In this study, we investigated the properties of proteolytic enzymes of two species of Aspergillus, Aspergillus flavus 1 (with a high degree of pathogenicity) and Aspergillus ochraceus L-1 (a conditional pathogen), and their effects on various components of the hemostasis system (in vitro) in the case of their penetration into the bloodstream. We showed that micromycete proteases were highly active in cleaving both globular (albuminolysis) and fibrillar (fibrin) proteins, and, to varying degrees, they could coagulate the plasma of humans and animals (due to proteolysis of factors of the blood coagulation cascade) but were not able to coagulate fibrinogen. The proteases of both Aspergillus fully hydrolyzed thrombi in 120–180 min. Micromycetes did not show hemolytic activity but were able to break down hemoglobin.

The high demand for keratinolytic enzymes and the modest presentation of fungal keratinase diversity studies in scientific sources cause a significant interest in identifying new fungal strains of keratinase producers, isolating new enzymes and studying their properties. Four out of the 32 cultures showed a promising target activity on protein-containing agar plates—Aspergillus amstelodami A6, A. clavatus VKPM F-1593, A. ochraceus 247, and Cladosporium sphaerospermum 1779. The highest values of keratinolytic activity were demonstrated by extracellular proteins synthesized by Aspergillus clavatus VKPM F-1593 cultivated under submerged conditions on a medium containing milled chicken feathers. The enzyme complex preparation was obtained by protein precipitation from the culture liquid with ammonium sulfate, subsequent dialysis, and lyophilization. The fraction of a pure enzyme with keratinolytic activity (pI 9.3) was isolated by separating the extracellular proteins of A. clavatus VKPM F-1593 via isoelectric focusing. The studied keratinase was an alkaline subtilisin-like non-glycosylated protease active over a wide pH range with optimum keratinolysis at pH 8 and 50 °C.

The effect of extracellular proteinases of A. flavipes A17, A. fumigatus D1, and A. sydowii 1 on proteins of the human haemostasis system was studied. It was shown that A. fumigatus D1 proteinases are able to hydrolyze a wide range of chromogenic peptide substrates of specific human proteinases of the haemostatic system. Proteinases formed by A. flavipes A17 and A. sydowii 1 have a narrow specificity, mainly to thrombin and plasmin substrates. It was first shown that proteinase of A. flavipes A17 is capable to activate protein C and Factor X. Extracellular proteinase produced by A. sydowii 1 has greater fibrinolytic activity as compared with proteinases produced by A. flavipes A17 and A. fumigatus D1.

Proteolytic cleavage of fibrin and fibrinogen under the action of proteases of microfungi Aspergillus alliaceus 7dN1 and A. terreus 2 was carried out. It was shown that both enzymes can have strong α-fibrinogenase and moderate β-fibrinogenase activity, practically without affecting the γ-chains of these molecules. The products of cleavage in the time interval up to 60 min are polypeptides with a molecular weight of 15 kDa or less.

Изучено антикоагулянтное действия in vitro протеазы, секретируемой микромицетом Aspergillus ochraceus L-1, и содержащихся в яде змей (препараты Protac® и RVV-X®) протеаз. Показана выраженность действия протеазы микромицета по отношению к плазмам человека и теплокровных животных, а также плазмам человека, дефицитным по определенным факторам системы гемостаза в сопоставлении с протеазами змей в реакциях с хромогенными пептидными субстратами активированного протеина C и фактора X, а также с помощью теста на активированное частичное тромбопластиновое время (АЧТВ). Были подобраны оптимальное время прединкубации протеазы микромицета А. ochraceus L-1 с плазмой крови человека (3 мин) и концентрация хромогенного пептидного субстрата активированного протеина C (от 0,1 до 0,5 мг/мл), необходимые для корректного определения протеина C.

Investigation of the Thrombolytic Potential of Exoproteinases Produced by the Micromycete Tolypocladium inflatum 62A Isolated from the Soils of the White Sea Исследование тромболитического потенциала экзопротеиназ, образуемых микромицетом Tolypocladium inflatum 62a, выделенным из грунтов Белого моря