The antithrombotic activities of superoxide dismutase and catalase are determined by their effects on reactive oxygen species. Modification of these enzymes with chondroitin sulphate enhances the effect due to accumulation of the derivatives on the surface of the vascular wall cells. We have shown that the effects of covalently modified biocatalysts exceed those of native enzymes, free chondroitin sulphate and their mixtures. The superoxide dismutase-chondroitin sulphate conjugate markedly reduced the thrombus mass, while the catalase-chondroitin sulphate conjugate predominantly preserved blood flow. The magnitude and duration of the antithrombotic activity of modified enzymes in a rat arterial thrombosis model allows one to expect a considerable protective effect after their combined application. A single-bolus intravenous injection of the combination between superoxide dismutase-chondroitin sulphate and catalase-chondroitin sulphate covalent conjugates had a significantly lower antithrombotic effect compared with that of the superoxide dismutase-chondroitin sulphate-catalase bienzymic covalent conjugate. This could be explained by different surface distribution of the conjugates in the circulation after their intravenous administration. Biomedical study of this approach promises a new therapeutic strategy of simple and effective protection of the vascular wall against various injuries with the use of the covalent conjugate superoxide dismutase-chondroitin sulphate-catalase. The review analyses the trends of combined application of enzyme preparations to enhance the effect of antioxidant therapy and to develop conjunctive courses of thrombolytic treatment.

The focus in antioxidant research is on enzyme derivative investigations. Extracellular superoxide dismutase (EC-SOD) is of particular interest, as it demonstrates in vivo the protective action against development of atherosclerosis, hypertension, heart failure, diabetes mellitus. The reliable association of coronary artery disease with decreased level of heparin-released EC-SOD was established in clinical research. To create a base for and to develop antioxidant therapy, various SOD isozymes, catalase (CAT), methods of gene therapy, and combined applications of enzymes are used. Covalent bienzyme SOD-CHS-CAT conjugate (CHS, chondroitin sulphate) showed high efficacy and safety as the drug candidate. There is an evident trend to use the components of glycocalyx and extra-cellular matrix for target delivery of medical substances. Development of new enzyme antioxidants for therapeutic application is closely connected with progress in medical biotechnology, the pharmaceutical industry, and the bioeconomy.

The progress in reperfusion therapy dictated the necessity for developing new tools and procedures for adjacent/additional therapy of acute cardiovascular disorders. The adjacent therapy is targeted on the damage of the microcirculation, leading to the unfavorable prognosis for the patients. The no-reflow phenomenon holds special place in the multifactorial etiology of the microcirculation disorders, offering a new challenge in treating the patients associated with ST-segment elevation on ECG at myocardial infarction. One of the numerous causes of no-reflow, the influence of the endothelial glycocalyx of the microcirculation, is analyzed. The results obtained in the studies of the endothelial glycocalyx ultrastructure are generalized, the effect that the fragments of the glycocalyx glycosaminoglycans have on the function of the vascular wall is demonstrated. The trends in searching for correlations between the thickness of the capillary glycocalyx and the cardiovascular disease risk are noted.

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Vascular wall protection can be achieved by preventive attachment to the vascular wall of antioxidants and elimination/neutralization of toxic products after their disproportioning. For this purpose we have prepared covalent conjugates between the vascular wall glycosaminglycan chondroitin sulfate (CHS) and the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). The following conjugates were obtained: SOD-CHS, CAT-CHS and SOD-CHS-CAT. Their anti-thrombotic activity was compared in a rat model of arterial thrombosis by measuring the time of occlusion emergence and thrombus mass. It is noteworthy that the effectiveness of single bolus injections of SOD-CHS/CAT-CHS mixture was much lower than that of the bienzymic SOD-CHS-CAT conjugate. The conjugate SOD-CHS-CAT proved to be anti-thrombotically effective in doses two orders of magnitude lower than the native biocatalysts and an order of magnitude lower than SOD-CHS and CAT-CHS derivatives. For effective anti-thrombotic protection in oxidative conditions it is important to maintain the stable connection of SOD and CAT activity on the vascular wall and the large size of these conjugates. Covalent conjugate SOD-CHS-CAT is the best prospect for pharmaceutical development.

In acute cardiovascular lesions, functional recovery of the myocardium occurs when adequate reperfusion of the infarct-related artery and an adequate level of microcirculation and tissue flow in the lesioned area are achieved. The major regulators of these effects are the glycosaminoglycan component of the endothelial cellular coating (glycocalyx, extracellular matrix, interstitial material) and its catabolic enzymes and their inhibitors. The causes and consequences of glycocalyx abnormalities in vascular lesions are considered, along with the potential for glycocalyx reconstruction. The glycoprotein environment of cells has been found to regulate the biomechanical properties of vessels, tissue assembly and repair; it was also able to bind low-and high-molecular-weight ligands. Hydration of glycosaminoglycans determines the development of tissue edema and mediates the anticoagulant activity of the extracellular matrix. Binding of chemokines, growth factors, other proteins, and lipoproteins to glycosaminoglycans has been noted in relation to execution of their regulatory functions. The existence of special structural biding sites for such reactants has been demonstrated, along with a relationship between the biological effects induced by glycosaminoglycans and their molecular weights. The involvement of glycosaminoglycans in the pathophysiological processes occurring in vascular lesions is reviewed, as are promising approaches to regulating the state of the pericellular coatings via the precise and effective control of the level of glycosylation of biological substrates. 553 0091-150X/08/4210-0553

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Properties of native and aldehyde dextran-modified hyaluronidase (with surface amino group modification about 98%) were investigated. Optimal endoglycosidase activity of the native enzyme was observed at 0.15 M NaCl and pH 5.5 and electrostatic interactions influenced the enzyme activity. The inhibitory effect of heparin on hyaluronidase activity slightly differed at pH 5.5 (1.5-fold inhibition) and 7.5 (1.2-fold inhibition). Ionic strength of the reaction medium only slightly influenced the effect of heparin. Modification of hyaluronidase with dextran increased hydrophobic interactions and steric hindrance. Conjugation with dextran increased the resistance of hyaluronidase activity to denaturing agents (urea, guanidinium hydrobromide) and extended the optimal conditions for maximal endoglycosidase activity (pH 4.5-6.5, the range of NaCl concentration from 0.1 to 0.3 M). The conjugation also reduced electrostatic effects on the active site of hyaluronidase and efficacy of heparin inhibition. At pH 7.5 the enzyme was almost insensitive to heparin. The resistance of dextran-modified hyaluronidase to heparin points to approaches for subsequent studies of the heparin-binding site of this enzyme and biomedical trial of the stabilized enzyme for the treatment of acute cardiovascular lesions.