1. Gene Analysis, Cloning, and Heterologous Expression of Protease from a Micromycete Aspergillus ochraceus Capable of Activating Protein C of Blood Plasma
Sergei K Komarevtsev, et al. Microorganisms. 2021 Sep 11;9(9):1936. doi: 10.3390/microorganisms9091936.
Micromycetes are known to secrete numerous enzymes of biotechnological and medical potential. Fibrinolytic protease-activator of protein C (PAPC) of blood plasma from micromycete Aspergillus ochraceus VKM-F4104D was obtained in recombinant form utilising the bacterial expression system. This enzyme, which belongs to the proteinase-K-like proteases, is similar to the proteases encoded in the genomes of Aspergillus fumigatus ATCC MYA-4609, A. oryzae ATCC 42149 and A. flavus 28. Mature PAPC-4104 is 282 amino acids long, preceded by the 101-amino acid propeptide necessary for proper folding and maturation. The recombinant protease was identical to the native enzyme from micromycete in terms of its biological properties, including an ability to hydrolyse substrates of activated protein C (pGlu-Pro-Arg-pNA) and factor Xa (Z-D-Arg-Gly-Arg-pNA) in conjugant reactions with human blood plasma. Therefore, recombinant PAPC-4104 can potentially be used in medicine, veterinary science, diagnostics, and other applications.
2. Serine protease specificity for peptide chromogenic substrates
L E Mattler, N U Bang Thromb Haemost. 1977 Dec 15;38(4):776-92.
Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160 (N-Bz-Phe-Val-Arg-pNA, HCl), S-2238 (H-D-Phe-Pip-Arg-pNA, 2HCl), S-2222 (N-Bz-Ile-Glu-Gly-Arg-pNA, HCl), and S-2251 (H-D-Val-Leu-Lys-pNA, 2HCl) from AB Kabi Peptide Research and Chromozym TH (Z-Gly-Pro-Arg-pNA, HCl) from Pentapharm Limited were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha thrombin, and bovine factor Xa. S-2160, S-2238, and Chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All 3 substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin. In contrast, S-2222 is sensitive to Xa and insensitive to thrombin. S-2251 is relatively plasmin-specific, being resistant to the clotting enzymes thrombin and Xa. S-2251 exhibits even greater sensitivity to the SK-plasmin complex than to plasmin. In addition, the substrate Chromozym PK (N-Bz-Pro-Phe-Arg-pNA, HCl) was evaluated and found to be relatively specific for plasma kallikrein. Assays for antithrombin III and heparin using S-2222 as the substrate and factor Xa as the enzyme, plasma plasminogen and plasmin inhibitors using S-2251 as the substrate, and plasma prekallikrein and kallikrein inhibitors using Chromozym PK as the substrate have been developed. Synthetic peptides mimicking amino acid sequences adjacent to proteolytic activation cleavage of plasma serine protease precursors appear to be sensitive and relatively specific tools applicable to kinetical and clinical studies of these enzymes and their inhibitors.
3. Isolation of anticoagulant from the venom of tick, Boophilus calcaratus, from Uzbekistan
Toshio Motoyashiki, Anthony T Tu, Djaloliddin A Azimov, Kazakov Ibragim Thromb Res. 2003 Jun 1;110(4):235-41. doi: 10.1016/s0049-3848(03)00409-2.
Boophilus calcaratus is a tick found in Central Asia and a common parasite to domestic animals. Venom from this tick was fractionated by two-step column chromatography, Sephadex G-75, and DEAE-Sephadex A-25. The homogeneity of the anticoagulant was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified component is named calcaratin and has a molecular weight of 14,500. The effect of the purified anticoagulant component (calcaratin) on various sites of the blood coagulation cascade scheme was examined and compared with crude venom. The chromogenic substrates S-2238 (H-D-Phe-Pip-Arg-pNA 2HCl) for thrombin and S-2765 (N-alpha-Z-D-Arg-Gly-Arg-pNA 2HCl) for factor Xa were also investigated. Activated partial clotting times were all prolonged, suggesting the anticoagulation nature of the purified component and crude venom. Prolongation of fibrinogen clotting time (FCT) is highly suggestive of the antithrombin property of the purified component and its original venom.
