Alpha 2-macroglobulin Activity Research Articles

Alpha-2 macroglobulin activity in SARS-CoV-2 induced infection and in the post-COVID-19 period.

The universal proteinase inhibitor α2-macroglobulin (α₂-MG) exhibiting antiviral and immunomodulatory activities, is considered as an important participant in the infectious process. The activity of α₂-MG in the new coronavirus infection and post-covid syndrome (long COVID) has not been studied yet. We examined 85 patients diagnosed with community-acquired bilateral polysegmental pneumonia developed under conditions of a new coronavirus infection SARS-CoV-2. For assessment of the post-COVID period, 60 patients were examined 5.0±3.6 months after the coronavirus infection. Among these patients, 40 people had complications, manifested in the form of neurological, cardiological, gastroenterological, dermatological, bronchopulmonary symptoms. The control group included 30 conditionally healthy individuals with a negative PCR result for SARS-CoV-2 RNA and lack of antibodies to the SARS-CoV-2 virus. The α₂-MG activity in serum samples of patients with coronavirus infection dramatically decreased, up to 2.5% of the physiological level. This was accompanied by an increase in the activity of the α₁-proteinase inhibitor, elastase- and trypsin-like proteinases by 2.0-, 4.4- and 2.6-fold respectively as compared with these parameters in conditionally healthy individuals of the control. In the post-COVID period, despite the trend towards normalization of the activity of inhibitors, the activity of elastase-like and especially trypsin-like proteinases in serum remained elevated. In overweight individuals, the increase in the activity of trypsin-like proteinases was most pronounced and correlated with an increase in the antibody titer to the SARS-CoV-2 virus. In the post-COVID period, the α₂-MG activity not only normalized, but also exceeded the control level, especially in patients with dermatological and neurological symptoms. In patients with neurological symptoms or with dermatological symptoms, the α₂-MG activity was 1.3 times and 2.1 times higher than in asymptomatic persons. Low α₂-MG activity in the post-COVID period persisted in overweight individuals. The results obtained can be used to monitor the course of the post-COVID period and identify risk groups for complications.

Augmented rescue of macroglobulins by supplementation of anti-snake venom with methanolic extract of Andrographis paniculata in Naja naja envenomation

Proteins of the macroglobulin family are prime targets of venom enzymes in snake bite. A massive reduction in the active concentration of these multifunctional proteins in snake bite, makes the living system vulnerable to dysregulation. This study investigates the ability of Indian polyvalent anti-snake venom (ASV), methanolic extract of Andrographis paniculata (MAP) and their combination in rescuing human alpha 2-macroglobulin (A2MG) and its homologues in rat plasma, from inactivation by Naja naja (N.N) venom enzymes. In-vitro experiments were conducted with heparinized human plasma and in-vivo experiments with female Wistar rats. Along with appropriate controls, there were 3 test groups in in-vitro and 8 test groups in in-vivo experiments. The in-vitro test groups were exposed to N.N venom for zero, 30 or 90 min prior to incubation with ASV or MAP or reduced ASV supplemented with MAP and incubated for 16 h at 37 °C. Chymotrypsin-bound esterase (CTBE) activity of A2MG was estimated. Rats were administered the venom intramuscularly and treated with ASV/MAP/ASV + MAP. CTBE activity of macroglobulin homologues was measured on day 1, 7 and 14. Survival of animals was noted. In human plasma, addition of ASV or MAP or ASV + MAP prevented loss of A2MG activity maximally to the extent of 88–100% (p = 0.001). In rats, reduced concentration of ASV supplemented with MAP showed complete rescue of macroglobulin homologues and 90% survival. The compulsive evidence from this study, underscores the merits of using this multipronged strategy in rescuing the macroglobulins and improving survival in envenomation due to N.N.

The Effects of Fasciola hepatica Infection on the Total Antioxidant Status (TAS) and the Activity of Proteases and Their Inhibitors in Rat Serum

Fasciola hepatica infection results in increased production of reactive oxygen species (ROS) and changes the activity/level of antioxidants in the host organism, which leads to oxidative stress formation and oxidative modifications of lipids and proteins. Taking this into account, the aim of this study was to assess the antioxidant potential and the activity of proteases and their inhibitors in the serum of rats infected with F. hepatica. Wistar rats were infected per os with 30 metacercariae of F. hepatica. The total antioxidant status (TAS) and the activity of cathepsin G and elastase and their inhibitors (alpha1-antitrypsin and alpha2-macroglobulin) were determined at 4, 7, and 10 weeks post infection (wpi). It was confirmed that F. hepatica infection leads to a decrease in the antioxidant capacity of serum, which was manifested as a reduction in total antioxidant status by about 24, 39, and 27%, respectively, at 4, 7, and 10 wpi. At the same time, the activity of proteases increased significantly: cathepsin G by about 25, 37, and 30%, and elastase by about 18, 16, and 9% during the course of F. hepatica infection, compared with the control group. However, the activity of alpha1-antitrypsin was significantly reduced, by 36, 55, and 25%, while alpha2-macroglobulin activity was reduced by about 14, 17, and 8% during the same period of fasciolosis. These results indicate that the shift in protease/antiprotease balance towards protease action observed during the course of fasciolosis may result in a decrease in host antioxidant capacity.

Binding of monofluorophosphate to alpha2-macroglobulin and C3.

After administering an oral dose of monofluorophosphate (MFP) to human beings or rats, a fraction of the drug appears in plasma that is bound to proteins, establishing a previously undetected compartment of nondiffusible fluoride. This article documents experiments performed in vitro, describing the binding of MFP to two plasma globulins: alpha2-macroglobulin and C3 (a beta-globulin). MFP binds irreversibly to these proteins through a stable bond. MFP binds to purified alpha2-macroglobulin or to C3 with a molar ratio MFP: protein close to unity. MFP binding reduces significantly the biological activity of these proteins, which share in common a macrocyclic 4-residue ring thiolactone (Cys-Gly-Glu-Glu). The binding site of MFP is as yet unknown. Protein-bound MFP appeared in the plasma of volunteers during the 5-7 hours following intake. Peak concentration of protein-bound MFP and maximal reduction of alpha2-macroglobulin activity was observed 2 hours after intake. Clearance of protein-bound MFP coincided with the return of alpha2-macroglobulin to basal levels.

Oxidative dissociation of human alpha 2-macroglobulin tetramers into dysfunctional dimers.

Human alpha 2-macroglobulin is a broad-spectrum, homotetrameric antiproteinase that can maximally bind up to two proteinase molecules in a ternary complex. Proteinases cleave the inhibitor within a peptide stretch termed the bait region and induce the emergence of internal thiol esters whose nucleophilic scission precede a major conformational change which entraps enzymes within molecular cages. In a previous study, leukocyte-generated hypohalous acids and N-haloamines were identified as the first examples of physiologically relevant inactivators of the antiproteolytic activity of alpha 2-macroglobulin (Reddy, V. Y., Pizzo, S. V., and Weiss, S. J. (1989) J. Biol. Chem. 264, 13801-13809), but the mechanisms whereby the oxidants damaged the inhibitor remained undefined. We now demonstrate that N-chloramines (RNCl) destroy the antiproteolytic activity of alpha 2-macroglobulin in an unusual biphasic process that results in the formation of inactive alpha 2-macroglobulin half-molecules. In the first phase, 8 eq of RNCl reacted with each alpha 2-macroglobulin subunit to generate a partially oxidized antiproteinase containing 8 methionyl sulfoxide residues/monomer. Structure-function analyses demonstrated that the oxidized inhibitor retained its homotetrameric structure as well as its ability to entrap proteinases. In marked contrast, the oxidation of an additional 6 methionyl residues and a single tryptophanyl residue fractured the alpha 2 M homotetramer across its non-covalent axis into two pairs of disulfide-linked dimers. Despite the fact that the oxidized dimers displayed normal bait regions whose cleavage by proteinases initiated thiol ester scission, all antiproteolytic activity was lost. Furthermore, the oxidized dimers were unable to undergo the critical conformational changes normally associated with bait region cleavage or thiol ester scission. Together, these results demonstrate that chlorinated oxidants destroy the antiproteolytic activity of alpha 2-macroglobulin by attacking a subset of methionyl and tryptophanyl residues whose oxidation mediates the dissociation of the native homotetramer into conformationally locked dimers.

Blood Coagulation, Fibrinolytic and Inhibitory Profiles in Renal Transplant Recipients: Comparison of Cyclosporine and Azathioprine

Renal transplant recipients treated with cyclosporine (CS) have been reported to be at increased risk of thrombotic complications. The present study was intended to examine the blood coagulation, fibrinolytic, and inhibitory systems in such patients. Eight transplant recipients on maintenance immunosuppression with CS and prednisone were studied. Five transplant recipients maintained on azathioprine (AZA) and prednisone and 32 normal volunteers served as controls. Plasma antigen concentrations and/or activities of various proteins in the above pathways were measured. Both the CS and AZA groups exhibited significant elevations of factor IX activity, von Willebrand factor (vWF), D-dimer, protein C and tissue type plasminogen activator (t-PA) levels when compared with the normal controls. In addition, CS group showed a significant elevation of alpha 2-macroglobulin activity and AZA group showed a significant reduction in factor XII activity when compared with the normal controls. Comparison of data from CS and AZA groups revealed higher factor XII activity and vWF concentration in the former group. In conclusion, transplant recipients treated with long-term cyclosporine and prednisone exhibited significant elevation of plasma vWF, D-dimer and protein C concentrations. In addition, both CS and AZA-treated transplant recipients showed increased plasma concentrations of D-dimer and t-PA. The latter observations suggest in vivo thrombin generation, fibrin formation and degradation.

4499186 Diagnosing autoimmune rheumatoid arthritis by measuring proteolytic activity of alpha2-macroglobulin: Marius C Teodorescu, Alexander M Gaspar, Gregory T Spear, Doina Ganea, John Skosey assigned to University of Illinois Board of Trustees

4499186 Diagnosing autoimmune rheumatoid arthritis by measuring proteolytic activity of alpha2-macroglobulin: Marius C Teodorescu, Alexander M Gaspar, Gregory T Spear, Doina Ganea, John Skosey assigned to University of Illinois Board of Trustees

Kinetics of the conformational alterations associated with nucleophilic modification of alpha 2-macroglobulin.

The effect of nucleophilic modification of alpha 2-macroglobulin (alpha 2M) with methylamine on the kinetics of sulfhydryl exposure was investigated. The generated sulfhydryl groups were detected with 4,4'-dithiodipyridine. The bimolecular rate constant for sulfhydryl exposure was determined to be 11.6 +/- 0.8 M-1 s-1 at 30 degrees C and pH 8.0. Treatment of alpha 2-macroglobulin with methylamine or proteases, such as plasmin and trypsin, results in a substantial increase in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonic acid. This probe was used to monitor the kinetics of the conformational change occurring in alpha 2-macroglobulin upon treatment with methylamine. It was found that the conformational change did not occur simultaneously with the cleavage of the thiol ester bonds by the nucleophile but, rather, the conformational alterations occurred following a lag phase. The data are consistent with a mechanism requiring the random cleavage of two thiol ester bonds of a dimeric unit in the molecule prior to the unimolecular process representing the conformational change. According to this model, the two dimeric units present in alpha 2M act as independent entities. On the basis of the best fit with the model, the bimolecular rate constant, for hydrolysis of the thiol ester bonds, was determined to be 11.9 +/- 0.7 M-1 s-1, while the rate constant for the conformational change was (9.7 +/- 2.0) X 10(-3) s-1. The measured rate of conformational change is rate limited by thiol ester cleavage at lower methylamine concentrations. The conformational change, measured with this fluorescence probe, approximately parallels the loss of trypsin binding activity of alpha 2-macroglobulin, measured by resistance of the bound trypsin to soybean trypsin inhibitor. A much slower loss of plasmin binding activity was observed than was found for trypsin, suggesting that the structural requirements on alpha 2M for the interaction with plasmin are disrupted much more slowly than the structural requirements for trypsin binding upon methylamine treatment of the molecule.

Amidolytic and immuno-nephelometric determination of alpha 1-proteinase inhibitor and alpha 2-macroglobulin in serum with calculation of specific inhibitor activities in health and disease.

In sera of healthy persons (n = 50) and patients with a variety of diseases (n = 197) the two major proteinase inhibitors, alpha 1-proteinase inhibitor (alpha 1-antitrypsin) and alpha 2-macroglobulin, were measured by two methods: a chromogenic (amidolytic) substrate assay to assess the functional activities, and a laser nephelometric method to determine the immunoreactive concentrations of the respective proteins. The specific proteinase inhibitor activities defined as the number of inhibitor units per g inhibitor protein were calculated. The precision and accuracy of both assays were found to be similar, showing a satisfactory correlation of results for the sera of healthy persons (r = 0.916 for alpha 2-macroglobulin and 0.988 for alpha 1-proteinase inhibitor). In diseased individuals the correlation was lower than in normal persons (0.862 for alpha 2-macroglobulin and 0.907 for alpha 1-proteinase inhibitor). A poor correlation was obtained in patients with liver diseases (r = 0.586 for alpha 1-proteinase inhibitor and 0.852 for alpha 2-macroglobulin). Reference ranges were established for functional and immunological concentrations and for specific inhibitor activities, respectively. Normal values followed a Gaussian distribution. In patients with various diseases including those with acute phase response, the specific inhibitor activities of alpha 1-proteinase inhibitor are reduced significantly; this is because inhibitor activity shows a smaller relative increase than immunoreactivity. Among the various diseases, no significant differences were noted. The specific inhibitor activity of alpha 2-macroglobulin changed significantly only in patients with carcinoma, liver diseases and trauma. Follow up of some patients shows also intraindividual variation of specific proteinase inhibitor activities.

Trypsin binding activity of alpha 2-macroglobulin in cystic fibrosis and other lung diseases.

Abnormalities of the interaction between alpha 2-macroglobulin and proteases have been suspected in cystic fibrosis. We measured the binding activity for trypsin of alpha 2-macroglobulin in 65 patients with cystic fibrosis, 41 obligate heterozygotes for cystic fibrosis, 18 children with asthma, 21 adult patients with chronic obstructive pulmonary disease, and 21 healthy control subjects. The assay was based on the observation that, once it is bound to alpha 2-macroglobulin, trypsin is no longer inhibited by soybean trypsin inhibitor. The bound trypsin retains activity against synthetic substrates such as N-alpha-benzoyl-arginine-p-nitroanilide. We found a moderately increased molar binding ratio of trypsin to alpha 2-macroglobulin in all patient groups and heterozygotes compared to healthy control subjects. The absolute concentration of alpha 1-macroglobulin was related to age rather than to the disease of the patient. Our data argue against a defect in protease-binding activity of alpha 2-macroglobulin in cystic fibrosis. The moderately increased binding activity observed could have been related to a structural difference in alpha 2-macroglobulin or to binding of ligands. This finding however, was not unique to patients with cystic fibrosis; the binding activity was increased in heterozygotes and in patients with other pulmonary diseases.

The Reversible Antithrombin Activity of Incubated Plasma

The thrombin time of normal citrated plasma is progressively prolonged on incubation in open glass tubes at 37 degrees C. The phenomenon is dependent on the temperature and duration of incubation, on the pH, and on the concentration of calcium ions present. Platelet-rich citrated plasma fails to exhibit augmented antithrombin activity when similarly incubated, and the addition of washed platelets to platelet-poor plasma inhibits this activity. The clot retarding action of incubated plasma against thrombin is also manifested against Arvin (Ancrod), but not against Reptilase-R. This thrombin time lengthening may be inhibited by incubation with anti-antithrombin III antiserum thus indicating that the phenomenon of thrombin time lengthening is consistent with enhanced activity of antithrombin III. It is unlikely that alterations in the activity of alpha2-macroglobulin, is important in the reduced thrombin-coagulability of incubated plasma. Interference with the polymerisation of fibrin monomers by the physico-chemical changes may contribute to the observed phenomenon.