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. 2015 Dec 30;14(1):7.
doi: 10.3390/md14010007.

Determination of FVIIa-sTF Inhibitors in Toxic Microcystis Cyanobacteria by LC-MS Technique

Affiliations

Determination of FVIIa-sTF Inhibitors in Toxic Microcystis Cyanobacteria by LC-MS Technique

Andrea Roxanne J Anas et al. Mar Drugs. .

Abstract

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.

Keywords: aeruginosins; anticoagulant; blood coagulation cascade; cyanobacteria; fVIIa-sTF inhibitors; peptides; toxic Microcystis.

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Figures

Figure 1
Figure 1
Compounds active against fVIIa.
Figure 2
Figure 2
LC-MS profiles of M. aeruginosa K139 and NIES-89 cyanobacteria fVIIa-sTF active fractions: (a) Total ion chromatogram (TIC) of M. aeruginosa K139–60% MeOH; (b) TIC of M. aeruginosa NIES-89–40% MeOH; (c) TIC of M. aeruginosa NIES-89–45% MeOH; (d) Mass spectrum (MS) of M. aeruginosa K139–60% MeOH, retention time (tR) 7.3–8.5 min; (e) MS of M. aeruginosa NIES-89–40% MeOH, tR 8.8–9.7 min; (f) MS of M. aeruginosa NIES-89–40% MeOH, tR 6.0–7.9 min; (g) MS of M. aeruginosa NIES-89–45% MeOH, tR 7.9–11.3 min.
Figure 3
Figure 3
LC-MS profiles of fVIIa-sTF active cyanobacterial extracts from algal blooms. (a) Extracted ion chromatogram (EIC m/z 600–800) of JX-1-5 (from Ibaraki)–40% MeOH; (b) EIC m/z 600–800 of JX-1-5–60% MeOH; (c) EIC m/z 600–800 of Koyaike site 2–60% MeOH; (d) EIC m/z 600-800 of Koyaike site 3–40% MeOH; (e) ESI-full MS of JX-1-5–40% MeOH, tR 8.2–10.2 min; (f) ESI-full MS of JX-1-5–60% MeOH, tR 8.1–11.7 min; (g) ESI-full MS of Koyaike site 2–60% MeOH, tR 5.4–14.5 min; (h) ESI-full MS of Koyaike site 3–40% MeOH, tR 0.01–13.7 min.
Figure 4
Figure 4
Aeruginosins detected by LC-MS.

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