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The rise of bleeding and bleeding complications caused by oral anticoagulant use are serious problems nowadays. Strategies that block the initiation step in blood coagulation involving activated factor VII-tissue factor (fVIIa-TF) have been considered. This study explores toxic Microcystis aeruginosa K-139, from Lake Kasumigaura, Ibaraki, Japan, as a promising cyanobacterium for isolation of fVIIa-sTF inhibitors. M. aeruginosa K-139 underwent reversed-phase solid-phase extraction (ODS-SPE) from 20% MeOH to MeOH elution with 40%-MeOH increments, which afforded aeruginosin K-139 in the 60% MeOH fraction; micropeptin K-139 and microviridin B in the MeOH fraction. Aeruginosin K-139 displayed an fVIIa-sTF inhibitory activity of ~166 µM, within a 95% confidence interval. Micropeptin K-139 inhibited fVIIa-sTF with EC50 10.62 µM, which was more efficient than thrombin inhibition of EC50 26.94 µM. The thrombin/fVIIa-sTF ratio of 2.54 in micropeptin K-139 is higher than those in 4-amidinophenylmethane sulfonyl fluoride (APMSF) and leupeptin, when used as positive controls. This study proves that M. aeruginosa K-139 is a new source of fVIIa-sTF inhibitors. It also opens a new avenue for micropeptin K-139 and related depsipeptides as fVIIa-sTF inhibitors.

Bioassay-guided investigation of the cyanobacterium Anabaena compacta extracts afforded spumigin J (1) and the known thrombin inhibitor spumigin A (2). The absolute configuration of 1 was analyzed by advanced Marfey's methodology. Compounds 1 and 2 inhibited thrombin with EC(50) values of 4.9 and 2.1 μM, and 0.7 and 0.2 μM in the cathepsin B inhibitory assay, respectively. The MM-GBSA methodology predicted spumigin A with 2S-4-methylproline as the better thrombin inhibitor.

The blood coagulation cascade involves the human coagulation factors thrombin and an activated factor VII (fVIIa). Thrombin and fVIIa are vitamin-K-dependent clotting factors associated with bleeding, bleeding complications and disorders. Thrombin and fVIIa cause excessive bleeding when treated with vitamin-K antagonists. In this research, we explored different strains of toxic Microcystis aeruginosa and cyanobacteria blooms for the probable fVIIa-soluble Tissue Factor (fVIIa-sTF) inhibitors. The algal cells were subjected to acidification, and reverse phase (ODS) chromatography-solid phase extraction eluted by water to 100% MeOH with 20%-MeOH increments except for M. aeruginosa NIES-89, from the National Institute for Environmental Studies (NIES), which was eluted with 5%-MeOH increments as an isolation procedure to separate aeruginosins 89A and B from co-eluting microcystins. The 40%–80% MeOH fractions of the cyanobacterial extract are active against fVIIa-sTF. The fVIIa-sTF active fractions from cultured cyanobacteria and cyanobacteria blooms were subjected to liquid chromatography-mass spectrometry (LC-MS). The 60% MeOH fraction of M. aeruginosa K139 exhibited an m/z 603 [M + H]+ attributed to aeruginosin K139, and the 40% MeOH fraction of M. aeruginosa NIES-89 displayed ions with m/z 617 [M − SO3 + H]+ and m/z [M + H]+ 717, which attributed to aeruginosin 89. Aeruginosins 102A/B and 298A/B were also observed from other toxic strains of M. aeruginosa with positive fVIIa-sTF inhibitory activity. The active fractions contained cyanobacterial peptides of the aeruginosin class as fVIIa-sTF inhibitors detected by LC-MS.