonal antibodies (mAbs) to these neoantigenic determinants, which don't react with fibrinogen, can be obtained. The aim of our investigation was to obtain fibrin-specific mAbs, to study their influence on fibrin polymerization and to use them for quantification of soluble fibrin in human blood plasma. MATERIALS AND METHODS: Human fibrin desAABB in 2 M urea was used as an antigen. Standard hybridoma technique was used for production of mAbs. Turbidity analysis and transmission electron microscopy were used to study the effect of mAbs and their Fab-fragment on fibrin polymerization. The localization of epitope for mAb in fibrin molecule was determined using ELISA and immunoblot analysis with fibrinogen, fibrin desAA, fibrin desAABB and various fibrin(ogen) fragments. RESULTS: A mAb FnI-3C has been obtained that doesn't bind to fibrinogen and reacts with fibrin desAA and fibrin desAABB with K(D) value of 9.7610(-10) M. The epitope for this mAb proved to be localized in the fibrin fragment Bbeta118-134. MAb FnI-3C and its Fab retarded specifically the stage of fibrin protofibril lateral association. CONCLUSIONS: A fibrin-specific mAb FnI-3C has been obtained to fibrin fragment Bbeta118-134 which may be a contact site taking a part in protofibril lateral association. MAb FnI-3C was used as a "catch"-one in double-sandwich ELISA for soluble fibrin quantification in human blood plasma.

Functional role of Bbeta-chain N-terminal fragment in the fibrin polymerization process.

Four mAbs of the IgG(1) class to the thrombin-treated N-terminal disulfide knot of fibrin, secreted by various hybridomas, have been selected. Epitopes for two mAbs, I-3C and III-10d, were situated in human fibrin fragment Bbeta15-26, and those for two other mAbs, I-5G and I-3B, were in fragment Bbeta26-36. Three of these mAbs, I-5G, I-3B and III-10D, as well as their Fab-fragments, decreased the maximum rate of fibrin desAA and desAABB polymerization up to 90-95% at a molar ratio of mAb (or Fab-fragment) to fibrin of 1 or 2. The fourth mAb, I-3C, did not influence the fibrin desAABB polymerization and inhibited by 50% the maximum rate of fibrin desAA polymerization. These results suggest that these mAb inhibitors block a longitudinal fibrin polymerization site. As the mAbs retard both fibrin desAABB and fibrin desAA polymerization, one can conclude that the polymerization site does not coincide with polymerization site 'B' (Bbeta15-17). To verify this suggestion, the polymerization inhibitory activity of synthetic peptides BbetaSARGHRPLDKKREEA(12-26), BbetaLDKKREEA(19-26), BbetaAPSLRPAPPPI(26-36), BbetaAPSLRPAPPPISGGGYRARPA(26-46) and BbetaGYRARPA(40-46), which imitate the various sequences in the N-terminal region of the fibrin Bbeta-chain, have been investigated. Peptides Bbeta12-26 and Bbeta26-46, but not Bbeta40-46, Bbeta19-26, and Bbeta26-36, proved to be specific inhibitors of fibrin polymerization. The IC(50) values for Bbeta12-26 and Bbeta26-46 were 2.03 x 10(-4) and 2.19 x 10(-4) m, respectively. Turbidity and electron microscopy data showed that peptides Bbeta12-26 and Bbeta26-46 inhibited the fibrin protofibril formation stage of fibrin polymerization. The conclusion was drawn that fibrin fragment Bbeta12-46 took part in fibrin protofibril formation simultaneously with site 'A' (Aalpha17-19) prior to removal of fibrinopeptide B. A model of the intermolecular connection between fragment Bbeta12-46 of one fibrin desAA molecule and the D-domain of another has been constructed.

[Soluble fibrin and D-dimer at normal pregnancy and pregnancy with risk of miscarriage].

ELISA for soluble fibrin (SF) quantification has been elaborated on the basis of our fibrin-specific monoclonal antibodies (mAb). Epitope for these mAb is localized in fibrin fragment Bbeta118-134. The method was used on the blood plasma of healthy pregnant women (control group) and pregnant women with the risk of fetal loss (RFL). The increased mean values of SF concentrations were observed at pregnancy with RFL as compared to the normal pregnancy at the terms from 4 to 24 weeks (17.87 +/- 3.15 mkg/ml and 9.03 +/- 1.58 mkg/ml accordingly, p < 0.05). A weak negative correlation between SF concentration and pregnancy term was found at RFL (r = -0.201, n=35), while there was no correlation between these variables in control group (r = 0.004, n=28). The mean values of SF concentration estimated by semiquantitative test (by phosphates salting out of SF) were also higher at the pregnancy with RFL as compared to the normal pregnancy. However, the absolute values of SF concentrations determined by salting out method were essentially higher than in the case of ELISA. Immunoblot analysis with mAb 2d-2a (epitope for which in fibrin molecule encompasses peptide bond Bbeta14-15), showed that the main molecular component of SF at normal pregnancy and RFL was oligomeric fibrin desAA with possible incorporation of fibrinogen and/or fibrin desA which was not stabilized by factor XIIIa. D-dimer concentrations determined in blood plasma samples of pregnant women by ELISA varied in the range of 1-224 ng/ml at the pregnancy period from 4 to 37 weeks. There was positive correlation between D-dimer concentration and pregnancy term both at normal pregnancy and pregnancy with RFL (r = 0.765, n=33 and r = 0.712, n=44 correspondingly). The mean values of D-dimer concentration at various terms of normal pregnancy and pregnancy with RFL did not vary considerably. Thus SF but not D-dimer quantification may give useful diagnostic information at the pregnancy with RFL.

Two monoclonal antibodies to D-dimer-specific inhibitors of fibrin polymerization.

D-dimer of human fibrin was used as antigen to obtain monoclonal antibodies (mAbs). We have obtained 16 hybridomas producing mAbs of different specificity. Only two of these mAbs inhibited fibrin polymerization. They are of the IgG-class. One mAb (II-4d) inhibited fibrin polymerization to 100% and another (II-3b) to 60% at a molar ratio mAb/fibrin=1.0. Fab-fragments of these mAbs inhibited fibrin polymerization completely at the same molar ratio. The epitopes for the mAbs studied are situated in the NH2-terminal part of the gamma-chain in fibrin D-domain. Electron microscopy showed that fibrin was in monomeric form in the presence of these mAbs or their Fab-fragments. Thus, these mAbs stop the initial step of fibrin polymerization, i.e. protofibril formation. Only one site of protofibril formation is known now in COOH-terminal half of the D-domain gamma chain named "a" site, which is complementary to the "A" site in the central E-domain of fibrin molecule. Our experiment with immobilized GPRP showed that the "a" site in fibrin D-fragment preserved its binding activity to GPRP when the D-fragment was complexed with mAbs-inhibitors of fibrin polymerization. Thus, these two mAbs inhibit fibrin polymerization not by blocking the sites "a" but either by blocking another (not "a") specific site in D-domain or by steric hindrance of highly organized fibrin polymerization process.

[Quantification of D-dimer and soluble fibrin in blood plasma of people with ischemic heart disease and hypertension]. / Kolichestvennoe opredelenie D-dimera i rastvorimogo fibrina v plazme krovi cheloveka pri ishemicheskoi bolezni serdtsa i gipertonicheskoi bolezni.

The method of D-dimer quantification in the human blood plasma has been developed using monoclonal antibodies 111-3b and II-4d. The method has been verified on the blood plasma of the patients with ischemic heart disease with and without stenocardia and with hypertension. The results showed that at ischemic heart disease with and without stenocardia and at hypertension the quantities of D-dimer in the blood plasma were generally less than the highest normal level 500 ng/ml (64.3%, 76.2% and 95%, correspondingly). The semiquantitative measurements of soluble fibrin levels in blood plasmas of the patients with ischemic heart disease and hypertension have been performed. It has been shown that the quantity of soluble fibrin at these diseases range greatly from < 0.03 mg/ml to 0.15 mg/ml. There was no correlation between the quantities of D-dimer and soluble fibrin in blood plasmas of the patients. Electrophoresis in PAAG with SDS showed that the soluble fibrin at these diseases had the mo- lecular mass of the fibrin (ogen). Thus the soluble fibrin in blood plasmas analysed consisted mainly of fibrin desAA oligomers (may be with fibrinogen incorporation) which are not stabilized by the factor XIIIa.

A neoantigenic determinant in the D-dimer fragment of fibrin.

Monoclonal antibody (mAb) III-3b binds D-dimer with K(d)=1.4 x 10(-10) M without cross-reaction with fibrin(ogen). The epitope for this mAb is in Bbeta134-190, presumably in Bbeta155-160. The latter site is buried in the coiled coil structure of fibrin(ogen) but it is exposed as a neoantigenic determinant in D-dimer upon plasmic lysis of fibrin. mAb III-3b may be used as a tool for immunodiagnostic quantification of D-dimer in blood plasma.

Polymerization sites in the D-domain of human fibrin(ogen).

The present work deals with localization of previously unknown polymerization sites of the fibrin DD-fragment. D-dimer we obtained has a pronounced inhibitory effect on fibrin polymerization (IC50 = 0.06 microM). The inhibitory effect of the DD-fragment disappeared after reduction and carboxymethylation. However, polypeptide chains betaDD (Bbeta134-461) and gammagammaDD (gamma63-411)2 of the DD-fragment, isolated by preparative electrophoresis, displayed their inhibitory activity. For instance, the rates of fibrin protofibril lateral association were decreased twice in the presence of betaDD and gammagammaDD chains at their molar ratios to fibrin of 0.40 and 0.15, respectively. The IC50 values for betaDD and gammagammaDD were 0.24 and 0.10 microM, respectively. Highly specific inhibition of protofibril lateral association suggests that the protofibril lateral association sites are located in Bbeta134-461 and gamma63-411 regions of the fibrin D-domain. Our data confirm those reported by Doolittle et al. regarding the gamma-chain and a hypothesis about beta-chain of fibrin D-domain (Yang, Z., Mochalkin, I., and Doolittle, R. F. (2000) Biochemistry, 97, 14156-14161).

[The role of calcium ions in fibrin polymerization]. / Rol' ionov Ca v polimerizatsii fibrina.

The article reviews the literature and authors' own data on the role of Ca ions in blood coagulation, namely, in the process of the formation of highly ordered fibrin clot. It has been shown that the main role of Ca2+ is the timely formation and stabilization of fibrin polymerization sites at all successive stages of the fibrin polymerization process.