Level of overall hemostasis potential in donor and patient plasma in pathology.

Coagulation potential (CP), overall hemostasis potential (OHP) and fibrinolysis potential (FP) in plasma of donors and patients with myocardial infarction (MI), stroke (St) and hip joint diseases (HJD) have been investigated using M. Blomback's global hemostasis assay. Plasma samples of the patients were analyzed with APTT reagent in the presence or absence of t-PA. It was found that the ratio of values of СP, OHP and FP in plasma of patients to those of donors plasma were 78, 60 and 123% at MI; 157, 155 and 162% at St; 128, 131 and 124% at HJD. CP to FP ratio that indicated balance between coagulation and fibrinolytic systems activities were 4.13, 2.5, 4.0 and 4.26 in plasma of donors and MI, St and HJD patients, respectively. These results are evidence of increased fibrinolytic activity in plasma of MI patients. Lag-periods of plasma clotting of MI, St and HJD patients were prolonged by 2.3, 7.2 and 1.5-fold, respectively. Pearson's correlation analysis between parameters, obtained in vitro studies using global hemostasis assay, and concentrations of the molecular markers (soluble fibrin and D-dimer), which formed in vivo in plasma of MI, St and HJD patients, did not reveal any relationship between them.

[Simultaneous quantification of soluble fibrin and D-dimer in blood plasma for the assessment of the threat of thrombosis].

Soluble fibrin and D-dimer are the most specific markers of blood coagulation cascade and the threat of thrombosis. Two immunoassay test systems were designed using the fibrin-specific and D-dimer-specific monoclonal antibodies. The clinical trials of the test systems were carried out in Ukraine. The high informative value of soluble fibrin quantification as a prognostic indicator of the threat of thrombosis associated with hip replacement and endoprosthetics of the abdominal aorta was shown. Independent D-dimer quantification is less informative. Simultaneous quantification of soluble fibrin and D-dimer before operation and at different time intervals after it is required for the prediction of postoperative thrombotic complications and monitoring the efficiency of antithrombotic therapy. Only in this case it is possible to get information about the state of the balance between blood coagulation and fibrinolytic systems, and determine the degree of the threat of thrombosis.

[Isolation and characteristics of the protein C activator from Agkistrodon halys halys snake venom]. / Vydelenie i svoistva aktivatora proteina S iz iada shchitomordnika obyknovennogo (Agkistrodon halys halys).

Protein C activator from Agkistrodon halys halys venom has been isolated and purified by ion-exchange and gel-filtration chromatography. The purified enzyme consists of the single peptide-chain with molecular weight of 34 kDa. Its pH-optimum was in 7.5-8.0 range. The enzyme was inhibited by DFP, benzamidine, PMSF, EGTA. Protein C activator was as effective as "Protac" (Pentapharm AG, Switzerland).

[Enzymes of snake venoms]. / Fermenty zmeinykh iadov.

Snakes' venom is a mixture of biologically active substances, containing proteins and peptides. A number of these proteins interact with haemostasis system components. Activators and inhibitors affecting blood coagulation and fibrinolysis systems are of special interest. Venom components can be classified into three main groups, such as procoagulants, anticoagulants and fibrinolytic enzymes according to their action. This review is focused on enzymes from Agkistrodon halys halys venom. They are thrombine-like enzyme, named Ancystron-H, flbrinogenolytic enzyme, protein C activator and platelet aggregation inhibitor. Ancystron-H is used for determination of fibrinogen level in blood plasma of patients undergoing heparin treatment and blood coagulation inhibitors accumulation. The fibrinogenolytic enzyme can be used as the instrument for protein-protein interactions in fibrinogen-fibrin system. The protein C activator is used for protein C level determination in blood plasma with different pathologies. Functions of the platelet aggregation inhibitor, belonging to disintegrins group, can be used for development of antithrombotic preparations. Information about the use of snake venoms in science and medicine is presented.

[Isolation and properties of the protein C activator from Agkistrodon halys halys venom]. / Vydelenie i svoistva aktivatora proteina C iz iada shchitomordnika obyknovennogo (Agkistrodon halys halys).

Protein C activator from Agkistrodon halys halys venom has been purified by ion-exchange and gel-filtration chromatography. The purified enzyme consists of a single peptide chain with molecular weight of 34 kD. Its pH-optimum was the range of 7,5-8,0. The enzyme was inhibited by DFP, benzamidine, PMSF, EGTA. Protein C activator was as effective as Protac (Pentapharm AG, Switzerland) for determination of protein C level in blood plasma using APTT test and protein C chromogenic substrate.

[Application of the phase transition theory to the description of fibrinogen-fibrin transition]. / Primenenie teorii fazovykh perekhodov dlia opisaniia prevrashcheniia fibrinogena v fibrin.

A physical model of the kinetics of the fibrinogen--fibrin transition was constructed using the theory of phase transitions. It was shown that the model is consistent with the experimental data.

[Purification and characterization of the fibrinolytic enzyme from Agkistrodon halys halys venom]. / Vydelenie i svoistva fibrinogenoliticheskogo fermenta iz iada shchitomordnika obyknovennogo (Agkistrodon halys halys).

By Q-sepharose column ion-exchange chromatography, alkyl-sepharose column hydrophobic chromatography the purified fibrinogenolytic enzyme was obtained from Agkistrodon halys halys venom. It is a single peptide-chain with molecular weight about 28 kDa. It was founded that this enzyme cleaved A alpha-chain of fibrinogen, pH-optimum was determined in the range of 7.5-8.0. Its fibrinogenolytic activity was estimated 15.6 mM fibrinogen/min per mg protein; caseinolytic activity was estimated 7.5 c.u., and amidolytic activity was 0.325 mM pNA/min/mg and 0.175 mM pNA/min/mg for S2238 and S2251 respectively; K(m) was 5.6 mM. The enzyme activity was inhibited by DFP and benzamidine. These results suggest that the enzyme is serine protease. It inhibited the platelet-aggregation.

[Coagulation factors and fibrinolytic system in subretinal liquid in patients with rheumatogenous retinal detachment]. / Faktory svertyvaiushchei i fibrinoliticheskoi sistem v subretinal'noi zhidkosti u bol'nykh s regmatogennym otsloniem setchatki.

The work deals with estimation of some factors of blood coagulation and fibrinolytic systems, which include antithrombin III, factor X, prothrombin, plasminogen, protein C concentrations in the subretinal fluid of the patients with rhegmatogenous retinal detachment retina. The tendency to increase of the blood coagulation and fibrinolysis factor levels, except protein C, was revealed in the patients with complicated forms of the disease. The investigations mentioned above are capable of serving as a diagnostical and forecasting test characterizing the rhegmatogenous retinal detachment retina and surgical treatment proceeding.

[Hemostasis in patients operated for abdominal bleeding. I. Estimation of the relationship of hemostasis parameters according to data of the correlation coefficient and factor analysis]. / Gemostaz u bol'nykh, operirovannykh po povodu abdominal'nykh krovotechenii. I. Otsenka sviazi pokazatelei gemostaza po dannym koéffitsienta korreliatsii i faktornogo analiza.

The work was aimed to estimate the interrelation and indexes which define the main alterations of hemostatic deviations in the patients with abdominal bleeding. For this reason the authors have analysed interrelations between 31 hemostasis indexes in 30 patients after operations. Fourteen groups of interrelated indications have been revealed. They are as follows: 1. Prothrombin index, activated partial thromboplastin index; 2. Prothrombin's inactive forms (prethrombin-1, decarboxylated prethrombin etc.), ecamulin index; 3. Velocity bend of distraction of fibrin clot, velocity bend of forming of fibrin clot, height of clot using turbidimetric method; 4. Half-lysis time, lysis time, time of fibrin clot using turbidimetric method; 5. Velocity, Intensity, remainder of platelet aggregation induced by ADP 10 mcg/ml, 2.5 mcg/ml, 1.9 mcg/ml; 6. Thrombin time; 7. Protein C; 8. Fibrinogen; 9. Platelet count; 10. Soluble fibrin; 11. Ancystron time; 12. X factor; 13. Antithrombin-III; 14. Fibrin(ogen) degradation products. It was shown how different groups affect hemostasis. The authors have suggested to use the data of mathematical analysis and laboratory tests for the estimation of hemostatic deviations.

[Hemostasis in patients operated on for abdominal hemorrhage. 2. Characterization of the process of activation of blood coagulation system factors. Coagulation inhibitors]. / Gemostaz u bol'nykh, operirovannykh po povodu abdominal'nykh krovotechenii. 2. Kharakteristika protsessa aktivatsii faktorov sistemy svertyvaniia krovi. Ingibitory svertyvaniia.

Haemostasis indexes were estimated in four groups of patients which were isolated by the cluster analysis and operated for abdominal haemorrhages. A tendency or explicit hyperfibrinogenemia have been fixed which are connected with the syndrome of the system inflammation response of the patient in the critical state. The correlation between the indexes of the prothrombin time, activated partial prothrombin time and protein C activity is shown. An analysis of anticoagulants effect suggests it possible to consider the antithrombin III to be the main, stable blood anticoagulant, while protein C is responsible for the operational response of the organism on the part of haemostasis. The decrease of ancystron and thrombin time was recorded in 50% of patients notwithstanding the hyperfibrinogenemia and presence of fibrin fibrinogen degradation products. It is supposed that the acceleration of fibrin polymerization in the examined groups of patients is the mechanism of the organism protection from the blood loss under the given pathology.

[Hemostasis in patients operated on for abdominal hemorrhage. 3. Boundary deviations of hemostasis]. / Gemostaz u bol'nykh, operirovannykh po povodu abdominal'nykh krovotechenii. 3. Krainie varianty otkloneniia gemostaza.

The fibrinolytic process in the plasma of patients operated for abdominal haemorrhages have been investigated. The results allowed to conclude that the blockade of fibrinolysis did not effect on the course of the disease. The high level of the inhibitors and of the platelets hypoaggregation can be considered as a cause increased of the recurring gastrointestinal haemorrhages. It was demonstrated that the probability of DIC-syndrome development increased at the aggravation of the patient's state after the operation.

[Hemostasis in patients operated on for abdominal hemorrhage. 4. Conception of hemostasis changes]. / Gemostaz u bol'nykh, operirovannykh po povodu abdominal'nykh krovotechenii. 4. Kontseptsiia izmenenii gemostaza.

Studies of haemostasis changes in the dynamics of early post-operational period permitted revealing the tendency to the growth of fibrinogen concentration, decrease in the fibrinogen self-assembling rate, weakening of thrombinemia, disturbances in fibrinogen degradation products (FDP) elimination, increase of inhibitors activity and/or weakening of blood coagulation factors activity, intensification thrombocytes aggregation. Hypercoagulation has been registered under acute haemorrhage and the haemorrhage time exceeding 24 h before the operation, the weakening of hypercoagulation response was observed, notwithstanding the possibility of haemorrhage continuation. The letter is underlined by the changes in the balance between the coagulation factors and inhibitors up to the absence of typical hypercoagulation response to surgical interference.

[Isolation and characteristics of ekamulin--a prothrombin activator from multiscaled viper (Echis multisquamatus) venom]. / Vydelenie i kharakteristika ékamulina--aktivatora protrombina iz iada éfy mnogocheshuichatoi (Echis multisquamatus).

Ecamulin, a novel prothrombin activating enzyme, has been isolated and purified 63-fold with a 57% yield from the venom of the Middle-Asian sand viper Echis multisquamatus using three-step ion-exchange chromatography. The enzyme was shown to activate prothrombin similarly to Ecarin, a prothrombin-converting enzyme from Echis carinatus venom, however, differing from the latter by structural and physico-chemical properties. The enzyme is a Zn-proteinase: it contains 1 mol Zn per 1 mol of protein. The molecular mass of the enzyme as determined by Sephacryl S-200 chromatography is 93 +/- 2 kDa. Upon SDS-PAAG electrophoresis ecamulin produces two bands with Mr of 67 and 27 kDa under non-reducing conditions, and three bands with Mr of 67, 14 and 13 kDa in the presence of DTT. During native PAGE without SDS, the activator yields one slow mobility band: two bands are observed after addition of DTT or EDTA. Carbohydrates containing N-acetyl-alpha-D-glucosamine residues are localized in the 67 kDa chain. Ecamulin has two isoforms, S2 and S3, that are distinguished by the charge and partial coagulation activities: form S2 has 250 NIH units/mg, while the S3 form has 524 NIH units/mg. The amino acid sequences of the both isoforms are similar but the more active S3 form has 4 times higher content of Gln and 4 times less of Gly than the S2 form. The isoelectric point is 4.3-4.5; E280 of 1% solution is 10.2. Forms S2 and S3 of ecamulin hydrolyze chromogenic substrates of plasma kallikrein S2302 and glandular kallikrein 2266. Ecamulin does not hydrolyze BAEE, TAME, LEE, thrombin substrates Chromozym TH and S2160, factor Xa-S2222, protein Ca-Chromozym PCa and Plasmin S2251. The amidase activity is nonreversibly inhibited by EDTA, o-phenanthroline (the activity is recovered by addition of Zn2+), Cys or DTT, EGTA, DFP, PMSF or pCMB do not inhibit the enzyme activity. Ecamulin converts prothrombin to alpha-thrombin passing by a shunt via the meizothrombin stage. The reaction of prothrombin activation does not require Ca2+, phospholipids of factor Va. Part of this work was presented at the International Conference "Fibrinogen and fibrinolysis", Yalta, September 23-28, 1995.

[Determination of the general prothrombin level and detection of its functionally inactive forms using the enzyme ecamulin purified from Echis multisquamatus venom]. / Opredelenie obshchego urovnia protrombina i vyiavlenie ego funktsional'no neaktivnykh form s pomoshch'iu fermenta ékamulina, vydelennogo iz iada éfy mnogocheshuichatoi.

The method for determination of the general prothrombin level and detection of its inactive forms (prothrombin-1, decarboxylated prothrombin, etc.) has been worked out. Enzyme ecamulin was purified from the venom of Echis multisquamatus. Ecamulin test was used for plasma of practically healthy people and patients with hypertension. The results obtained proved that this test is informative and precise. Comparison of two tests, prothrombin and ecamulin ones, permits characterizing the state of blood coagulation and finding functionally inactive prothrombin forms which are the markers of some pathological states.

[Comprehensive laboratory diagnosis of intravascular blood coagulation syndrome in late gestosis]. / Kompleksnaia laboratornaia diagnostika sindroma vnutrisosudistogo svertyvaniia krovi pri pozdnykh gestozakh.

A chronic form of intravascular blood coagulation (the DIC syndrome) is an obligatory pathogenetic mechanism in the organism of pregnant women with late gestoses. In order to find the DIC-syndrome and to provide urgent protective measures comprehensive laboratory diagnostics of the hemostatic pathologies is necessary. Analysis of clinician data has shown an optimal quantity and sequence of tests, among which some are traditional and several have been worked out by the authors. This approach can be recommended for corresponding medical institutions as such that provides obtaining exact and informative data about the state of blood coagulation with minimal amounts of human plasma.

[An analysis of a disorder in the fibrin-forming phase in virtually healthy middle-aged and elderly subjects by using the ancistron test]. / Analiz porushennia fazy fibrynoutvorennia u praktychno zdorovykh osib pokhyloho ta starechoho viku za dopomohoiu antsytronovoho testu.

Damages in the fibrin-formation phase initiated by activation of the blood coagulation system and fibrinolysis and promoted by interaction between fibrinogen and its derivatives (dissolved fibrinogen, products of fibrin and fibrinogen lysis) and by the presence of various blood coagulation inhibitors were found in 34% of practically sound old people and in 47% of people of senile age. It is shown very significant to apply the "ancistrone time" test developed on the basis of enzyme ancistrone-H isolated from the venom of snake Agkistrodon halys halys for estimating the fibrin-formation phase both on the model system and in practically sound people of old and senile age. The ancistrone test permits rapidly (for 30-60s) obtaining qualitative and quantitative characteristic of the fibrinogen level in the blood plasma, of the products of fibrin and fibrinogen lysis, of the blood coagulation inhibitors.