Cleavage of prothrombin bound in immune complexes results in high thrombin enzymatic activity.

Thrombin plays a pivotal role in blood clotting as well as in the regulation of vascular remodeling and oxidative stress. Recent evidence suggests that auto-antibodies directed against prothrombin, may play an important role in the pathogenesis of atherosclerosis. It is however not clear, if prothrombin bound in an immune complex retains its clotting and regulatory properties or acts solely by increasing vascular inflammation. In order to answer this question, we used a newly developed stain for the detection of thrombin activity of such complexes. Plasma and serum samples were subjected to rocket immunoelectrophoresis in an anti-prothrombin antiserum containing agarose gel. Gel plates, covered with a nitrocellulose membrane were soaked with chromogenic thrombin substrate. The product of thrombin activity was diazotized to red azo dye bound to nitrocellulose. Activity stain revealed barely discernible rockets in plasma, but heavily stained ones in serum. Pre-incubation with trypsin enhanced activity of immunoprecipitates deriving from plasma, but not from serum. Densitometric analysis showed, that the trypsin-enhanced activity in plasma derived immune complexes was twice as high as in serum derived immunoprecipitates. Thrombin active centre is not blocked by anti-prothrombin antiserum allowing to retain thrombin activity. Moreover, prothrombin in immunoprecipitate is readily cleaved by proteolytic enzymes. This cleavage could potentially be enhanced by antibody binding, although these results need to be confirmed using different antibodies.

[Aspirin esterase: biochemical and clinical aspects of aspirin catabolism]. / Esteraza aspirynowa--biochemiczne i kliniczne aspekty katabolizmu aspiryny.

Aspirin, one of the most commonly used drugs, when entering human body is partially subjected to autolytic degradation to salicylic acid. However, hydrolytic degradation through enzymatic reaction, involving aspirin esterase is much more effective. It is obvious, that native drug, aspirin (acetylsalicylic acid) differs in respect of pharmacological properties from its catabolite, salicylic acid, a molecule without functionally important acetyl residue. Therapeutic implications of this simple esterolytic reaction are not yet fully understood. The paper presents current data on the biochemistry of aspirin esterase and clinical aspects of its activity.

[Comparative testing of aspirin esterase and cholinesterase activities in sera of healthy blood donors]. / Porównawcze badanie aktywnosci esterazy aspirynowej i esterazy cholinowej w surowicy zdrowych dawców krwi.

Activities of aspirin esterase, EC 3.1.1.55 and cholinesterase, EC 3.1.1.8 were measured in sera of 106 healthy blood donors. Surprisingly high correlation of these two activities has been observed. Aspirin esterase exhibited about 10 times lower activity than cholinesterase, when expressed in mkat/L.

Enhancement of fibrinolytic activity of human plasma in the presence of acetone.

The fibrinolytic system was studied in normal human plasma containing increasing concentrations of acetone up to 23.4 mmol l-1. Fibrinolytic activity measured as euglobulin clot lysis time [ECLT] and amidase activities toward chromogenic peptide substrates H-D-Valyl-L-Leucyl-L-Lysine-p-nitroanilide 2 HCl [S-2251], designed for plasmin determination, H-D-Valyl-L-Phenylalanyl-L-Lysine-p-nitroanilide 2 HCl [S-2390], designed for the determination of t-PA in plasma via plasminogen activation and H-D-Prolyl-L-Phenyl-Alanyl-L-Arginine-p-nitro-anilide 2 HCl [S-2302], designed for the determination of kallikrein and activated Hageman factor, increased when 15.7 mmol l-1 concentration of acetone was reached. A parallel increase of esterolytic [substrate: naphthol-AS-acetate] activity was observed in euglobulin fractions. Crossed immunoelectrophoresis [CIE] revealed changes in fibrinogen profiles of plasma enriched with acetone as compared to native plasma. These findings suggest that acetone present in plasma in concentrations comparable to those found in some pathological states might activate fibrinolytic system.

[Clinical significance of heat shock proteins. Influence of heat shock proteins on the pathomechanism of selected diseases]. / Kliniczne znaczenie bialek szoku cieplnego. Udzial bialek szoku cieplnego w patomechanizmie rozwoju wybranych jednostek chorobowych.

The presentation shows the survey of diseases the pathogenesis of which might be connected with the existence of Heat Shock Proteins (HSPs). We discuss the data referring to the influence of the HSPs upon the occurrence and progression of the following diseases: systemic lupus erythematosus, reactive arthritis, rheumatoid arthritis, insulin dependent diabetes mellitus, schizophrenia and Alzheimer's disease. There is also indicated a possible activity of HSPs in the pathogenesis of neoplasia, organ ischaemia and inflammation or degeneration.

[Reaction of dental methacrylate material in in vivo tests--selected hematologic and immunologic indices]. / Oddzialywanie dentystycznego tworzywa metakrylanowego w testach in vivo--wybrane wskazniki hematologiczne i immunologiczne.

Wistar rats were implanted subcutaneously with 2.5 x 6 mm cylindrical matrices, made from the home-made metacrylate material Akpol S, and, for comparative purposes, with the imported materials Trevalon and Superacryl. At different time intervals quantitative and qualitative analyses of the picture of leucocytes and of blood plasma proteins were carried out in groups of animals implanted with the above-mentioned materials and in a control one. No differences in the values of the above parameters were found in any of the examined groups of animals. The carried out tests were aimed at making a thorough study of general reaction of the living organism to the new, home-made metacrylate material Akpol S, used for making removable dentures.

Alpha-1-antitrypsin and alpha-1-antitrypsin-neutrophil elastase complex in bronchoalveolar lavage fluid of patients with pulmonary diseases (pilot study).

A simple method based on rocket and crossed immunoelectrophoresis was adapted for detection of alpha 1 AT-HNE complexes in BALF: Agarose gel with anti-alpha 1 AT combined with intermediate gel containing anti-HNE was used for detection and evaluation of the ratio of complexes with HNE to the free form of alpha 1 AT. BALF samples from 31 patients divided into 5 groups suffering from various respiratory tract diseases were tested. It was found that 3 out of 8 patients with inflammatory pulmonary diseases showed the presence of both forms of alpha 1 AT, 7 out of 13 patients suffering from cancer showed both forms, whereas the complexed form was not detected in any of 8 patients with asthma bronchiale. It is suggested that the presence of alpha 1 AT-HNE complex may reflect, to some extent, the proteolytic status of pulmonary tissue.

Inhibitory effect of human recombinant interferon gamma on synthesis of acute phase proteins in human hepatoma Hep G2 cells stimulated by leukocyte cytokines, TNF alpha and IFN-beta 2/BSF-2/IL-6.

Supernatants from endotoxin-stimulated human leukemic cells and human recombinant interferon-beta 2 similarly enhance synthesis of alpha 1-antichymotrypsin and haptoglobin but suppress synthesis of albumin in cultured Hep G2 cells. Human recombinant tumor necrosis factor only slightly affects production of alpha 1-antichymotrypsin and albumin in a similar manner as leukocyte cytokines. In distinction, recombinant human interferon-gamma profoundly inhibits synthesis of alpha 1-antichymotrypsin, and especially of haptoglobin, but stimulates production of alpha 2-macroglobulin thus modulating the acute phase response of these cells.

Serum trypsin inhibitory activity and alpha-1-antitrypsin level in liver diseases.

Alpha-1-antitrypsin level and serum trypsin inhibitory activity were measured in patients with viral hepatitis, chronic hepatitis and liver cirrhosis. The most pronounced discrepancy between these two parameters were observed in patients with liver cirrhosis: the increase of alpha-1-At level was not accompanied by adequate increase of trypsin inhibitory activity. Some mechanisms potentially responsible for this discrepancy are discussed.

Detection of inhibitory inactive fraction of alpha-1-antitrypsin in human serum.

Pseudomonas aeruginosa elastase is not inhibited by human serum alpha-1-antitrypsin, it damages inhibitory activity of alpha-1-antitrypsin activity instead. A simple method, based on electrophoresis in urea containing polyacrylamide gel, is described for the separation of active part of the inhibitor from that inactivated by the bacterial enzyme.

Comparison of antiproteolytic activities of alpha-1-proteinase inhibitors from the plasma of some mammalian species.

Alpha-1-proteinase inhibitors isolated from plasmas of horse, ox, pig, rabbit and man were used for determination of some kinetic parameters of interaction with three horse leucocyte proteinases and bovine pancreatic trypsin and chymotrypsin. Effective molar ratio of enzyme-to-inhibitor, inactivation rate constant and inhibition constant were measured. In horse, ox, pig and rabbit two principal electrophoretic forms of alpha 1-PI could be distinguished. Both forms effectively inhibited trypsin but usually only one form reacted promptly and stoichiometrically with chymotrypsin and leucocyte elastases. It appears that genetic variability and functional heterogeneity of multiple forms of alpha 1-PI as well as lack of other tissue inhibitors of proteinases may be responsible for lung emphysema occurring in man and horse.

Detection of plasma proteins during sequential ultrafiltration/dialysis.

Elimination of low molecular weight proteins during sequential ultrafiltration/dialysis was studied in 29 uremic patients. Beta-2-microglobulin, retinol binding protein, free light chains lambda and kappa, Zn-alpha-2-glycoprotein, hemopexin, prealbumin, hemoglobin, albumin, acid alpha-1-glycoprotein, haptoglobin, alpha-1-antichymotrypsin, ribonuclease, lysozyme, amylase, non-specific esterase, and proteolytic activity were detected in all ultrafiltrates tested. The level of total protein and ribonuclease was determined in 36 crude ultrafiltrates from 23 patients. Concentrated ultrafiltrates were used to quantitate retinol binding protein, prealbumin, albumin, lysozyme, and amylase. Other proteins identified in the ultrafiltrates are present in trace amounts. The question was discussed whether ++inextensive but systematic loss of proteins during hemofiltration in chronic RDT might be the cause of patient homeostasis disturbances.

Complexed forms of alpha 1-proteinase inhibitor in human sera.

Crossed immunoelectrophoresis in polyacrylamide/agarose system was applied to compare profiles of free and complexed forms of alpha 1-proteinase inhibitor (alpha 1-antitrypsin) present in human serum in normal and pathological conditions. Two to six complexed forms were found in sera of patients with some proliferative and liver diseases compared to only one present in normal sera.

Heterogeneity of acid phosphatase, beta-glucuronidase, and alpha-naphthyl-acetate esterase in normal and leukaemic lymphocytes.

Comparative study of isoenzymic patterns of acid phosphatase, beta-glucuronidase and alpha-naphthyl acetate esterase of normal and chronic lymphocytic leukaemia (CLL) lymphocytes was carried out by means of polyacrylamide gel electrophoresis. The isoenzymograms of acid phosphatase and beta-glucuronidase of leukaemic cells were similar to that of normal lymphocytes. However, different patterns were found in the case of alpha-naphthyl acetate esterase. Two prominent anodal bands were present in CLL lymphocytes; these bands were either absent or very weak in normal cells.