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Biomedical subjects

E M Makogonenko

Publications and source records attributed to E M Makogonenko.

At least 19 recordsLinked to original sources
PubMed1997

Studies of the t-PA-catalyzed Glu-plasminogen activation on different models of polymeric fibrin.

The rate of activation of Glu-plasminogen by t-PA on fibrin clot and on the complexes of fibrinogen, its fragment X2, fibrin fragment D-D with N-DSK of fibrin has been studied. The reaction rate constant of (kcat/Km) increased and Km decreased in series of effectors X2-->X2 - N-DSK-->D-D-->D-D - N-DSK = fibrinogen - N-DSK-->fibrin clots. The structure of activator complexes of reaction of Glu-plasminogen activation by t-PA on fibrin clot has been suggested.

Fibrin↗
PubMed1996

Thermal stability of individual domains in platelet glycoprotein IIbIIIa.

Thermal denaturation of platelet glycoprotein IIbIIIa (integrin alpha IIb beta 3) was investigated by spectrofluorimetry and differential scanning calorimetry (DSC). Two forms of the protein were compared: active IIbIIIa, i.e., that fraction that binds to RGD-Sepharose, and inactive IIbIIIa, the non-binding fraction. At pH 8.5 in the presence of octyl glucoside and Ca2+ both forms exhibited a broad complex endotherm consisting of a well expressed low-temperature heat-absorption peak in the range of 40-65 degrees C followed by a broad peak stretching over 65-110 degrees C. Each endotherm could be deconvoluted into at least eight transitions reflecting the melting of at least this many independently folded domains. The first two transitions in the region of the low-temperature peak had similar positions in both forms while at least some of the other transitions occurred at higher temperature in the active protein suggesting that some of the domains are more stable in the latter. When both fractions of IIbIIIa were heated in the fluorometer a sigmoidal transition was observed in the region of the first endothermic peak where the two thermolabile domains melt. This transition was destabilized by 15 degrees C in the presence of EDTA, suggesting that these domains are formed by the 243-468 region of the IIb subunit which contains four Ca(2+)-binding motifs. It was further stabilized by 3 degrees C upon addition of the GRGDSPK peptide in the presence of Ca2+ while in EDTA the peptide had no effect. This is consistent with the involvement of Ca(2+)-binding region in the formation of the ligand-binding site. A 66-kDa chymotryptic fragment, containing the 17-kDa NH2-terminal portion of the IIIa subunit disulfide-linked to its 50-kDa COOH-terminal portion including the cysteine-rich core, exhibited a fluorescence-detected Ca(2+)-independent transition in the region where the higher temperature DSC-detected transitions occur suggesting that some of the latter may be connected with the melting of the corresponding portions of IIbIIIa.

Amino Acid Sequence↗
PubMed1991

The study of fibrin polymerization with monoclonal antibodies.

Three kinds of monoclonal antibody (Mab) of different specificity have been obtained against the N-terminal disulphide knots of fibrinogen and fibrin. Their effects on different phases of fibrin polymerization have been studied. These antibodies were shown to be directed against different epitopes of the B beta(1-53) fragment of the fibrinogen molecule. The different Mab had different effects both on the rate of protofibril lateral aggregation and on the final turbidity of fibrin clots. The Mab were of three specificities: (1) those from clone 2d-2a inhibited the rate of lateral aggregation of protofibrils and decreased the turbidity of the final clot; (2) those from clone B-4C accelerated the polymerization step but did not affect clot turbidity: and (3) those from clone D-IB did not have any effect on either fibrin polymerization or final clot turbidity. The localization of the epitopes recognized by all three kinds of Mab and analysis of our own data and those of others allow us to conclude that one of the active loci involved in protofibril lateral association is situated in the B beta(15-53) fragment of the fibrinogen molecule. Fibrinopeptide B does not need to be split off for this site to function. Fibrin polymerization can occur when one of the two sites of protofibril lateral aggregation in dimeric fibrin molecules is blocked by Mab, and the final clot turbidity is then reduced. The splitting off of one of the two fibrinopeptides B in fibrinogen molecules by thrombin can take place even when the second B beta(Arg14-Gly15) bond is blocked by an antibody molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

[The effect of heparin on the proteolytic and fibrinolytic activity on plasmin(ogen) and fibrin clot lysis].

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.

Animals↗
PubMed1988

[Plasminogen activators in malignant tissues and lungs of mice during metastatic spreading and administration of various pharmacological agents].

The method of fibrin plates and electrophoresis in PAAG was used to study the activity and forms of plasminogen activators (PA) in extracts of normal tissues (muscle, lungs) as well as of the malignant tumours and the lungs of mice during metastatic spreading and under certain drugs (trielin and cyclophosphane). It was stated that determination of the specific activity level of plasminogen activator in a target organ (lungs) may be used as one of the indices during evaluation of the antimetastatic effect.

Animals↗
PubMed1987

[Kinetics of glu- and lys-plasminogen activation by the tissue activator in a fibrin clot].

Using a modified procedure for measuring the time of fibrin clot lysis, the kinetics of Glu- and Lys-plasminogen activation by the tissue activator was studied. Within the plasminogen concentration range of 0.4-100 nM the rate of activation of both protein forms obeys the Michaelis-Menten kinetics. At Lys-plasminogen concentration equimolar to that of fibrin, the rate of activation of the former decreases down to that of Glu-plasminogen activation. The kinetic constants for Glu- and Lys-plasminogen activation (Km) are equal to 0.055 and 0.013 microM; k = 0.19 and 0.21 s-1, respectively. The Km values for fibrin-bound Glu- and Lys-plasminogen are equal to 0.25 nM and 8 nM, respectively (k = 0.08 and 0.26 s-1, respectively). It is assumed that the tissue activator exhibits a higher affinity for the Glu-plasminogen--fibrin complex than for the Lys-plasminogen-fibrin complex.

Fibrin↗
PubMed1983

[Comparative evaluation of fibrinogen and fibrin hydrolysis with plasmin].

A high-sensitive method is developed for determining the degree of plasmin-catalyzed fibrinogen hydrolysis by the released amino groups stained with trinitrobenzene sulphoacid. The method permits determining 0.02-0.08 casein units of plasmin. The method made it possible to establish that after streptokinase activation plasmin hydrolyzes equally fibrinogen and fibrin in solution and as gel. When a tissue activator is used, fibrin intensifies significantly the plasminogen activation. Inhibition of plasmin by an inhibitor produced from soya is considerably slowed down in fibrin gel.

Animals↗
PubMedDate not supplied

[Features of the structure of fibrin clots, connected with conditions of gel formation].

Fibrinogen to fibrin conversion and then fibrin clot three-dimensional network formation is one of the final steps in the coagulation system activation. Different factors, such as the environment temperature and pH, ions, so on, render an effect on the fibrin gel formation. Recently, the presence or absences of interface between two phases influence on the fibrin gel structure during its formation have been shown. Studies of fibrin gel structure peculiarities formed at different conditions (between two phases and without one phase) are demonstrated in this article. The plasmin enzymatic hydrolysis of fibrin clots both with surface film and without it was investigated. Experimental data allow to make a conclusion that the fibrin clot structure changes depend on its essential influence on the plasmin hydrolysis process of these clots.

Electrophoresis, Polyacrylamide Gel↗
PubMedDate not supplied

[Solubility of the Mg2+-dependant Ca2+-activated ATPase fraction of the plasma membranes of synaptosomes].

Mg2+-dependent Ca2+-activated ATPase (Mg2+,Ca2+-atpase, EC 3.61.4) in the fraction of synaptosome plasmatic membranes is activated with 0.05-0.08% solutions of digitonin and sodium deoxycholate. At higher concentrations of digitonin the activating effect lowers, sodium deoxycholate in the increasing concentrations inactivates the enzyme. 0.08% digitonin activates Mg2+,Ca2+-ATPase of synaptosome membranes, without demanding for its transition into solubilized state. Separation of non-active 0.08% digitonin extract from the deposit results in a decrease in the enzymatic activity of the latter and addition of the extract to the deposit activates the enzyme. At least two components separable from each other are likely to be necessary for the enzyme activity manifestation. A solubilized preparation of Mg2+,Ca2+-ATPase with the maximum activity and the ratio of total ATPase to Mg2+-ATPase equal to 3.5-4.0 may be obtained by extraction with 0.15-0.20% digitonin solution. Maximum quantily of protein is extracted by means of digitonin in the same concentrations. The extracted protein is divided in 7.5% polyacrylamide gel into eight-ten electrophoretic zones.

Adenosine Triphosphatases↗
PubMedDate not supplied

[Role of the K4 and K5 plasmin heavy chain kringles in the fibrin clot structure destruction].

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.

Aminocaproic Acid↗
PubMedDate not supplied

[Hydrolysis of polymeric fibrin by plasmin, miniplasmin, microplasmin and trypsin].

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.

Biopolymers↗
PubMedDate not supplied

[Functional and diagnostic significance of A-, Bbeta 1-42 and Bbeta 15-42 peptide fragments of fibrin(ogen)].

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.

Blood Coagulation↗
PubMedDate not supplied

[Role of lysine binding sites in activation of plasminogen by streptokinase].

The function of lysine-binding sites in kringle domains K1-4 and K5 of plasminogen (Pg) during its activation by streptokinase (SK) was studied. Activation rates of Glu- and Lys-Pg exceed activation rate of mini- and micro-Pg 26 and 40 times, respectively. 6-Animohexanoic acid (6-AHA) in concentrations from 10(-5) to 10(-2) M inhibits activation of Glu-, Lys- and mini-Pg and does not impact the activation of micro-Pg. Complete inhibition of Lys-Pg activation occurs with presence of 10(-3) M 6-AHA while 90% inhibition of mini-Pg activation and 70% inhibition of Glu-Pg activation occur with 10(-2) M 6-AHA. Isolated kringles K1-3 and K4 of Pg inhibit activation of Glu-Pg by SK and concentrations [I]50 are 4.0 and 8.1 x 10(-6) M, respectively. Catalytic activity of Glu-Pg-SK, Lys-Pg-SK and Pm-SK complexes with respect to S 2251 is not inhibited by 6-AHA in concentrations from 10(-5) to 10(-2) M. Activation of substrate Pg by Pm-SK complex is also inhibited by 6-AHA in concentrations from 10(-5) to 10(-2) M; however, this effect of inhibition is significantly weaker than that with activation by SK. Cleavage of C-terminal Lys or chemical modification of NH2-groups of amino acid residues in SK molecule also results in the decrease of the Glu-Pg activation rate. Lysin-binding sites in K1-4 and K5 of Pg molecule are important at different steps of Pg activation process which includes formation of equimolar complex; structural reorganizations resulted in formation of active center in Pg; and binding of substrate Pg with Pg-SK complex. Lysin-binding sites in K1-4 of Pg are necessary for maintenance of high rate of Pg activation by SK.

Aminocaproic Acid↗
PubMedDate not supplied

[Modifying effect of antiplasminogen monoclonal antibody IV-1c on human plasmin catalytic properties].

Antiplasminogen monoclonal antibody IV-1c (IV-1c) binds to Val 709-Gly 718 site of plasminogen (Pg) protease domain, which is far removed from the active site. Pg-IV-1c complex formation induces catalytic activity in proenzymes active site. Influence of IV-1c binding to plasmin (Pm) on Pm catalytic properties has not been investigated yet. Data on catalytic properties of Pm in equimolar Pm-IV-1c complex are presented. It was found that Pm and mini-Pm amidolytic and caseinolytic activity was twice as high as in Pm-IV-1c and mini-Pm-IV-1c complexes. 20 mM 6-AHA and 100 mM arginine did not influence this rise. The increase of amidolytic activity is connected with reduction of K(m) of S 2251 hydrolysis reaction for Pm and mini-Pm from 0.125 and 0.43 to 0.05 and 0.23 mM, correspondingly. Kcat remains almost the same. Fibrinolytic and fibrinogenolytic activity of Pm in Pm-IV-1c complex decreased to 20% of initial value alpha 2-Antiplasmin inhibited Pm activity in complex Pm-IV-1c by 80%. Pm-IV-1c complex did not activate free Pg, but activated equimolar Pg-IV-1c complex. Affinity of IV-1c to Pm and Pg was the same as C50 approximately 1.5 nM. Binding of Pm with IV-1c in a complex: a) leads to increase of Pm active site affinity to LMW substrates; b) causes steric hindrances for fibrin/fibrinogen access to Pm active site; c) proceeds with the same affinity for Pm and Pg, that indicates to invariable Val 709-Gly 718 site conformation after Pg transition in Pm.

Aminocaproic Acid↗
PubMedDate not supplied

[Plasminogen activation by a tissue activator and effector properties of fibrinogen-N-terminal disulfide (N-DSK) fibrin complex].

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.

Disulfides↗