Dissertation is devoted to development of technique for platelet aggregation inhibitor and acid phospholipase A2 obtaining from Gloydius (G.) blomhoffii brevicaudus venom, with condition of its integration into one cycle of the general venom processing technology. Manufacturing of pharmaceutical products based on snake venom biologically active compounds requires incoming quality control because of the diversity of venom composition. At the same time, opposite functions of target proteins in the mixture complicate their identification. We developed new methods for incoming quality control for effective identification of biologically active compounds in Gloydius snake venom. Using the method, we provided comparative characteristic for venoms of several Gloydius species. Prothrombin activator was found in A. blomhoffii ussuriensis venom for the first time. Manufacturing incoming quality control of G. blomhоffii brevicaudus venom proved it to be a source for fibrino(geno)lytic enzyme, thrombin-like enzyme, protein C and prothrombin activators, platelet aggregation inhibitor and acid phospholipase A2 production. For the effective production of these proteins, it was targeted to purify all of them in one cycle of venom processing technology. This study describes development of obtaining method for platelet aggregation inhibitor and acid phospholipase A2 integrated to the general technology used. Processing of complex protein mixtures requires separation of proteins with similar physical - chemical characteristics. We developed experimental techniques for effective fractionation of such molecules with ion exchange chromatography. In the result of 15 different variants of ion exchange separation, we revealed optimal process conditions for separation of several biological active compounds from snake venom. It was shown, that contrary to the existing conceptions, decrease of the ratio of sorbent layer geometric parameters (diameter to height) increases effectiveness for separation of proteins with similar physical - chemical characteristics. Obtained results are important for complex manufacturing technologies development for multi-protein/peptide mixtures. Furthermore, highest possible protein separation achieved in the minimal sorbent layer in industrial applications is used for step-gradient elution calculations. Using received data we developed a new chromatographic method for obtaining of highly purified platelet aggregation inhibitor and acid phospholipase A2 from G. blomhоffii brevicaudus venom. The method integrated into general venom processing technology including several other proteins purification, such as fibrino(geno)lytic enzyme, thrombin-like enzyme and protein C activator. Physical - chemical properties of purified platelet aggregation inhibitor and acid phospholipase A2 were investigated with 1D-, 2D-electrophoresis and MULDI-TOF mass spectrometry. Obtained platelet aggregation inhibitor was a single chain protein with molecular weight - 12,00 kDa, isoelectric point - 8.03, effectively inhibiting ADP-induced platelet aggregation with IC50% = 3.26?10-6 g or 5,42?10-10 M and exposing no enzymatic activity. Purified phospholipase A2 has molecular weight - 13,76 kDa, isoelectric point - 4.17, pH optimum - 8.5, activity - 2876 IU?mg of protein, dose-dependently inhibiting ADP-induced platelet aggregation with IC50% = 6,85 ?10-6 g +or 1?10-9 M and showing strong toxic effect on PC-12 cell line. General cytotoxity of Gloydius venoms correlated with phospholipase A2 content and could be used as a characteristic for incoming quality control.
