FVIIa-sTF and Thrombin Inhibitory Activities of Compounds Isolated from Microcystis aeruginosa K-139

Abstract

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.

Keywords: LC-MS; Microcystis; aeruginosin K-139; blood coagulation cascade; fVIIa-sTF inhibitors; micropeptin K-139; microviridin B; thrombin.

Figure 1

FVIIa-sTF and thrombin inhibitors isolated from toxic M. aeruginosa K139. (A) aeruginosin K139: EC₅₀, µM: thrombin, 0.66; fVIIa-sTF, ~166. Reproduced with permission from Nakano and Harada, Study on non-ribosomal peptide synthesis of peptides by cyanobacteria, BS Thesis; Meijo University, 2003 [30]; (B) micropeptin K139:EC₅₀, µM: thrombin, 26.94; fVIIa-sTF, 10.62. Reproduced with permission from Nishizawa et al. Characterization of the locus of genes encoding enzymes producing heptadepsipeptide micropeptin in the unicellular cyanobacterium Microcystis. The Journal of Biochemistry; Oxford University Press, 2011 [25]; and from Nakano and Harada, Study on non-ribosomal peptide synthesis of peptides by cyanobacteria, BS Thesis; Meijo University, 2003 [30]; (C) microviridin B: EC₅₀, µM: thrombin, 4.58; fVIIa-sTF, no activity. Reproduced with permission from Nakano and Harada, Study on non-ribosomal peptide synthesis of peptides by cyanobacteria, BS Thesis; Meijo University, 2003 [30].

Figure 2

2D-NMR correlations of aeruginosin K139.

Figure 3

Advanced Marfey analysis of Leu in aeruginosin K139 using LC-MS in 25% acetonitrile with 0.1% formic acid to 65% acetonitrile with 0.1% formic acid over 45 min, TSKgel SuperODS (TOSOH) 100 × 2.0 mm, capillary temp 250 °C, 25 µL injection of 1 mg/mL. (A). Extracted Ion Chromatogram (EIC) with m/z 426–427 of dl-Leu-l-FDLA; (B). EIC of l-Leu-l-FDLA; (C). EIC of aeruginosin K139-l-FDLA; (D). m/z of dl-Leu-l-FDLA with a retention time (t_R, min) 12.1; (E). m/z of dl-Leu-l-FDLA with a retention time (t_R, min) 20.3; (F). m/z of l-Leu-l-FDLA with a retention time (t_R, min) 12.2; (G). m/z of aeruginosin K139-l-FDLA with a retention time (t_R, min) 12.5.

Figure 4

Rotational-frame nuclear Overhauser Effect SpectroscopY (ROESY) correlation of Choi.

Figure 5

ROESY correlation of argininal in hemiaminal form.

Figure 6

The structure of aeruginosin K139: l-Hpla-l-Leu-l-Choi-l-Argal.