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.