Studies on thrombolytic enzyme nattokinase

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Nattokinase (NK) was prepared from the saline extract of natto as reported previously (H. Sumi et al., Experientia 43: 1110, 1987; Thromb. Haemostas. 62: 5409, 1989). The mol. wt. and pI were about 20,000 and 8.6, respectively. NK not only digested fibrin (with or without plasminogen) but also the plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251). While not reacting with the plasmin titrator p-nitrophenyl-guanidnobenzoate, purified NK reacted with N-trans-cinnamoylidazole, a papain or chymotrypsin titrator, 53.0 % active per molecule.The amino acid sequence of the purified preparation was determined from 9 peptides obtained by Lys-endopeptidase treatment ; NK was found to be a serine protease consisting of 275 residues with Ala at the N-terminal, and possessing no S-S molecular bond, in which it differs from previously known fibrinolytic enzymes, including urokinase, tissue plasminogen activator (t-PA) and plasmin. It had no "Kringle structure" seen in ordinary fibrinolytic enzyme molecul … More es. NK antibodies prepared in rabbits failed to respond to proteases (subtilisin) thus far isolated from Bacillus subtilis by Ouchterlony and ELISA methods.By WBCLT method, the poly-Glu rich fraction of natto was found to potentiate the plasma fibrinolysis. A crude material, containing 20,000 NK units and 113 mg/g poly-Glu, prepared from a high-NK natto product, was fed to 15 male Wister rats at 48,000 NK units/kg b.w. daily for 3 months. After dosing or in comparison with the non-dosed group, plasma ELT shortened significantly (p<0.01),and plasma amidolysis (pyro-Glu-Gly-Arg-pNA and H-D-Val-Leu-Lys-pNA,p<0.01) increased. Renal and pulmonary PA activities (p<0.1) increased, whereas APTT and Re-Ca^<++>T unchanged. The enteric coated capsules of NK (2.13 CU/mg of protein) were prepared. EFA was gradually increased from the 1st day to the 8th day after administration. FDP was statistically higher (p<0.001) on the 1st day of NK administration in comparison with pre-administration. The significant increase of t-PA antigen (p<0.005) was also proved over the long te rm (Clinical applications of NK capsule are now in progress under the permission of University Committee for Ethics).These results demonstrate that NK or NK-rich natto will be useful not only for the treatment of embolism but also for prevention of the disease,since they are safety and also canbemass-produced. Less