[Effects of Lys-form of plasminogen on platelet actin cytoskeleton].

The effects of several forms of plasminogen on the state of actin cytoskeleton of human platelets were studied. A ratio between various actin pools, which were detected by immunoblotting, was taken as indicator of platelet cytoskeleton reorganization. It was revealed that intact platelets contain globular (G) actin and membrane cortex (MC) actin in amounts that are 56 and 40% of filamentous (F) actin level, respectively. In both thrombin- and collagen-activated platelets, actin is almost entirely presented in F-form. Incubation of resting platelets with Lys-plasminogen causes elevation of MC-actin level up to 79% in respect to F-form content. In addition, Lys-plasminogen inhibits reorganization of actin cytoskeleton typical for activated platelets. In contrast to Lys-form, Glu-plasminogen affects neither platelet aggregation nor redistribution of actin pools. Thus, these data indicate that cytoskeletal structures of platelets are involved in realization of anti-aggregating effects of Lys-plasminogen.

[Development and optimization of the methods for determining activity of plasminogen activator inhibitor-1 in plasma].

The activity and content of plasminogen activator inhibitor-1 (PAI-1) are important indicators of pathological processes, because its content in plasma increases at acute myocardium infarction, tumor, diabetes mellitus, etc. The present work is dedicated to the development and optimization of the methods of PAI-1 activity definition, which can be used in clinical practice. We have proposed the modification of the method COATEST PAI with the usage of chromogenic substrate S2251. According to our modification, the cyanogen bromide fragments of human fibrinogen have been changed into bovine desAB-fibrin. We have also developed the method with the usage of fibrin films. In this method fibrin is used as a stimulator of activation reaction and as a substrate at the same time. Using fibrin, the native substrate of plasmin, we provide high specificity of the reaction and exclude the cross-reaction with other plasma enzymes.

[Regulation with alpha-2-antiplasmin of Glu-plasminogen activation by tissue activator on fibrin].

Interaction of tissue plasminogen activator with alpha-2-antiplasmin and its influence on tissue activator binding to fibrin was studied. Alpha-2-Antiplasmin decreases the binding of tissue activator to fibrin by 20%. The inhibitor formed a complex with tissue plasminogen activator (Kd 78.2 nM) and had no effect on amidolytic activity of the activator. The tissue activator binding to alpha-2-antiplasmin decreases by 20-35% in the presence of 6-aminohexanoic acid. It indicates that not only kringle 2 of the tissue activator molecule takes part in complex formation with alpha-2-antiplasmin, but also other activator domains. Two models were proposed to explain the alpha-2-antiplasmin effect on the Glu-plasminogen activation by tissue activator on fibrin. In the first place, the inhibitor binds to fibrin in the site where the activator complex is localized. It can create steric hindrances for the proenzyme interaction with its activator on fibrin. In the second place, alpha-2-antiplasmin in a complex with tissue plasminogen activator can bring to a change in the activator conformation and a decrease of its functional activity.

[Inhibition of the process of Glu-plasminogen activation by tissue activator with fibrin, DDE-complex, and D-dimer using alpha-2-antiplasmin].

The alpha-2-antiplasmin influence on the Glu-plasminogen activation by tissue activator both on fibrin and fibrin(ogen) fragments was investigated. The kinetics of activation was studied and velocity of this process in the absence and presence of the inhibitor was calculated. It was established that alpha-2-antiplasmin decreased the velocity of Glu-plasminogen activation on desAABBfibrin, DDE-complex and DD-dimer and did no influence upon proenzyme activation on fibrinogen fragment--Ho1-DSK. In the presence of fibrin plasminogen activation linear related to the amount added tissue activator in limit concentration from 5 before 50 units/ml. It was shown that alpha-2-antiplasmin reduced the activation velocity with used concentration of tissue activator. Fibrin hydrolysis by plasmin, forming on its surface during the plasminogen activation by tissue activator, was also inhibited with alpha-2-antiplasmin. The obtained results are explained by the influence of the inhibitor on formation of the triple complex between plasminogen, tissue activator and fibrin, and competition of the alpha-2-antiplasmin for lysine-binding sites of tissue activator kringle 2 or for binding sites of the activator on fibrin.

[Binding of alpha-2-antiplasmin with fibrinogen/fibrin and their fragments independent of factor XIII]. / Zv'iazuvannnia alfa-2-antyplazminu z fibrynohenom/fibrynom ta ïkhnimy frahmentamy bez uchasti faktora XIII.

Possible interaction of alpha-2-antiplasmin with fibrinogen, fibrin and their fragments independent of factor XIII as well as the inhibitor effect on the Glu-plasminogen activation by tissue activator were studied. It was shown that alpha-2-antiplasmin is adsorbed on desAA- and desAABBfibrin films (Kd 69.0 +/- 1.0 nM 68.6 +/- 5.3 nM, respectively). Glu-Plasminogen has no effect on the inhibitor binding with desAABBfibrin. Alpha-2-antiplasmin shows strong affinity for fibrin D-dimer (Kd 65.0 +/- 4.0 nM) and D-fragment of fibrinogen (Kd 119.0 +/- 21.0 nM), but it does not interact with E-fragment. The inhibitor inside the fibrin clot decreases 10 times the activation rate of Glu-plasminogen by the tissue activator both is the presence and without factor XIII at physiological ratio of Glu-plasminogen, tissue activator, fibrin and alpha-2-antiplasmin. Thus we have shown that fibrinogen/fibrin binds alpha-2-antiplasmin independent of the factor XIII. Binding sites of the inhibitor are localized in D-fragment of fibrinogen and/or fibrin D-dimer. Alpha-2-antiplasmin inhibits the Glu-plasminogen activation by tissue activator on fibrin.

[Degradation of streptokinase and the catalytic properties of the plasmin-streptokinase complex]. / Dehradatsiia streptrokinazy ta katalitichi vlastyvosti plazmin-streptokinaznoho kompleksu.

Streptokinase (SK) interacts with human plasminogen (Pg) or plasmin (Pm) with formation of Pg-SK or Pm-SK complex. Pm-SK complex manifests a fibrinolytic, amidolytic and Pg activator activity. SK in complex with Pm isn't stable and so capable to be hydrolysed rapidly. We investigated a correlation between molecular form of SK and catalytic properties of equimolar Pm-SK complex during preincubation at 20 degrees C. It was found out that amidolytic activity of Pm-SK complex was not changing for 5 hours and decreased to the initial Pm value after 24 hours. During this time alpha 2-antiplasmin (alpha 2-AP) has any effect on amidolytic activity of the complex. Fibrinolytic activity of Pm-SK complex makes up 20% of the initial Pm value and wasn't changing within the investigated period. Pg activator activity was decreasing rapidly to 30-40% of the initial one within few minutes from the moment of Pm-SK complex formation. It was 10-20% of that initial after 24 hours. The decrease in Pg activator activity of Pm-SK complex correlated with the initial very rapid conversion of 47 kDa SK to 36 kDa SK within few minutes and following more slow conversion of SK in 31, 25 and 15 kDa fragments after 5 hours. alpha 2-AP didn't influence on the Pg activator activity of Pm-SK complex but eliminated its fibrinolytic activity completely. It was supposed that alpha 2-AP inhibited fibrinolytic activity of Pm-SK complex similarly to 6-aminohexanoic acid by preventing Pm-SK complex binding to fibrin polymer.

[Features of the interaction between alpha2-antiplasmin and plasminogen/plasmin]. / Osoblyvosti vzaémodiï alpha2-antyplazminu z plazminohenom/plazminom.

The kinetic of plasmin, Va1442-plasmin, Lys530-plasmin inhibition reaction by alpha 2-antiplasmin as well as interaction of the inhibitor with different derivatives of the plasminogen and its fragments were studied. It was shown that plasmin, mini- and micro-plasmin activity decreased by 97, 88 and 85%, respectively, for equimolar ratio 1:1 of the inhibitor. The value of the inhibition reached its maximum in 1-2, 5-10 and 10-15 min, respectively. The constants of the complex formation rate were 1.4 x 10(6); 1.7 x 10(5) and 6.2 x 10(4) M-1s-1 for the plasmin, mini- and micro-plasmin with alpha 2-antiplasmin, respectively. Both 10(-2) M 6-aminohexanoic acid and 10(-1) M arginine reduced the complex formation rate between plasmin, mini-plasmin and alpha 2-antiplasmin to the value of the rate reaction between micro-plasmin and inhibitor. alpha 2-Antiplasmin bound with all investigated derivatives and fragments of plasminogen. The amount of inhibitor decreased in the series: plasmin, kringle 1-3, kringle 4, mini-plasminogen, micro-plasminogen. The kringle 1-4 and kringle 5 were determined to control the rate of reaction between enzyme and inhibitor, being not necessary for the inhibition. The comparison of the inhibitor interaction with DPP-plasmin, mini-plasminogen and micro-plasminogen displayed the possibility of the additional region existence in catalytic domain. This region participated in the complex with alpha 2-antiplasmin formation. It is supposed that the multisite interaction between plasmin and alpha 2-antiplasmin provides for the specificity and efficiency the inhibitor action.

[Plasminogen activation by antiplasminogen monoclonal antibody IV-1C. Properties and mechanism of reaction]. / Aktyvatsiia plazminhenu antyplazminohenovym monoklonal;nym antytilom IV-1c. Vlastyvosti ta mekhanizm reaktsiï

In review the results of investigation of plasminogen(Pg) activation by antiplasminogen monoclonal antibody IV-1c have been presented. Antigenic determinant of IV-1c was localized in Val709-Gly718 site of Pg protease domain. IV-1c completely inhibited the Pg activation by streptokinase, but increased the rate of Pg activation by t-PA and urokinase. Catalytic properties of plasmin in complex with IV-1c were studied. It was found that IV-1c induced catalytic activity in Pg-IV-1c complex. It was shown that Pg and IV-1c interacts in complex by two-centre mechanism: IV-1c binds with Pg by paratope and by N-terminal lysine of gamma-chain and Pg binds to IV-1c by one of the lysine binding sites and by V709-G718 site of protease domain. The influence of pH, temperature, 1.5 mM Ca2+, Mg2+, Sr2+, Ba2+, Co2+, Ni2+ cations and 10 mM Cl-, F-, Ac-, SO4(2-), HPO4(2-) anions on lag and fast phases of Pg activation by VI-1c was investigated. It was revealed that Val709-Gly718 site was determining in Pg activation by IV-1c and streptokinase.