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The condition of proteinase-inhibitor balance was studied in blood plasma of patients with obstetric sepsis. Hypoproteinemia (due to a massive blood loss and toxic effect) was shown to result in a 2-5-fold decrease of the activity of endogenous proteinases in blood plasma versus the control group. The activity of alpha-macroglobulins was also by 2 times lower versus the controls, while the activity of alpha 1-antichymotrypsin and of an acid-stable inhibitor of trypsin was found to be higher by 20-30%. A degree of imbalance of proteinase/inhibitor depends on a disease severity.

The review is devoted to modification of lipoproteins (Lp) connected with various biological processes. Possible mechanisms of Lp oxidation and the nature of oxidants involved in to these processes are considered. Specific attention is paid to the participation of proteolytic enzymes in modification of Lp.

Aim: Development of a protocol of multiple-modality treatment of patients with nonspecific aortoarteritis (NAA) making use of plasmapheresis and specific plasma adsorption of proteinases., Materials and Methods: Six patients with NAA aged 15 to 58 years were examined using ultrasonic dopplerography, magnetic imaging, and angiography of the large vessels. Red cell sedimentation rate was assessed, cathepsin G activity, antitryptic activity, and content of C-reactive protein in the blood measured. Drug therapy was supplemented by repeated sessions of plasmapheresis and specific plasma adsorption on immotin., Results: After sessions of plasmapheresis (n = 17) and plasma adsorption (n = 13), increased cathepsin G activity dropped by at least 30% (in 3 patients it normalized), the content of C-reactive protein decreased in the presence of normal antitryptic activity (in patients with decreased activity it normalized and in those with increased values a tendency to normalization was observed). Red cell sedimentation rate decreased, particularly so 1 and 2 months after treatment; the patients felt better., Conclusion: Multiple-modality treatment of NAA patients making use of plasmapheresis and plasma adsorption decreases the activity of the inflammatory process.

Some components of plasma and granulocyte proteolytic system, granulocyte activation degree were evaluated in 15 patients with nonspecific aortic arteritis (NAA) and 12 hereditary hypercholesterolemia (HHC) patients. Catepsin G activity in blood plasma of NAA and HHC patients was higher than in donors 8-9 and 6-fold, respectively (p < 0.001). Granulocyte collagenase activity, spontaneous NBT test values in them were higher than in donors 6 and 4-fold (p < 0.02 and p < 0.01, respectively), 2-fold (p < 0.005 and p < 0.001, respectively). The data obtained confirm the suggestion on the presence of an inflammatory component not only in pathogenesis of NAA, but also in HHC causation.

Three MAb M2, B6, P1 (IgG1 type) against human urinary trypsin inhibitor (UTI), a glycoprotein with antiinflammatory properties, have been produced by hybridization of mouse myeloma cells P3O1 with spleen cells of immunized mice BALB/c. Competitive ELISA-examination of the peroxidase conjugates of M2, B6, and P1 MAb in the presence of the trypsin binding domain shows the M2 antibody to possess the highest affinity for this domain. On the basis of the MAb M2, a competitive ELISA of UTI concentration in urine is proposed. ELISA-detectable changes in the UTI content of urine from patients with nephritis without renal failure can be considered as an early index of renal parenchyma damage.

Prevailing fibrin-formation in the pleural cavity entails hypoactivity of trypsin-like proteinases and a high inhibitory potential in the serum and pleural exudate. Streptokinase preparations appeared an effective means of pharmacological pulmonary decortication in patients with high pleural levels of plasminogen. The authors obtained higher efficacy of conservative therapy for pyothorax when they used a specially designed technique of intrapleural administration of streptokinase-activated fresh frozen plasma of the same group. The outcomes of the disease were also improved noticeably.

Using plasminogen-Sepharose 4B chromatography, a 50-70% reduction in the alpha-2-antiplasmin (alpha-2-AP) content in human and dog blood plasma was reached. The decrease in the alpha-2-AP concentration in human and dog blood plasma markedly enhanced the lysis of fibrin clots formed from the plasma under the action of urokinase and streptokinase. Thus, the lysis of model clots for a definite period of time for human and dog blood plasma depleted for alpha-2-AP by 50-70% required 5-8 and 3-6 times as low urokinase and streptokinase concentrations as those needed for the clot lysis in the native plasma. The decrease in the fibrinogen concentration in human blood plasma caused by plasmosorption on plasminogen-Sepharose enhanced the model fibrin clot lysis by urokinase and streptokinase, however, by no more than 5-10%. Hence, the increased efficiency of plasminogen activators in the given model system can be accounted for by the decline of the antiplasmin potential of the blood plasma.

A novel procedure for isolation of an acid-resistant trypsin inhibitor from human urine (UTI) is described. The method is based on the use of immobilized on an insoluble matrix monoclonal antibodies specific for the given inhibitor. The use of Immotin (which represents an immobilized on Sepharose 4B UTI) as precolumn during affinity chromatography makes it possible to significantly prolong the immunoadsorbent life and to increase the inhibitor yield up to 92% of its original content in the urine. The preparation obtained with the use of an immunoadsorbent contains predominantly the native form of the inhibitor whose molecular mass is about 44 kDa.

A comparative study of thermo-acid stable proteinase inhibitor from rabbit serum and commercial drug contrykal was made on rats and in vitro experiments. Both inhibitors in equal doses effectively suppress proteolytic activity during initiation of lung inflammation. It was shown that the thermo-acid stable inhibitor possesses higher than contrykal, antielastase activity. A high effect of the acid stable inhibitors in the treatment of lung disease is suggested.

The authors treated destructive forms of acute pancreatitis for the first time by means of specific plasma sorption of proteinases with the use of a proteinase plasma sorbent which is an acid-stable proteinase inhibitor immobilized on sepharose (ASI-Sepharose). Specific plasma sorption of proteinases on ASI-Sepharose was applied to the treatment of 29 patients with various forms of acute destructive pancreatitis. At the end of specific plasma sorption procedure the activity of blood plasma proteolytic enzymes in the patients reduced by 60-75%, which was attended by marked improvement of the condition in most cases; aggravation of the patients' condition was not encountered. The mortality in this group was 20.7% and was due to complications occurring during the development of the main disease. The mortality rate among patients treated by specific proteinase plasma sorption is much lower than that recorded in the USSR for destructive forms of acute pancreatitis which ranges from 30 to 75%.

Changes of proteinase-inhibitory system values in blood plasma of dogs with experimental acute pancreatitis (EAP) were studied in dynamics. Their diagnostic and prognostic values was appraised. Essential shifts were revealed in the proteinase-inhibitory balance in blood plasma of dogs with EAP of various severity. The concentration of trypsin- and chymotrypsin-like proteinases exceeded the normal values 1.8-3.3 times in these animals. Coefficients expressing the relation of antitryptic and antichymotryptic activities to the activities of the corresponding proteinases in the blood plasma of animals with EAP were indicative of diminished blood plasma inhibitory potential as compared to that in intact animals. The authors are the first to show the diagnostic and prognostic value of the antichymotryptic activity/chymotrypsin in blood plasma of dogs with EAP.

The dynamics of acid phosphatase and granulocytic elastase activities in bronchoalveolar washings of 40 patients with chronic catarrhal bronchitis was studied. In case of effective cure, the activities of these enzymes were increased shortly on the 10-13 days from beginning of the cure. In order to study the nature of this enzymes, the additional researches were conducted. These researches demonstrated that the macrophages were the source of the acid phosphatase, and the neutrophils--elastase. The transitory increase of the activity of both enzymes is favourable prognostical sign of disease's course.

Concentration of acid stable inhibitors (ASI)--metabolites of plasma inter-alpha-trypsin inhibitor--was measured in urine samples of healthy persons and of 6 patients with nephrotic syndrome by means of modified cross immunoelectrophoresis. There was obtained 2-25 mg of ASI (Mr 22,000 and 32,000) in diurnal urine samples of patients with nephrotic syndrome, which were 10-20 times higher as compared with these inhibitors level in the urine of healthy persons. The reverse correlation was established for the value of glomerular filtration and the lever of ASI (Mr 32,000) in the urine of patients with nephrotic syndrome.

The inhibitory effect of thermo- and acid-resistant inhibitor of trypsin, chymotrypsin and leukocyte proteinases (TASPI) from rabbit serum on the kininogenase activity of cathepsins D from different organs and tissues (human spleen and liver, chicken liver, spleen leukemic infiltrate from patients with myeloid leukemia) was revealed. The progressive mechanism of TASPI and cathepsins D complexation dependent on time and temperature was revealed. The rate constant of inhibition (ki) of chicken liver cathepsin D by TASPI at 37 degrees was 4,25.10(3)M-1 min-1. It was shown that the kininogenase activity of chicken liver cathepsin D was slightly inhibited by the basic pancreatic trypsin and kallikrein inhibitor from bovine organs (Kunitz type) and by soya bean trypsin inhibitor. The role of TASPI as regulator of cathepsins D activity under pathological conditions accompanied by lysosomal disintegration is discussed.

Proteinase-inhibitor balance in human plasma before and after artificial blood circulation was examined. It was shown that trypsinlike and chemotrypsinlike proteinases activities after this procedure were 1.6 and 2.8 times higher and antitryptic activity 1.2 times lower than in the same patients before the procedure. The 40-50% decrease of proteinase activity in human plasma after artificial blood circulation was observed as a result of in vitro plasmasorption on immobilized human urinary trypsin inhibitor.

The authors studied the level of secretory Ig, activity of elastase and locally produced acid-resistant inhibitors (ARI) of proteinases in 63 new cases of destructive pulmonary tuberculosis complicated by nonspecific endobronchitis. Functional abnormality of the local defense systems (immune and inhibitory) was found in patients with purulent bronchial inflammation associated with a considerable rise of granulocyte proteinase content. This makes it necessary to carry out corrective drug therapy of these alterations. A significant positive correlation was revealed between SIgA concentration and ARI in catarrhal patterns of endobronchitis.

Preparative isoelectrofocusing used for fractionating the whole human granulocyte lysate serine proteinases revealed multiple forms of elastase, cathepsin G, kininogenase, human granulocytes plasminogen activator (pI 6.2-10.75). Kinetic characteristics of their substrate specificity were also obtained. It is shown that serine kininogenase of human granulocytes is not identical with elastase as it had been supposed before, it is of trypsin-like nature and is identical with plasminogen activator of these cells. The results obtained reveal new aspects in comprehension of the role of the granulocyte plasminogen activator in development of the inflammatory reaction. It is found that acid-stable proteinase inhibitors formed from blood plasma inter-alpha-inhibitor of trypsin, have an inhibitory effect on the granulocyte plasminogen activator, that supports an assumption on the anti-inflammatory function of these inhibitors.

The method for the preparation of a specific proteinase adsorbent (acid-resistant human urinary proteinase inhibitor--UPI--immobilized on sepharose) has been developed. Proteinase adsorption using this adsorbent has proved highly effective in the treatment of acute pancreatitis with plasmasorption. High adsorption capacity of UPI-sepharose from plasma samples of patients with acute nonspecific aortoarteritis has been demonstrated in vitro. Determination of UPI activity in the human urine is an informative test for early tissue damage, as compared to plasma middle molecules assay. The former test may be used as a diagnostic technique in patients with parenchymal kidney injury and increased arterial pressure.

It was shown that the plasminogen activator inhibitor, ZGlyGlyArgCH2Cl, inactivates the kininogenase and plasminogen activator activities in the whole human granulocyte lysate and human granulocyte proteinase fractions isolated by isoelectrofocusing from the granulocyte lysate (pH 3-10). The kinetics of irreversible inhibition of the ZGlyGlyArgpNA-amidase activity in granulocyte proteinase fractions (pI 10.75, 8.9 and 8.3) by ZGlyGlyArgCH2Cl was measured. These data confirm the earlier obtained results on the trypsin-like nature of the human granulocyte plasminogen activator and its identity to this cell kininogenase.

Role of granulocyte proteinases and their inhibitors, found in bronchial secretions, in pathogenesis of bronchopulmonary diseases is discussed. The acid-stable inhibitors, synthesized in bronchi, were responsible mainly for the antileukoproteinase effect in upper respiratory tract and bronchi. Influence of alpha 1-inhibitor of proteinases and other blood plasma labile inhibitors on destructive impairments in bronchopulmonary tissue is considered. Use of proteinase inhibitors as therapeutic drugs in bronchopulmonary pathologies is discussed.

The kininogenase activity of highly purified preparations of cathepsins D from human liver and spleen, leukemic infiltrate obtained from patients with myeloic leukemia, and from chicken liver was studied. It was found that pepstatin, a specific inhibitor of carboxylic proteinases, inhibits this activity of cathepsins D. Interaction of chicken liver cathepsin D with human plasma substrate, which is possibly a low molecular weight kininogen (Ks = 1.3.10(-7) M) results in a production of the bradikinin analog methionyl-lysyl-bradikinin. The role of cathepsins D as potent inflammatory agents responsible for the generation of biologically active peptides--mediators of inflammation from the protein substrates including kininogens under desintegration of lysosomes is discussed.

Thermo- and acid-stable serine proteases inhibitor from the rabbit blood serum (TASPI) was shown to inhibit the human peripheral blood lymphocytes transformation stimulated by phytohemagglutinin (PHA) or concanavalin A. The extent of inhibition depended on the concentration of the preparation and its specific activity. The maximal inhibition of lymphocytes proliferation constituted 50 to 70%. TASPI displayed no cytotoxic activity. Considerably more effective inhibition was demonstrated by TASPI addition to the culture medium 24 hours after the addition of PHA. The antiprotease activity of crude human serum and that inactivated under different conditions is described. The results obtained suggest the participation of TASPI in the control of biological activity of the lymphoid tissue cells.

The literary and experimental data on some new natural proteinase inhibitors derived from their precursor, plasma inter-alpha-trypsin inhibitor, or synthesized by secretory glands and mucous membranes of various mammalian organs and referred to as "acid-stable inhibitors" are discussed. The physico-chemical properties, primary structure, specificity and biological functions of these inhibitors are described. The mechanism of interaction of the acid-stable inhibitors with human granulocyte proteinases is discussed in terms of their pathophysiological importance. The antiinflammatory role of locally synthesized acid-stable inhibitors from genital and respiratory tracts and the role of these inhibitors in fertilization are discussed. The barrier function of the acid-stable inhibitors which, similar to that of the plasma labile inhibitors (alpha 1-antitrypsin, alpha 2-macroglobulin, etc.), restricts the destructive action of leukocyte proteinases under normal and pathological conditions is considered.

Using an inhibitory analysis, the different enzymatic nature of kininogenase and elastase activities in serine proteinase fractions (pI 8.3-10.75) isolated from human granulocyte lysates by isoelectrofocusing was demonstrated. The thermo- and acid-stable serine proteinase inhibitor from rabbit serum was shown to completely inhibit the kininogenase activity in these fractions but to have no inhibiting action on the elastase activity. On the contrast, the specific granulocyte elastase inhibitor, N-3-carbomethoxypropanoyl-L-alanyl-L-alanyl-L-prolyl-L-valyl-chloromethylketone , inhibits granulocyte elastase and does not inhibit the kininogenase activity in lysate fractions. The efficiency of granulocyte elastase inhibition by this chloromethylketone is evaluated by the kinetic parameters k3, Ki. The values of k3/Ki for granulocyte elastase forms with pI of 10.75, 8.9 and 8.0 are 1430, 670 and 360 M-1 S-1, respectively and show effective inhibition of the three forms by this inhibitor. Based on the different degree of inhibition of the three elastase forms by chloromethylketone inhibitor the existence of the family of elastaselike enzymes in human granulocytes is postulated.

The activity of granulocyte elastase (GE) was discovered in the preparations of leukocyte thermostable alpha-glycoprotein (LTG) isolated from pus by means of ion-exchange chromatography. The activity of GE was determined according to MeoSuc(Ala)2ProValpNa hydrolysis. The antibodies against LTG were isolated from monospecific antisera. Sepharose with immobilized fraction of pus proteins was utilized as immunosorbent. Isolated antibodies to LTG inhibited the GE activity. An inhibitory effect of antibodies increased with the increase in their concentration. The identity or binding of LTG and GE was suggested. The binding of LTG with pus protein component was discovered, the biological meaning of this phenomenon being unknown.

Two highly purified forms of acid-stable proteinase inhibitor from urine of pregnant women with nephropathies (Mr 22000 and 32000, ASI-22 and ASI-32, respectively) were obtained. The preparations were homogenous in molecular mass and polymorphous in molecular charge (pI from 3.9 to 4.2). ASI-22 an ASI-32 effectively inhibited trypsin and chymotrypsin (Ki approximately 1 x 10(-8)-1 x 10(-9) M, ka approximately 10(5)M-1 sec-1, kd approximately 3 x 10(-4) sec-1) by the permanent mechanism of action. Both forms inhibited the esterase activity of pancreatic pig elastase by the progressive mechanism of action (ki approximately 1 x 10(4)M-1 min-1 at 37 degrees). Rabbit monospecific antiserum to total ASI-22 and ASI-32 preparation was obtained 1 ml of the anti-ASI-serum contained 95 micrograms of antibodies. Total ASI preparations was immunochemically homogenous and had antigenic similarity to inter-alpha-trypsin inhibitor from human plasma.

The use of a "Cell-Saver" technique prevented hyperproteolysis and deletion of proteinase inhibitors in the blood plasma of patients during cardiopulmonary bypass. The technique also improved the course of the postoperative period, decreased the incidence of lung complications and prevented effectively postoperative bleedings. A prognostic value of alpha 1-proteinase inhibitor to trypsin activity ratio in the blood plasma of patients subject of cardiopulmonary bypass surgery has been demonstrated.

The thermo- and acid-stable trypsin, chymotrypsin and intracellular proteinases inhibitor (TAS-inhibitor) from rabbit serum was digested by trypsin, and its domain (Mr 6200) with antitryptic activity was obtained in homogeneous state. The N-terminal amino acid sequence of this domain was established by automatic Edman degradation: Thr-Val-Ala-Ala-Cys-Asx-Leu-Pro-Ile-Val-Pro-Gly-Pro-X-Arg-Gly-Ile-Phe-X- Leu-X-Ala-Phe-X-Ala-Val-X-Gly. A high degree of homology of the primary structures rabbit, human and bovine TAS-inhibitors was demonstrated.

Several human granulocyte proteinases sensitive to the thermo- and acid-resistant proteinase inhibitor from rabbit serum (TASPI) were revealed, using TASPI-Sepharose 4B. It was found that TASPI inhibits the following human granulocyte proteinases: granules-localized kininogenase and chymotrypsin-like kininase (serine proteinases), elastase-like proteinase and benzoyl arginine ethyl ester esterase, as well as chymotrypsin-like kininase from the post-granule supernatant. These enzymes were compared to known granulocyte proteinases. Some carboxylic kininogenase sensitive to TASPI was identified in the granulocyte membrane debris fraction. The capability to inhibit neutral kininogenase suggests that TASPI is a first natural proteinase inhibitor, which can differentiate granulocyte and blood plasma kininogenases. Using trypsin-Sepharose 4B in the granulocyte post-granule supernatant, the acid-resistant trypsin and chymotrypsin inhibitor was identified. The data obtained are indicative of an antiinflammatory function of TASPI in mammals.

An effective method of plasma proteinases (trypsin, chymotrypsin. elastases) removal in dogs with acute pancreatitis has been suggested. The method is based on plasmasorption, using human urine acid-stable proteinase inhibitor immobilized on sepharose. The procedure of plasmasorption reduces proteinase levels in dogs with acute pancreatitis to 35-70%, with the animal life-span prolonged 8-fold.

A modification is described of the previously developed procedure for isolation of acid stable proteinase inhibitor (ASI) from urine of patients with nephritis. The modified procedure enabled to increase the yield of the inhibitor and to obtain its highly purified preparations. These preparations possessed a property to inhibit effectively the activity of human granulocyte elastase (ki = 10(4) M-1 min-1). The modified procedure is based on affinity chromatography on chymotrypsin-Sepharose 4B, substitution of dialysis by gel filtration on Sephadex G-25, use of alternative methods for concentration of protein fractions instead of membrane ultrafiltration and chromatography on polyamide for ASI depigmentation. The inhibitor yield amounted up to 75% of the initial activity in urine. The possibilities of the human urine ASI use in medical practice are discussed.

The determination of urine acid-stable proteinase inhibitor activity proved to be more informative at the early stages of renal tissue lesions, as compared to measurement of intermediate molecule blood level. It can be used as a diagnostic test in patients with parenchymatous renal diseases accompanied by blood pressure elevation, and essential hypertension.