[Role of carboxypeptidase in regulation of fibrinolysis].

The paper is devoted to analysis of the literature data about a recently described component of haemostasis system that posseses carboxypeptidase activity and is activated by thrombin. The history of investigation and isolation of the new carboxypeptidase, their properties and participation in inhibition of fibrinolysis reactions was considered. Data are cited about carboxypeptidase B level changes under different physiological conditions and possible methods of fibrinolysis reactions strengthening by means of influence on activity of that carboxypeptidase.

[Methods and prospects of development of the affinitive sorbents for plasminogen isolation from human blood plasma]. / Puti i perspektivy sozdaniia affinnykh sorbentov dlia vydeleniia plazminogena iz plazmy krovi cheloveka.

The review paper was dedicated to development of the promising photochemical synthesis of affine sorbents for plasminogen isolation from the human blood plasma. Some most interesting, from the viewpoint of practice, types of sorbents and carriers based on high-molecular compounds of natural (organic) or synthetic origin have been considered. The advantages of the use of photochemical synthesis of biospheric sorbent as compared with thermochemical method have been shown. The most promising methods of creation of affine sorbents on the basis of oligomer-polymeric photoinitiators and oligomerpolymeric photosensibilizing (donor-acceptor) systems are presented.

[Photochemical synthesis of a bioaffinity sorbent for plasminogen extraction]. / Fotokhimicheskii sintez bioaffinnogo sorbenta dlia izvlecheniia plazminogena.

Conditions for affine sorbents creation by means of linear photocopolymer photochemical synthesis, its thermochemical granulation-crosslinking, and grafting by L-lysine hydrochloride as an sorbent affinant (ligand) were investigated. Linear photocopolymer properties depending on concentration ratio of photopolymerisation components, chemical nature of reaction media (solvent), and UV irradiation time were studied. Dioxane is shown to be the optimal solvent. Dependence of molecular mass on UV irradiation time (energy) is extremal with maximum at 5 hours and Mm = 243,300. Biospecific properties of several created affine sorbents were investigated.

[Effect of chemical modification of arginine residues of fibrinogen and its DH-fragment on the rate of hydrolysis of these proteins by plasminogen]. / Vliianie khimicheskoi modifikatsii ostatkov arginina fibrinogena i ego DH-fragmenta na skorost' gidroliza étikh belkov plazminom.

Plasminolysis of the fibrinogen arginine and its DH-fragment residues was sufficiently lower in contrast to that of initial proteins. It is supposed that the decrease of the speed of the process is the result of the blocking of centres, adequate to arginyl-binding sites of plasmin molecule.

[Rapid turbidimetric micromethod for the simultaneous estimation of parameters of coagulation and fibrinolysis in blood plasma]. / Turbidimetric heskii ékspress-mikrometod odnovremennoi otsenki parametrov svertyvaniia i fibrinoliza v plazme krovi.

A method has been developed to determine six parameters of coagulation and fibrinolysis by means of turbidimetry; 0.1 ml of dissolved plasma after its activation with thrombin and streptokinase is used. Testing conditions which permit performing express diagnostics of haemostasis disturbances and controlling their correction have been optimized.

[Functional and diagnostic significance of A-, Bbeta 1-42 and Bbeta 15-42 peptide fragments of fibrin(ogen)]. / O funktsional'nom i diagnosticheskom znechenii A-, Bbeta 1-42 i Bbeta 15-42 peptidnykh fragmentov fibrin(ogen)a.

A scheme of in vitro formation and hydrolysis of the fibrin clot is suggested. This scheme considers the data concerning a cofactor role of fibrin in activation of polymerization and fibrinolysis. Such parameters of a number of components as concentration of A-, B beta 1-42-and B beta 15-42 peptides and of other fragments of fibrinogen which allow characterizing a state of hyperfibrinolysis, hyperclotting or dynamic equilibrium of these systems are selected in the scheme.

[Hydrolysis of polymeric fibrin by plasmin, miniplasmin, microplasmin and trypsin]. / Osobennosti gidroliza polimernogo fibrina plazminom, miniplazminom, mikroplazminom i tripsinom.

125I-labeled polymeric fibrin hydrolyzed with plasmin, Val442-plasmin (miniplasmin, Lys530-plasmin (microplasmin) and trypsin has been studied for radioactivity of its separate electrophoretic bands. The reaction of hydrolysis was stopped at a moment of a two-fold decrease of the fibrin clot turbidity (t1/2) at the wave length 350 nm. For plasmin, miniplasmin, microplasmin and trypsin taken in the same caseinolytic activities t1/2 was 12.4, 40.0 164.1 and 76.8 min, respectively. Differences in composition of fibrin digests taken at t1/2, are demonstrated: the content of high-molecular components of digests decreases in the order of plasmin greater than miniplasmin greater than microplasmin greater than trypsin, thus showing differences in the processes of fibrin clot structure disruption by the enzymes.

[Features of the interaction of Glu- and Lys-forms of plasminogen with native and partially hydrolyzed fibrin]. / Osobennosti vzaimodeistviia Glu- i Lys-form plazminogena s nativnym i chastichno gidrolizovannym fibrinom.

Glu- and Lys-plasminogen interaction with native and desAABB-fibrin obtained from fibrinogen partially hydrolyzed by plasmin was studied. It was found that native fibrin adsorbs 6 times more Lys-plasminogen as compared to the native form of the proenzyme. The range of the Lys-plasminogen binding does not change, if part of the fibrinogen molecules hydrolyze down to X-fragments. At the same time, the appearance in the system of 1% Xi-fragments leads to a 6-fold increase in the Glu-plasminogen binding. The amount of adsorbed Glu-plasminogen reaches the level of Lys-plasminogen adsorption both in the native and partially hydrolyzed fibrin. It was found that kringle K 1-3 or 6-aminohexanoic acid at saturating for high-affinity lysine-binding sites concentrations do not influence the Glu-plasminogen binding to native fibrin but inhibit it when the partially purified form is used. It is assumed that the manyfold increase of the Glu-plasminogen binding to partially hydrolyzed fibrin is due to the alteration of the proenzyme conformation at the initial steps of fibrin hydrolysis during the formation of Xi fragments.

[Role of the K4 and K5 plasmin heavy chain kringles in the fibrin clot structure destruction]. / Rol' kringlov K4 i K5 tiazheloi tsepi plazmina v razrushenii struktury fibrinovogo sgustka.

A comparative analysis of the rates of polymeric fibrin structure destruction by plasmin (Pm) and its proteolytic derivatives such as Val354-plasmin (c-Pm), Val442-plasmin (m-Pm) and Lys530-plasmin (mu-Pm) has been undertaken. It was shown, that Pm, c-Pm, m-Pm and mu-Pm at equal proteolytic activity, have dissolved fibrin clots with relative rates 40.3:38.0:4.6:1.0 correspondingly. The Pm, m-Pm and mu-Pm relative rates were changed by epsilon-aminocaproic acid to 4.6:1.5:1.0 correspondingly. In this case fibrin clot destruction time was increased for Pm and m-Pm and was not changed for mu-Pm. The rates of fibrinogen hydrolysis were nearly equal for these forms of enzyme. It was suggested, that the specific interactions between plasmin K4 and K5 kringles and solid phase fibrin substrate determine the polymer fibrin structure destruction rate.

[The effect of arginine on hydrolysis of fibrinogen by plasmin and mini-plasmin]. / Vliianie arginina na gidroliz fibrinogena plazminom i mini-plazminom.

The rate of plasmin or Val442-plasmin catalyzed hydrolysis of fibrinogen decreases several times as affected by arginine in high concentrations. The enzyme is shown to be not inhibited by arginine. The observed effect is supposed to depend on saturation of the protein-proteins interaction sites located between 442 and 790 amino acid residues.

[Plasminogen activation by a tissue activator and effector properties of fibrinogen-N-terminal disulfide (N-DSK) fibrin complex]. / Aktivatsiia plazminogena tkanevym aktivatorom i éffektornye svoistva kompleksa fibrinogen-N-kontsevoi disul'fidnyi uzel (N-DSU) fibrina.

Fibrinogen-NDSK complex is a model of protofibril having some features of the fibrin polymer structure. This complex has been studied for its ability to stimulate the plasminogen activation by t-PA. The fibrinogen-NDSK complex have increased the rate of plasminogen activation by t-PA as compared to fibrinogen or NDSK taken separately. This acceleration had slow and fast phases. Lys-plasminogen was activated more effectively as compared to glu-plasminogen. The kinetic parameters of glu- and lys-plasminogen activation at fast phase were: Km--0.18 and 0.015 mu/M, Kkat--0.27 and 0.06 s-1, respectively. Fibrinogen X2--fragments, deprived of alpha C-domains and NH2-end peptides of bB-chains, formed complexes with NDSK, which however did not stimulate the plasminogen activation by t-PA. These findings have shown that the fibrinogen-NDSK complex is an effective stimulator of the plasminogen activation by t-PA. The activating ability of the complex may be due to structures formed in the course of fibrinogen and NDSK polymerization as a result of alpha C-domain interaction.

[Proteolytic activity of the Glu-plasminogen complex with fibrinogen fragment E]. / Issledovanie proteolitcheskoi aktivnosti kompleksa Glu-plasminogena s fragmentom E fibrinogena.

Glu-plasminogen interaction with fibrinogen fragment E results in the alteration of its adsorptive capacity. During this interaction in the absence of plasmin and tissue activator of plasminogen, Glu-plasminogen is transformed into a partly degraded form. Glu-plasminogen complexes with soluble and immobilized fibrinogen fragment E. contain a serine proteinase-specific activity which is inhibited by diisopropylfluorophosphate. The complexes under study are active towards fibrin and the plasmin-specific tripeptide substrate, D-Val-L-Leu-L-Lys-p-nitroanilide. It is concluded that fibrinogen fragment E induces structural changes in the enzyme molecule which eventually result in the formation of an active center.

Fluorescence spectroscopic analysis of ligand binding to kringle 1 + 2 + 3 and kringle 1 fragments from human plasminogen.

The ligand binding of kringle 1 + 2 + 3 and kringle 1 from human plasminogen has been investigated by fluorescence spectroscopy. Analysis of fluorescence titration of kringle 1 + 2 + 3 with 6-aminohexanoic acid shows that this fragment, besides the high-affinity lysine-binding site with Kd = 2.9 microM, contains two additional lysine-binding sites which differ in binding strength (Kd = 28 microM and Kd = 220 microM). This strongly suggests the existence of a lysine-binding site in each of the first three kringles. 6-Aminohexanoic acid, pentylamine, pentanoic acid and arginine were used for investigation of the ligand specificity of isolated kringle 1 prepared by pepsin hydrolysis of kringle 1 + 2 + 3. It has been established that kringle 1 has high affinity to 6-aminohexanoicacid, pentylamine and arginine (Kd values are 3.2 microM, 4.8 microM and 4.3 microM, respectively). At the same time pentanoic acid did not bind with kringle 1. These facts indicate, firstly, a broad ligand specificity of kringle 1 and, secondly, the paramount importance of the positively charged group of the ligand for its interaction with lysine-binding site of this kringle.

[Two classes of lysine-binding sites of plasminogen molecule]. / Dva klassa lizinsviazyvaiushchikh uchastkov molekuly plazminogena.

Affinity of plasminogen fragments K1, K2-3, K4 and K5 for 6-aminophenyl-Sepharose was investigated to characterize the lysine-binding sites of the protein. K1 and K5 fragments were bound to the affinity column, whereas kringle 2-3 and kringle 4 were not. The results obtained and data known from literature have indicate that two types of lysine-binding sites are present in the plasminogen molecule. Both positively and negatively charged groups of the ligand are necessary for binding with the first-type sites (K4 and K2-3). The interaction between ligands and the second-type sites localized in kringles 1 and 5 is provided by their positively charged group only.

[The effect of heparin on the proteolytic and fibrinolytic activity on plasmin(ogen) and fibrin clot lysis]. / Vliianie geparina na proteoliticheskuiu i fibrinoliticheskuiu aktivnost' plazmin(ogen)a i na razrushenie fibrinovogo sgustka.

The effect of heparin on the proteolytic and fibrinolytic activities of plasmin and plasminogen was studied. Heparin at a concentration of 6.3.10(-6) M did not change the caseinolytic activity of plasmin and plasminogen stimulated by streptokinase but suppressed their fibrinolytic activity. At concentrations from 2.10(-8) to 0.5.10(-6) M heparin increased, whereas at 1.10(-6)-4.10(-6) M reduced the time of desAAfibrin clot half-lysis by plasmin. Within the concentration range of 2.10(-8) to 4.10(-6) M heparin did not change the time of the clot half-lysis by glu-plasminogen and slightly decreased the time of fibrin clot half-lysis by lys-plasminogen in the presence of the tissue activator. It was supposed that heparin inhibits the fibrinolytic effect of plasmin by way of formation of complexes with plasmin and reduction of plasmin specificity to the solid phase substrate, i. e., polymeric fibrin.

[Preparation and properties of trypsin from the pyloric caeca of Pacific Ocean salmon]. / Poluchenie i svoistva tripsina iz pilorichekikh pridatkov tikhookeanskikh lososei.

Trypsin from pyloric caeca of Pacific salmon was purified by affinity chromatography of the water extract on hexamethylenediamine-glycidylmethacrylate-cellulose. A protein band with a molecular weight of 22.5 kDa was found on SDS-electrophoresis in PAG. The protein band was homogeneous according to isoelectrofocusing in PAG (pI 4.0). The amino acid composition of the enzyme is typical of trypsin anionic forms; the major difference from the cationic forms is the lower content of lysine. The differences in properties caused by change of the enzyme molecule charge are similar to those observed in cationic trypsin when the lysine epsilon-amino groups of the latter are modified (change of pI, shift of the pH-optimum towards basic values, increase of stability to autolysis). Some natural trypsin inhibitors of the different origin suppressed the enzyme activity of trypsin from Pacific salmon in typical stoichiometric ratios. An unusual interaction of the enzyme with the specific inhibitor N-L-tosyl-L-lysine chloromethyl ketone was observed.

[Binding of Glu-plasminogen by fibrinogen and byproducts of its proteolysis]. / Sviazyvanie Glu-plazminogena s fibrinogenom i produktami ego proteoliza.

The ability of the native form of plasminogen (Glu-plasminogen) to form complexes with fibrinogen and its fragments immobilized on CNBr-agarose was studied. It was found that unlike Lys-plasminogen, the native form of the proenzyme does not bind to fibrinogen agarose. Limited proteolysis of fibrinogen by plasmin involving alpha C-domains results in the appearance of Glu-plasminogen binding sites at fibrinogen surface. The X2 fragment of fibrinogen binds to about 0.5 moles of Glu-plasminogen at an equimolar ratio of the interacting proteins. Under these conditions, the amount of bound Glu-plasminogen does not increase as a result of subsequent hydrolysis of fibrinogen down to end products, fragments E and D. It was found that Glu-plasminogen interacts with both E- and D-fragments of fibrinogen. Similar to Lys-plasminogen, Glu-plasminogen exhibits a high affinity for the E-fragment. The maximal quantity of the bound protein under the given experimental conditions is 2 moles per mole of the immobilized E-fragment. The interaction of Glu-plasminogen with the E-fragment is mediated by the lysine-binding sites of the proenzyme with a high and low affinity [Kd = 1.8.10(-6) and 7.5.10(-5) M, respectively]. Glu-plasminogen, unlike Lys-plasminogen, shows a low affinity for the D-fragment (Kd = 2.10(-5) M). Glu-plasminogen cannot be adsorbed by arginine-binding sites at the DH fragment-agarose.

Analysis of ligand binding to kringles 4 and 5 fragments from human plasminogen.

The interaction of the isolated kringles 4 and 5 from human plasminogen with 6-aminohexanoic acid, pentylamine, pentanoic acid and arginine has been quantitatively characterized by scanning calorimetry and fluorescent spectroscopy. It has been found that the ligands with the positively charged group have a good binding ability while pentanoic acid in comparison with 6-aminohexanoic acid being devoid of amino group does not interact with the kringles under study. The positively charged group of the ligand is suggested to play a crucial role in ligand binding with the lysine-binding site.

[Binding of lys-plasminogen to E-fragment of fibrinogen]. / Sviazyvanie liz-plazminogena s E-fragmentom fibrinogena.

The interaction of Lys-plasminogen and its fragments with fibrinogen fragment E was studied by equilibrium affinity binding. A quantitative analysis of binding parameters revealed two types of binding sites responsible for Lys-plasminogen interaction with the immobilized fragment E, i.e., with a high (Kd = 1.5 x 10(-6) M) and low (Kd = 82 x 10(-6) M) affinity ones. Among plasminogen fragments, only miniplasminogen and KI-3 bound immobilized fragment E and were eluted by epsilon-aminocaproic acid. Hence, two lysine binding sites may be involved in the binding of Lys-plasminogen to fragment E; they are localized in the KI-3 and K5 kringle structures.

[Binding of K 1-3 and K 4 fragments of plasminogen containing lysine-binding sites with fibrinogen and its fragments]. / Sviazyvanie K 1-3- i K 4-fragmentov plazminogena, soderzhashchikh lizinsviazyvaiushchie uchastki, s fibrinogenom i ego fragmentami.

Interaction of plasminogen K 1-3 and K 4 fragments containing lysine binding sites with fibrinogen and its fragments has been investigated. It has been established that K 1-3 fragment binds to fibrinogen and its E and DL fragments. K 4 fragment does not bind to E and DL fragments, but it interacts with fibrinogen. K 4 fragment does not interact with early fibrinogen proteolysis X2 fragment which differs from the native molecule of fibrinogen in the alpha C domain absence. The results obtained indicate that lysine binding sites located at plasminogen K 1-3 and K 4 fragments correspond to different fibrinogen molecule centres. The centre complementary to K 4 fragment lysine binding sites could be located at the fibrinogen alpha C domain.